a a ‘ . :31 v 5... yak. L ..m.. .. 2+... ' . AMWHWQ- : nfli‘ ‘ .n r b. n. n F. 53.22: . , ‘ . A. .. . .. . Lg . n u . . ‘ .. . .. . . . . , , , ... ‘ MpnwWN. . x am“... I .. c ‘ . smgbfi. m: 2% 3.. 4 J u’bikt I ' .utctvill. . \ttcli‘n urflllIltKvli ‘10! vi \: d.|~3“¢. .: Illlllllllllllllllll11H“lllllllllllllIllllllllllllllllll 3 1293 02080 / L DICK 1 This is to certify that the thesis entitled CHARACTERIZATION AND IDENTIFICATION OF AN ENTEROCOCCAL ISOLATE AND ITS BACTERIOPHAGE presented by Sally Elizabeth Burns has been accepted towards fulfillment of the requirements for M.S. degree in Animal Science ,/ / / 4/ / Majoflmr U Date _S£.p1'_emhe.r_].0,_l.9.99 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution LlBRARY Michigan State Universlty TO AVOID FINB return on or before date due. MAY BE RECALLED with earlier due date if requested. PLACE IN RETURN BOX to remove this checkout from your record. DATE DUE DATE DUE DATE DUE moo mummmspu -——-—-——_—— CHARACTERIZATION AND IDENTIFICATION OF AN ENTEROCOCCAL ISOLATE AND ITS BACTERIOPHAGE By Sally Elizabeth Burns A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE College of Agriculture and Natural Resources Department of Animal Science 1999 ABSTRACT CHARACTERIZATION AND IDENTIFICATION OF AN ENTEROCOCCAL ISOLATE AND ITS BACTERIOPHAGE By Sally Elizabeth Burns The purpose of this study was to Characterize and identify the isolate BAW#1, and to Characterize and Classify a bacteriophage associated with that isolate. Identification and metabolic profile analysis using BIOLOGTM, the API-STREP kit, and conventional testing identified the isolate as Enterococcus casseliflavus. Analysis of 16srRNA sequence data supported the identification of the isolate BAW#1 as a member of the E. casseliflavus-E. gallinarum group., The major fermentation product of the isolate under anaerobic conditions was lactic acid. The bacteriophage was examined with a transmission electron microscope after two different preparation methods. The phage has a noncontractile tail and an elongated head. Tail fibers and a base plate were visible. The size of the phage capsid measured 90 nm by 36 nm, the tail was 136 nm by 9 nm. Nucleic acid was extracted from the phage and a restriction digest carried out. Therefore, the phage has double stranded DNA. The phage belongs to the Siphoviridae family. Acknowledgments I gratefully acknowledge the support Dr. James Jay and the Office of Diversity and Pluralism, the Department Of Animal Science, the College of Agriculture and Natural Resources. I would like to thank Dr. Mel Yokoyama for facilitating my entry into graduate school after a long absence and for going the distance with me. I would like to thank Dr. Julius Jackson and Dr. Paul Coussens for their patience and support. Dr. Karen Klomparens and Dr. John Heckman have inspired and nourished my aspiration to become an electron microscopist. I appreciate the help of Dr. Bob Walker of AHDL, and Dr. Loren Snyder and Dr. John Urbance of Microbiology. I My cohort in the lab, Faith Gandiya, Ruby Bato and Sunghe Park were comic relief and tremendous support. I would most especially like to thank my dear friends Karen Quinn, Mary Burns and Ann Flescher who have inspired and supported me. iii TABLE OF CONTENTS Page List of Tables vi List of Figures viii List of Abbreviations ix Introduction 1 Chapter One: Characterization and Identification of the Isolate BAW#1 5 Obj ectives 3 1 Materials and Methods 32 Results 40 Discussion 59 Summary 65 Chapter No: Characterization of an Enterococcal Bacteriophage 67 Objectives 75 Materials and Methods 76 Results 83 Discussion 99 Summary 104 Bibliography 105 iv Appendix I Appendix 1 1 Appendix III Appendix IV Media used for growth and examination of the isolate BAW# 1 Dendrogram showing phylogenetic relationship of the isolate BAW# 1 to organisms in the RDP database in 1995 Alignment of 16s rRNA sequence of isolate BAW#I with Closely sequences of E. casseliflavus and E. gallinarum strains. Endnotes 117 123 124 130 Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 1 1 LIST OF TABLES Transfers of species from the genus Streptococcus to the new genus Enterococcus 1984 to 1998. Reference Source for 16s rRNA Sequences of Species in the genus Enterococcus. Growth characteristics of the isolate BAW#l in GCS-RF broth: Optical Density and Colony Forming Units charted against time Fermentation Products of the isolate BAW # 1 and Streptococcus bovis as determined by High Pressure Liquid Chromatography BIOLOG TM SYSTEM Substrate utilization profile of the isolate BAW # 1. PART 1 Substrates the isolate was unable to utilize. (mean utilization value lessthan 2). BIOLOG TM SYSTEM Substrate utilization profile of the isolate BAW # 1. PART 2 Substrates the isolate was able to utilize weakly. (mean utilization value between 2 and 8). BIOLOG TM SYSTEM Substrate utilization profile of the isolate BAW # 1. Part 3. Substrates the isolate was able to utilize in the BIOLOG:TM system (mean utilization value greater than 8). Similarity and Distance of the E. casseliflavus isolate as determined by BIOLOG“M software for the isolate. The first data set is based on BIOLOG TM software release 3.5; the second is based on BIOLOG TM software release 3.7. Metabolic profile of the isolate BAW #1 and related species as determined by API StrepTM. Antibiotic Sensitivity of the isolate BAW # 1. Phylogenetic relationship of the enterococci and the isolate BAW#I based on 16S rRNA sequence analysis by the ARB Fast DNA ml program. vi Page 10 19 42 44 47 48 49 50 55 56 58 Table 12 Table 13 LIST OF TABLES-continued Phenotypic differentiation of E. casseliflavus from closely 60 related enterococci with test results of the BAW# 1 isolate Measurements of the phage infective of isolate BAW#l. 87 Phage stained with uranyl acetate and examined in Phillips CM 10 Transmission Electron Microscope. All measurements are in nanometers. vii Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 LIST OF FIGURES Dendrogram showing the similarities between strains based on sequence homologies and clustering by the unweighted pair group method. A 168 rRNA Derived Dendrogram of 14 species of the genus Enterococcus by Williams et al 1991. Figure 2. Growth data for isolate BAW #1 grown in GCS- RF anaerobically: Colony Forming Units and Time. BIOLOGTM generated dendrogram of the isolate BAW #1 showing metabolic proximity to E. casseliflavus. Phylogenetic relationship of the enterococci and the isolate BAW#I based on 16S rRNA sequence analysis by the ARB Fast DNA ml program. Figure 5. Bacteriophage DNA extracted from the isolate BAW# 1 and cut with the restriction endonuclease HindIII (4)) The DNA of the phage lambda is used as a standard for size determination (A). Phages harvested from the isolate BAW #1 and stained with uranyl acetate prior to examination in the transmission electron microscope Phages harvested from the isolate BAW #1 and stained with uranyl acetate prior to examination in the transmission electron microscope. The phages in these micrographs show positive and negative staining reactions. Phages harvested from the isolate BAW #1 and shadow cast with platinum prior to examination in the transmission electron microscope. The micrographs show the isolate BAW #1 prior to, during and after attack by the bacteriophage. All grids were stained with UA for 15 seconds. viii Page 20 42 51 57 85 92 94 96 98 List of Abbreviations ADM AHDL Bp C dH2O GCS-RF GCSX-RF HPLC Hz Kb m1 nm 0D600 rpm str TMV TSA TSB UA ul anaerobic dilution media Animal Health Diagnostic Laboratory base pair degrees centigrade distilled water Glucose-cellulose—starch-rumen fluid media Glucose-cellulose-starch-xylose-rumen fluid media high pressure liquid Chromatography Hertz kilobase IOOObase pairs Milliliter Nanometer Optical density measured at a wavelength of 600nm Revolutions per minute Strains Tobacco mosaic virus Trypticase soy agar Trypticase soy broth Uranyl acetate Microliter ix INTRODUCTION A bacterium and bacteriophage were recovered in a laboratory conducting research on rumen microbiology. The bacterium was thought to be a wild strain Of Ruminococcus albus (Tadese 1993). The Rumincoccus are well known gram-positive anaerobic rumen microorganisms. They are significant for their role in the breakdown of cellulose in the rumen. Workers at the University of Illinois had also been working on the phage: bacterium pair, and had sequenced the 16s rRNA of the isolate. Based on this, using the Ribosomal Database Project sequence analysis programs, they had identified the isolate as Enterococcus saccharolyticus (Lawes 1995). The isolate was entered into the 16s rRNA databases as str.BAW1. It has been assigned the ascension U30931. The species Of the genus Ruminococcus are gram positive, anaerobic, cellulolytic, cocci. The genus Enterococcus is composed of gram positive, facultative anaerobic cocci. The first objective of this study was to test the previous identification of the isolate as a wild isolate of R. albus or as E. saccharolyticus. A preliminary battery of tests was devised to answer this question. These tests were oxygen sensitivity and fermentation product analysis. Fermentation product analysis is an important tool in the identification of anaerobic bacteria. This analysis provides data indicative of the major metabolic pathways in bacteria. The members of the genus Ruminococcus produce acetate, ethanol and H 2 in pure culture (Iannotti et al 1973). Fermentation product analysis is not used in the identification of streptococcus, enterococcus and closely related species; in fact these species are all homofermentative. Anaerobically, the major fermentation product of the isolate was lactic acid. Aerobically, acetic acid, lactic acid and ethanol were produced. These are the fermentation products expected from members of the gram-positive facultatively anaerobic cocci. The isolate was tested for oxygen tolerance. It was found to be facultatively anaerobic. Based on these Observations it became clear that the isolate was not a member of the genus Ruminococcus. The identification of the isolate as E. saccharolyticus was tested. To determine if the isolate was actually E. saccharolyticus, or some other facultative cocci, a battery of phenotypic tests was used. The gram positive facultatively anaerobic cocci contains many genera, including Abiotrophia, Aerococcus, Camobacterium, Dolosigranumum, Enterococcus, Globicatella, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus and Vagaococcus. The isolate was tested with the microbial identification tools BIOLOGTM and API-STREP. The identification of the isolate as a member of the genus Enterococcus was affirmed, however, these tests named the isolate E. gallinarum and E. faecium respectively. The proprietary characterization schemes of API- STREP and BIOLOG TM were examined and compared with literature on the metabolic profiles of the various species. The above tests were supplemented with conventional tests suggested in Bergey’s manual. A review of the characterization schemes found in the literature was undertaken to help resolve the identity Of the isolate. The 16s rRNA based identification was compared to a more complete compliment of enterococcal sequences using the Ribosomal Database Project program Similarity Rank. Sequences of type strains of the E. gallinarum, E. saccharolyticus, E. faecium, E. ntundtii and E. casseltflavus were compared to the sequence of isolate BAW#l. Preliminary research on the phage concluded that the phage was lysogenic to the bacterial isolate. The phage was thought to be infective of gram positive and gram negative bacteria. The preliminary research showed a tailed icosahedral phage, similar in appearance to the well known phage lambda (Tadese 1993). Lawes found the phage lysate to contain two morphotypes, an icosahedral lambda like phage, and a phage with an elongated head and a noncontractile tail. Lawes tested the phage lysate on a battery Of rumen microorganisms, no evidence of infection was found(Lawes 1995). Because of the discrepancy in descriptions of the phage morphology, bacteriophage recovered from lytic infection of the isolate BAW#I was examined using the transmission electron microscope. The lytic nature of the phage was tested. CHAPTER ONE Characterization and Identification Of the Isolate BAW#l Literature review: The streptococci are a well known, if ill defined genera of the gram- positive cocci. Within the streptococci, the enterococci have historically been considered a sub generic grouping, well known members Of this group are Enterococcus (Streptococcus) faecalis and Enterococcus (Streptococcus) faecium. 16s rRNA sequences have been determined for most of the enterococcus as well as many other gram-positive cocci. Based on this 16S rRNA sequence data as well as earlier DNA/ DNA homology studies, the Enterococci have been established as a genus and placed in the clostridial subdivision of the gram-positive bacteria. This subdivision includes the genera Aerococcus, Camobacten’um, Globicatella, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, Vagaococcus, Dolosigranumum and Abiotrophia. Within the clostridial subdivision, the Enterococci form a distinct cluster with the Vagoooccus, Tetragenococcus and Camobacterium (Hardie and Whiley 1997) What 15 years ago was simply known as the genus Streptococcus has undergone significant phylogenetic Changes. In 1984 Enterococcus and Lactococcus were split Off as new genera from Streptococcus. S. faecium and S. faecalis of the group D Streptococci were redefined based on DNA-rDNA homology and Oligonucleotide cataloging of 165 rRNA in 1984 (Schleifer and Klipper-Balz 1984). The genus Enterococcus is defined as gram positive, spherical to ovoid facultative anaerobes occurring in Chains or pairs. The major fermentation product from utilizable carbohydrates is lactate (Holt et al. 1993). Key distinguishing characteristics include ability to grow in 6.5 percent NaCl, at pH 9.6, at 10° C and 45 ° C, and to hydrolyze esculin in the presence of 40% bile. However, several species including E. saccharolyticus do not conform to these Characteristics (Williams et a1 1991) Additionally, defining characteristics of the genus Enterococcus are: “Cells occur singly, in pairs, or in short chains, and are frequently elongated in the direction of the chain. Endospores are not formed. May be motile. Optimum growth temperature, ca. 35 ° C. Most strains survive heating at 60 ° C for 30 minutes. Hydrolyze pyrrolidonly-fS-napthylamide. Chemoorganotrophs. Metabolism fermentative. The predominant end product of glucose fermentation is L-lactic acid. Oxygen or other hydrogen acceptors may alter the end products of carbohydrate metabolism. Hydrogen peroxide may or may not accumulate in the presence of oxygen. Do not contain heme compounds. Benzidine negative and usually catalase negative, but some strains contains pseudo catalase. Some strains synthesize cytochromes or catalase or both when they are provided with hemin. The minimal nutritional requirements are generally complex. React with Group D antisera; Some strains also react with group Q antisera. Some strains posses respiratory quinones (menaquinones or dimethylmenaquinones). Long chain fatty acids are predominantly of the straight chain saturated or monounsaturated types; some strains produce cyclopropane ring acids. Peptidoglycan type: Lys- D-Asp or Lys-Ala2-3 . G+C content of the DNA ranges from 37 to 45%” (Schleifer and Klipper-Balz 1984). The type species for the newly defined genus was defined as E. faecalis. The enterococci are best known for inhabiting the gastrointestinal tract, where they are usually not pathogenic. They are extremely versatile, mesophilic bacteria, and are capable of living in water and on plants. They are formidable Opportunistic pathogens, and are implicated in disease Of man, livestock and birds. Some species are used in cheese and yogurt production. An important feature of the enterococci is their genomic plasticity. They are known to posses conjugative transposons, pheromone responsive plasmids and broad host range plasmids (Murray 1998). Many members of the Streptococcus have been reassigned to the Enterococcus over the last fifteen years. Newly discovered species have also been assigned to the genus Enterococcus (Table 1). Characterization of the species E. Saccharoluticus. Data from researchers at the University of Illinois determined the 16s rRNA sequence of the isolate used in this research. Analysis of this sequence using the software of the Ribosomal Database Project led to the identification of the isolate as E. saccharolyticus. For this reason, the defining characteristics of E. saccharolyticus are presented here. The species E. saccharolyticus was named in 1984. Analysis of DNA base composition and DNA / DNA homology studies were undertaken to determine the relationship of strains of S. bovis, S. equinus and related species. This work found many strains with low homology which were grouped as the same species, and others with high homology were being differentiated into separate species. Of 85 strains examined, six new groupings were distinguished. Two new species were defined, S. alactolyticus and S. saccharolyticus. Significantly the type strains of S. bovis and S. equinus were regrouped into the single species S. equinus, which was redefined. Strains isolated from straw bedding, cow belly swab, teat swab and rectal swab formed a tight genetic group with 80- 100% DNA/DNA homology. Physiological and biochemical criteria also distinguish this group from others in the study (Farrow et al. 1984). The species name S. saccharolyticus was proposed for the group which also is characterized as: “Colonies on blood agar or nutrient agar are circular, smooth and entire. Non-pigmented. Non hemolytic. Cells are gram-positive mostly in pairs or short chains. Non-motile. Facultatively anaerobic. Catalase-negative. Growth at 10C and 45C; optimum ca.37C. No growth at 50C: does not survive heating at 60C for 30 min. Weak growth in 6.5% NaCl. Chemoorganotroph: metabolism fermentative. Acid and clot produced in litmus milk. Does not react with Lancefield group D antiserum. G+C content of DNA ranges from 37.6 to 38.3 Non-hemolytic. Does not react with Lancefield Group D antisera. G+C of DNA from 37.6 to 38.3 mol% as determined by TM. Type strain is NCDO 2594” (Farrow et a1 1984). The species S. saccharolyticus phenotypically more closely resembles the new genus Enterococcus in growth at 10C and 45C and in 6.5% NaCl. It does not, however react with Lancefield Group D, lacks pyrrolidonylarylamidase (PYR) and is not hemolytic. 16s rRNA sequencing confirmed the phylogenetic position of S. saccharolyticus NCDO 2594 with the genus Enterococcus. The species was renamed Enterococcus saccharolyticus. (Rodrigues and Collins 1990). E. saccharolyticus is phenotypically distinguished from other enterococcus groups in its negative VP reaction. Its lack of arginine hydrolysis is shared by members of the avium species group, the cecorum species group and E. sulfureus (Devriese et al. 1993). «I. w .Eil. x J .v . Table 1. Transfers of species from the genus Streptococcus to the new genus Enterococcus 1984 to 1998. Old name Reference for change E. avium S. avium Collins et al 1984b E. casseliflavus S. casseliflauus and S. faecium subsp. Mobilis Collins et al 1984b E. cecorum S. cecorum Williams et al 1991 E. columbae Unclassified Devriese et al 1990 E. dispar Unclassified Collins et a1 1991 E. durans S. durans Collins et al 1984b E. faecalis S. faecalis Schleifer 8t Klipper-Balz 1984 E. faecium S. faecium Schleifer 8r. Klipper-Balz 1984 E. flavescens a variant of E. casseliflauus, status currently in question Pompei et al 1992 and Descheemaeker et a1 1997 E. gallinarum S. gallinarum Collins et al 1984b E. hirae atypical E. faecium Farrow and Collins 1985 E. malodoratus S. malodoratus Collins et al 1984b E. mundtii Unclassified Collins et al 1984a E. pseudoavium Unclassified Collins et a1 1989 E. raffinosus Unclassified Collins et al 1989 E. saccharolyticus S. saccharolyticus Rodrigues and Collins 1990 E. seriolicida unclassified Kusuda et a1 1991 E. solitan'us Unclassified Collins et a1 1989 E. sulfureus Unclassified Martinez-Murcia and Collins 1991 10 Characterization of the species E. casseliflavus The species S. avium, S. casseliflavus, S. durans, S. gallinarum and S. faecalis subsp malodoratus were transferred to the genus Enterococcus in 1984 based on biochemical, chemical and genetic data. The species E. casseliflavus was described as: ‘Coccoid cells usually in chains or short pairs. Motile. Surface colonies on blood agar or nutrient agar are circular, smooth and entire. Yellow pigment produced... Isolated from plants, silage and soil. In many phenotypic Characters E. casseliflavus resembles E. faecium, but strains of E. casseliflavus may be distinguished by some metabolic tests, by the production of yellow pigment and by the possession of respiratory quinones.” (Collins et al 1984b). The type strain is ATCC 25788 (=NCDO 2372). Metabolic tests results of E. cassehflavus are: hippurate production: (+), D-tagatose: H, and production of B-glucuronidase: H. E. gallinarum tested H, (+), and (+) respectively in these tests (Collins et al 1984b). The species E. flavescens has been described as very similar to E. cassellflavus (Pompei et a1 1992). E. flavescens does not produce acid from ribose and does not produce alpha hemolysis on sheep’s blood, beyond this it tests identically to E. casseliflavus. The species status of E. flavescens is being questioned at this time by several research groups ( Quednau et a1 1998, Carvalho et al 1998, and Descheemaeker et a1 1997) 11 Phenomic strategies to differentiate the Enterococcus, with a special emphasis on E. Saccharoluticus The enumeration and identification of enterococci from environmental samples has been problematic for many years. The classification Of the genus Enterococcus and related species is in a state Of flux, and many identification schemes found in 1990’s literature use the old nomenclature or leave the newer species out all together. Complicated by the presence of Closely related organisms in the same environment, no particular medium ensures recovery of all enterococci For example, as many as 80 selective media have been used for the enumeration of enterococci from foods (Garg and Mital 1991. A conventional test scheme for the identification of enterococcal species was proposed in 1989 by Facklam and Collins. The physiological identification scheme was constructed for group D Streptococcus species isolated from humans. ATCC strains, stored strains with atypical reactions and strains previously identifies as S. avium, S. durans or unidentified were retrieved from their culture collection and examined. Enterococcus was distinguished from Lactococcus and Leuconostoc species. Group D antigen was found unreliable as a differentiator. Lactococci were identified as Group N antigen. The Leuconostocs were identified by vancomycin resistance, gas from glucose, and PYR negative reaction. They noted that all enterococcus species are PYR positive (E. saccharolyticus is not), and that most enterococci are vancomycin 12 resistant (see Clinical Section below). They found gas production from glucose to be a very reliable test, since only the Leuconostocs and about half of the Lactobacillus species (which may be confused as gram positive cocci) produce gas from glucose. Of the enterococci studied, only the outlier E. faecalis produced sufficient gas to generate a positive test in this study. Differentiation of the enterococci into groups was based on fermentation of mannitol, sorbitol, sorbose and arginine. The paper did not include E. saccharolyticus. (Facklam and Collins 1989). The species E. faecalis, E. solitarius, E. gallinarum, E. faecium, E. casseliflavus and E. mundtii are placed together in a metabolic subgroup. These species were then differentiated based on production of acid from arabinose (+), sorbitol (+/ -), lactose (+), motility (+) and pigment (+). The test results shown in parenthesis are those for E. casseliflavus . (Facklam and Collins 1989). A 1991 review Enterococcus in Milk and Milk Products fails to mention E. saccharolyticus; though E. gallinarum and E. casseliflavus are mentioned. (Garg and Mital 1991). Knudston and Hartman (1993) proposed flow charts of key tests which may be used without nucleic acid analysis in food and clinical labs. This study examined 13 of the 18 known ATCC strains of enterococcus along with S. bovis and S. equinus. However, E. saccharolyticus, E. columbae, E. dispar, E. seriolocida and E. sulfureus 13 were not included in this study. A follow up study Of enterococci in pork processing using the flow chart identified one percent of isolates as E. casseliflavus (Knudston and Hartman 1993). More significantly, the four volume tome The Procarvotes, Second Mon, published in 1992 contains no reference to E. saccharolyticus either in the Enterococcus or Streptococcus chapters. E. casseliflavus is . differentiated from E. gallinarum and E. faecium based on its yellow pigment production. E. casseliflavus is listed as motile, in contrast to the other yellow pigmented species, E. mundtii. (Devriese et al. 1992a). E. sulfureus is another yellow pigmented species (Martinez-Murcia and Collins 1991), it is not listed in the review of the enterococcal genus by Devriese, Collins and Wirth (Devriese et al. 1992a). However, E. sulfitreus is easily differentiated from E. casseliflavus and E. mundtii based on the lack of acid production from L- arabinose, inulin, mannitol, rhamnose and d-xylose (Martinez-Murcia and Collins 1991). Testing for the motility Of an isolate to identify E. casseliflavus is problematic. The type species of E. casseliflavus is not motile (Morrison et a1 1997). Numerous other non-motile strains of E. casseliflavus have been cited (Teixeria et a1 1996, Tyrell et al 1997). The 1993 Cowan and Steele's Manual for the Identification of Medical Bacteria includes E. casseliflavus , E. faecium and E. gallinarum in its identification schemes (Cowan and Steele 1993). E. casseliflavus is 14 differentiated from E. gallinarum based on a yellow pigment and a negative hippurate test by E. casseliflavus . Devriese, Pot and Collins synthesized much of the data on the new genus in 1993. The discussion of the gallinarum species group was extended to include E. flavescens. In summary the authors stated that with the inclusion of new species, the simplified phenotypic tests Of earlier years were inadequate to isolate and identify the enterococci. Many of the new enterococcal species do not grow on enterococcal selective media. Isolation and identification methods adapted to the ”classical“ enterococci are still valuable for monitoring drinking water and identification of human pathogens (Devriese et al 1993). Identification schemes of gram positive cocci found in Clinical and veterinary literature are frequently based on Lancefield and hemolysis test criteria, however, this criteria is insufficient for differentiating the newer species. Studies in the last year have focused on differentiating E. casseliflavus and E. gallinarum from E faecium and E. faecalis. Atypical strains of these species are difficult to distinguish using standard techniques. Carvalho, Teixeira and Facklam (1998) suggest adding tests for susceptibility to efrotornycin and acidification of methyl-a-D- glucopyranoside. Use of these tests on 107 typical and atypical strains of 15 enterococci differentiated the strains correctly as determined by SDS- PAGE and DNA:DNA re-association experiments. To summarize the phenotypic identification and characterization of the Enterococcus, the following points should be considered: 0 Gram Positive facultative anaerobic cocci or ovoid cells growing in Chains-could be Streptococci, , Lactobacillus, Aerococcus, Leuconostoc, Pediococcus, Gemella or Enterococcus (Cowan and Steele’s 1993). o Isolates tolerant of azide (0.04%) and salt (6.5%) would most likely be an enterococci. However, E. cecorum and E. pseudoavium are not salt tolerant (Holt et a1 1993). The E. faecium species group members include E. faecium, E. casseliflavus, E. mundtii, E. gallinarum and E. faecalis. Members of this species group are differentiated from other enterococci by their ability to produce acid from mannitol and arginine, and inability to produce acid from sorbitol (Carvalho et a1 1998). Pigmentation is unique to E. cassehflavus, E. mundtii and E. sulfureus, however E. casseliflavus is variable in pigment production. The inability to hydrolyze hippurate separates E. casseliflavus from E. gallinarum and E. sulfureus. The ability to utilize raffinose and inulin also separates E. casseliflavus from E. mundtii and E. sulfureus. A summary of 16 phenotypic characteristics which may be used to identify the Enterococci is presented in the Results section of this thesis (Table 4) Considerable progress has been made in the development of DNA and 16s rRNA based identification methods. Small subunit rRNA is currently the most widely used molecule for determining phylogenetic relationships Of microbial species. 16S rRNA is a good tool for assessing relationships among species. It has universality, it is present in all organisms (not phage and virus). The molecule has experienced little selective pressure and lateral gene transfer. 16S rRNA has invariable sites with highly conserved structure which allows the phylogenetic separation Of distantly related organisms. 16s rRNA also has hypervariable sites. The variability allows differentiation down to the species and subspecies level. The phylogenetic theory is based on comparison of homology Of 168 rRNA of different species, on a nucleotide by nucleotide basis. The greater the number of differences between two sequences, the greater the evolutionary distance between the two species (VanDamme et al 1996a). The new system provides a greater ability to predict success in genetic engineering. Aside from energy source utilization, which formed the backbone of the old classification system, a close phylogenetic relationship predicts that two species are highly similar in fundamental biochemistry, nature of gene expression systems, main regulatory l7 properties, main biosynthetic and degradative pathways, and that individual genes have closer homology to close relatives. Greater knowledge of species provides a greater potential for genetic engineering. The entire 16s rRNA sequence has been determined for most enterococcus species (Table 2). Four species groups have been identified based on this 16s rRNA data. (See Figure 1). E. casseliflavus is grouped with E. gallinarum, and this group is adjacent to one containing E. mundtii, E. hirae, E. durans and E. faecium; E. saccharolyticus is quite distant (Williams et a1 1991). These groupings are quite similar to the species groups derived from metabolic data. rRNA targeted Oligonucleotide probes were synthesized and flourescently labeled for six species of gram positive facultative anaerobic cocci. Lysozyme treatment Of the enterococci, lactococci, and streptococci allowed strong and consistent whole cell hybridizations. Identification of these species in a milk sample was completed within about 8 hours using this method. E. saccharolyticus was not included nor were any members of Devriese’ gallinarum species group. The methodology is presented as a potential solution to difficulties identifying cocci with traditional metabolic test methods (Beimfohr et al 1993). 18 Table 2. Reference source for 16s rRNA sequences of species in the genus Enterococcus. Species Data source Publication for rRNA sequence E. avium, E. casseliflavus, SSU rRNA “Williams, A.M., Rodrigues, U.M. E. cecorum, E. columbae, E. and Collins, MD. 1991 durans, E. faecium,. E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. psuedoavium, E. raffinosa E. dispar SSU rRNA Collins, M.D., Rodrigues, U.M., Pigott, N. E. and Facklam, R. R. 1 991 E. faecalis RDP ** CR Woese Lab, U. of Illinois E. hirae RDP Sechi, L.A., Fazari, S. and Daneo- Moore, L. 1993 E. saccharolyticus RDP Rodrigues, U.M. and MD. Collins 1 990 E. seriolicida SSU rRNA Miller J .M.; Unpublished "1 OS rRNA gene sequence of Enterococcus seriolicida" E.sp SSU rRNA Cai J. and Collins M.D.; "Unpublished" E. sulfitreus SSU rRNA & Martinez-Murcia, A.J. and Collins, RDP MD. 1 991 E.BAW#1 RDP A.Lawes 1 995 19 Figure 1. Dendrogram showing the similarities between strains based on sequence homologies and clustering by the unweighted pair group method. A 16s rRNA derived dendrogram of 14 species of the genus Enterococcus by Williams et al 1991. —— Evam _[ Enflhosus Eavium — Emalodorctus J Egallinarum L Ecasseh'flavus F— E. mundtii Edmans Ejaecium £11m: E. saccharolyticus E. Iaecah's Ececorum Ecolumbae I I 1 I T I 0.95 0.96 0.97 0.98 0.99 1.00 20 Restriction Fragment Length Polymorphism (RFLP) analysis of 16S rDNA of 12 ATCC type species was used to create a data base to identify streptococcus and enterococcus species of bovine origin. Using this database, eleven biochemically atypical isolates from bovine mammary secretions including E. saccharolyticus were correctly identified. This use of 16S rDNA RFLP demonstrated the potential for the technique. Typing of the same E. saccharolyticus isolate by API STREP resulted in low discrimination and the Vitek system did not identify the organism. The species tested did not include any of Devriese et al’s gallinarum species groups, it did include E. faecium (Jayarao et al 1992). In a related study, Polymerase Chain ReactiOn (PCR) amplification using arbitrary primer 8.6d resulted in a characteristic pattern for each of 12 ATCC type strains including E. saccharolyticus. PCR based DNA fingerprinting was then used to successfully ID streptococcal and enterococcal species from bovine milk. The database, developed to identify important mastitis pathogens, did not contain information to identify lactococcal and aerococal isolates (Jayaroa 85 Oliver 1994). Species identification schemes have also been developed based on intergenic ribosomal PCR, this methods clearly differentiates many Of the important nosocomial enterococci including E. mundtii and E. casseliflavus (Tyrell et a1 1997). 21 A more recent PCR based study focused on medically important strains, the E. faecium species group in particular. The primer D11344 proved to be useful for species specific identification. The band patterns are identifiable visually (Descheemaeker et al 1997). Clinical significance of enterococci. Nosocomial infections are hospital acquired infections. The source of the infectious agent may be endogenous, commonly the patients gastrointestinal tract, female genital tract or skin. Exogenous sources include health care personnel, biomedical devices and the hospital environment (Hindler et al. 1994). The enterococci are frequently non pathogenic commensals when found in intestinal, vaginal, and oral tracts, however enterococci posses properties that allow them to take on a pathogenic role. Their natural ability to acquire, transfer and accumulate extrachromasomal elements confers a plasticity to their genome. Survival under stresses including antibiotic therapy is facilitated by this plasticity (Jett et al. 1994). Hospital wide infection data covers all sites Of nosocomial infection for all patients. From January 1990 to March 1996 the enterococci were found in ten percent of 101,821 nosocomial infections. The enterococci were most frequently found in urinary tract infections (UTI: 5613 cases) and surgical site infections (881: 2120 cases). Lower incidence was found in blood stream infections (1298 cases), pneumonia (267 cases) and 22 other sites (1061) (CDC 1996). Enterococci are second only to Escherichia coli in Intensive Care Unit (ICU) nosocomial isolates (De Vera et al. 1996). Enterococcus faecalis is the most frequently found enterococcal pathogen (Tailor et al. 1993). It has been implicated in endocarditis, bacteremia, UTI and intraabdominal infections. In addition, enterococci have been cultured from other infections. Enterococci have been isolated as the disease causing organism in 5.2% of Clinical cases of endocarditis (Watanakunakom and Burkert 1993} In necrotizing fascitis, a fast spreading necrosis of the fascia and subcutaneous tissue, enterococci are one of several pathogens found. The infections are usually polymicrobial (Ou et al.1993). Foumiers gangrene (gangrenous ulcers of the scrotum and or penis) is probably the same disease and a similar complement of pathogens which includes enterococci is found (Efem 1994). Septic arthritis due to enterococcal infection occurs with prosthetic and natural joints. In cases of prosthetic joint infection, antimicrobial therapy is successful. In two of 8 cases reported of native joint infections, amputation was necessary to cure the infection (Raymond et al. 1995). 23 o 29 percent of patients with pyogenic liver abscess had enterococci as the causal organism. This was the second most common organism cultured, after Klebsiella (Hansen and Vargish 1993). Patient risk factors for enterococcal infections are immuno- compromised state, significant medical problems, recent surgery, and hospitalization for extended periods (Boullanger et al 1991). Strains implicated in nosocomial infections are E. faecalis, E. faecium, E. gallinarum, E. avium, E casseliflavus, E. raffinosus, E. hirae, E. durans and E. mundtii (Vandamme et al 1996b, Pompei et a1 1992, Clark et a1 1993 ). Antibiotic therapy and resistance of enterococcal pathogens. Several classes of antibiotics have been utilized successfully against enterococcal infections. These are the B-lactams, aminoglycosides, glycopeptides and llouroquinones. Enterococci which are resistant to antibiotics are becoming an increasing problem in nosocomial infections. High level antibiotic resistance has been found to vancomycin (Ortega et al 1991), teicoplanin (Schmit 1992), gentamicin, kanamycin and streptomycin (Ismaeel 1992). Enterococci resistant to the B-lactams have been isolated from clinical specimens. There are two basic mechanisms of resistance to the B-lactams, which inhibit bacterial cell wall synthesis: These are the production Of B-lactamase and a change in the penicillin binding protein 24 (PBB) structures of the cell wall. Of special significance to the future Of antibiotic use is a recent publication on antibiotic resistance gene swapping. Restriction mapping of the structural part of B-lactamase genes isolated from enterococcal and staphylococcal were found identical, however, the surrounding areas were not identical (Zsckech et a1 1998). The fluoroquinones are an important class of antimicrobials in the treatment of nosocomial infections by inhibiting bacterial DNA gyrase. Ciprofloxacin is effective against most enterococcal strains and is useful for treatment of the elderly with urinary tract infections (UTI) (Wiseman and Balfour 1994). The aminoglycosides have been effective in treatment of enterococcal infections. This class, which includes gentamicin and streptomycin, are bacteriocidal and disrupt microbial protein synthesis. Enterococcal resistance to the aminoglycosides is a growing problem in clinical microbiology. High level resistance to the aminoglycoside gentamicin was found in 61% of isolates in 1989 and 1990 but in none of the isolates from 1969 to 1988 (Grayson et al 1991). E. faecalis is the most frequently found enterococcal pathogen in bacteremia (Tailor 1993). An examination of E. faecalis bacteremia in a hospital found 48% Of 199 isolates over a 32 month period were highly resistant to gentamicin. A mortality rate of 65% was found for patients with resistant and non 25 resistant infections. RFLP analysis of plasmid patterns found one plasmid pattern in 15 isolates. There was no evidence for direct patient to patient transfer. The authors suggested that E. faecalis is a marker for severe illness and the investigation did not find clinical factors associated with high level aminoglycoside resistance (Antalek et a1 1995). The glycopeptides vancomycin and teicoplanin have been important weapons against gram positive bacterial infections. Combination therapy of vancomycin and aminoglycosides have been effective in serious enterococcal infections where B-lactam therapy was not an option. (Hindler et al 1994). Because aminoglycosides alone are unable to penetrate the cell wall at clinically acceptable levels, their use is supplemented with a cell wall active agent, such as vancomycin which inhibits peptidoglycan synthesis (Hindler et al. 1994). National data from the US Center for Disease Control (CDC) and Belgium indicate incidence of vancomycin resistant enterococci (VRE) as an emerging threat. From 1989 through 1993, the proportion of enterococcal isolates resistant to vancomycin (VRE) reported to CDC’s National Nosocomial Infections Surveillance (NNIS) system increased from 0.3% to 7.9%. (CDC 1993). In one year the percentage of nosocomial enterococci reported as resistant to vancomycin increased from 11.5% in 1993 to 13.6% in 1994 among Intensive Care Unit (ICU) isolates. The resistance level rose from 26 4.9% to 9.1% among noncritical care unit isolates. The increase was more dramatic among isolates from noncritical care units, suggesting that vancomycin-resistant enterococci are spreading from their focus in ICUs. (CDC 1995). A nationwide study of enterococcal pathogens in Belgium examined nearly 500 strains. Of these, over 50 percent were resistant to the aminoglycoside streptomycin, while only 8.7 percent were resistant to gentamicin. Vancomycin resistance was found in 16.3 percent Of the E. faecium strains and 1.5 percent of all enterococcal strains (VanDamme et al 1996b). Single hospital based studies draw a picture of VRE as an opportunistic pathogen found in the most seriously ill patients, who have already undergone antibiotic therapy, and been hospitalized repeatedly or for long periods. An analysis of 71 patients hospitalized between 1991 and 1994 was conducted to describe the population afflicted with bacteremia] VRE. 73% were hospitalized in an intensive care unit, adult oncology or AIDS unit. These severely ill patients had received extensive antibiotic treatment during prolonged hospital stays (Montecalvo et a1 1996). A review of patients in cardiothoracic ICU with vancomycin resistant E. faecium found prior nosocomial infection and a prior exposure to vancomycin as important variables. The antimicrobial susceptibility of 27 the isolates was identical from the six patients (Karanfil et a1 1992). Evidence of a similar outbreak was found in an ICU which affected nine patients. Isolates were resistant to glycopeptides, penicillins and aminoglycosides. Restriction digests data of genomic DNA were consistent with the spread of a single isolate. Prior extensive antibiotic exposure, particularly vancomycin, as well as renal insufficiency and length Of hospital stay were risk factors for infection with the isolate (Handwerger et a1 1993). A great deal of research is currently underway examining the biochemistry and genetics of vancomycin resistance. The vanA gene has been implicated in high level resistance to vancomycin and is thought to be transferred by plasmid (Clark 1993 et al, Leclerq et a1 1988, Barbier et al 1996). The vancomycin resistance phenotype VanC is constitutive to E. gallinarum and E. casseliflavus. It confers low level vancomycin resistance. The majority of isolates reported to the CDC are E. faecium with the VanA phenotype (CDC 1993). The VanA and VanB genes may be transferred between strains or species of enterococci (Morrison et al 1997). Strains of E. casseliflavus have been isolated which harbor the VanA gene (Morrison et a1 1997, Tyrell et al 1997). Concerns have been raised that enterococcus may provide a reservoir for genetic elements 28 conferring resistance, and that these elements may be transferred to other genera. Incidence of Enterococci Associated with Farm Animals Enterococci have been found in and on many animals. As with the human animal they may be pathogenic or commensal. The enterococci have been isolated from healthy cats, dogs, cows, bird of various species, and pigs (Devriese et a1 1987, Devriese et al 1991, Devriese et al 1992a, Devriese et al 1992b, and Saika et a1 1994). E. saccharolyticus has been associated directly with bovine mastitis. 317 gram positive catalase negative cocci isolated from bovine mammary glands were characterized. The most frequently isolated organisms from teat canal swabs were S. dysgalctiae and S. saccharolyticus. One cow with a teat canal colonized by S. saccharolyticus developed Clinical mastitis 1 week later, demonstrating the pathogenic potential of this organism (Watts 1988). In a later study of 377 strains isolated from bovine IMI, 41 were identified as E. saccharolyticus (Watts 1993) In a study evaluating the popular Rapid STREP, Rapid STREP identification of 199 strains of streptococci and enterococci associated with bovine intramammary infections (IMI) was tested. Enterococcal species were correctly identified 83.3% of the time. 14 of 25 E. faecalis isolates were incorrectly identified as E. faecium. NO discussion was made 29 of E. casseliflavus. API 20 STREP is a commercial system in frequent use, identifying streptococci in 4 to 24 hours. One of four E. saccharolyticus strains was misidentified as S. bovis using the test system in combination with hemolysis on bovine blood agar plates. S. bovis and E. saccharolyticus are both frequently isolated from bovine mammary glands. (Watts 1989). Vancomycin resistant enterococci have been found in farm animals and pets (Devriese et al. 1996). Avoparcin is a glycopeptide antibiotic which is sometimes used in farm animals for growth promotion. VRE strains are cross resistant to avoparicin. Investigators surveyed farm animals in Europe and found six to eight percent positive for VRE. (Devriese et al 1996). In the US research is examining possible links between the spread of VRE and antibiotic usage. A study of hospitalized patients and over 50 farm animals failed to find any VRE with high level resistance to vancomycin (carrying VanA or VanB genes) except among hospital patients with risk factors associated with multiple hospitalizations (Coque et a1. 1996). 30 Objectives: 1) TO test the isolate BAW #1 and S. bovis ATCC strain 33317 91-08 for gram stain and ability to grow on aerobic and anaerobic media. 2) To determine fermentation products of the isolate BAW #1 using HPLC. TO test culture supernatants of the isolate grown in Trypticase Soy Broth (TSB) and Glucose-Cellulose-Starch-Xylose- Rumen Fluid (GCSX-RF). To determine the fermentation products of S. bovis ATCC strain 33317 91-08 grown in TSB. 3) TO determine the metabolic profile of the isolate BAW #1 . To identify the isolate BAW #1 using BIOLOGTM, API STREP’I'M and conventional testing. 4) To identify the isolate BAW#l using 16S rRNA sequence data. 31 Materials and Methods 1. Purification Of the isolate BAW #1 The anaerobic method was used in purification Of the isolate BAW #1. Standard anaerobic technique as described by Bryant (1972) was used to cultivate the isolate BAW #1 prior to its identification as an enterococcus. Host culture purification: This procedure was carried out using anaerobic technique. Media used were GCS-RF broth, slants, plates, roll tubes, soft agar and anaerobic dilution medium (ADM). Please see Appendix I for complete media formulations. Oxidized media was discarded. All transfers were made under C02 gas, plates were incubated in an anaerobic jar that was sealed and gassed for 30 minutes with C02. All incubations were at 39°C.Two tubes of GCS-RF broth were inoculated with the isolate BAW #1 and incubated overnight at 39 °C under anaerobic conditions. The broth was transferred by sterile loop under C02 to a slant of GCS-RF. Culture was transferred to broth grown overnight and the following day a dilution series was prepared in ADM. Roll tubes were inoculated under C02 at 39°C overnight. A well isolated colony was picked and used to inoculate a fresh broth. The broth culture was gram stained and examined microscopically. The isolation procedure was repeated three times to insure purity Of isolate. 32 2. Conventional Microbiological tests. The isolate BAW #1 was tested on anaerobic and aerobic medium, including several types of selective media. The isolate BAW #1 was tested for its ability to grow on aerobic media. Trypticase Soy Agar (TSA), TSA with 5% defibrinated sheep’s blood and BIOLOG TM BUGM were inoculated by loop from a log phase culture of the isolate. A growth curve study of the isolate BAW #1 in GCS-RF broth under anaerobic conditions was conducted to facilitate work with the phage. Duplicate tubes were inoculated with 0.1 ml from an overnight culture. Samples were withdrawn at regular time points and diluted into ADM. A dilution series was set up and plated. The study was continued until 0Deoo readings indicated the end of the log phase had been reached. The growth curve was determined, and correlation’s of population size, 0Dsoo and time were made. (Meynell and Meynell 1965). The relationship between the number of cells per chain and nutritional conditions was not determined, therefore the colony forming units may not correlate exactly to numbers of cells. The OD 600 was determined on a Spectronic 21D Spectrophotometer. The isolate BAW #1 was inoculated from a log phase TSA culture into the following selective media with a sterile loop. Selective media tests were run in duplicate. Complete media formulations are in Appendix I. 33 The isolate BAW #1 was tested on m-enterococcus agar which contains 0.04% sodium azide and 2,3,5, triphenyl tetrazolium chloride This is a standard medium for the isolation, culture and enumeration Of enterococci from water, sewage and feces. The isolate BAW #1 was tested for growth in TSB broth with 6.5% NaCl. The isolate BAW #1 was tested for growth on TSA slants with 15% glycerol. The isolate BAW #1 was tested on motility sulfide plates and slants which contain 0.2% ferric citrate and 0.2% L-cystine. At 48 hours plates were examined for presence, pigmentation and spreading of colonies. A darkened pigment is indicative of production of H28 from L-cystine. Colony spreading is a positive test for motility. The isolate BAW #1 was tested for pigment production. Sterile cotton swabs were used to pick up colonies off of TSA and BIOLOG TM BUGM. Control swabs were rolled across TSA and BIOLOG TM BUGM with no inoculate. 3. High-pressure liquid chromatography (HPLC) analysis of fermentation products of the isolate BAW #1 . Fermentation products were determined using HPLC. To prepare samples for HPLC, cultures Of the isolate BAW #1 were grown in triplicate in anaerobic (GSCX-RF) and aerobic (TSB) liquid media. S. bovis was grown in triplicate in aerobic (TSB) liquid media 34 After overnight incubation, cultures were centrifuged at 13000g for 30 minutes, the supernatants were transferred to a clean centrifuge tubes and respun as before. Supernatants were transferred to HPLC tubes and held at -20 ° C until analysis. Supernatants were analyzed for fermentation end products by ion exchange exclusion HPLC. The column temperature was 65° C. Mobile phase consisted of 0.005 N H2SO4 at a flow rate of 0.9ml/min. Twenty ul of the filtered samples were autoinjected and analytes detected by refractive index. Integration areas were quantified by commercial HPLC software and compared to standards of acetic, butyric, citric, isobutyric, lactic and propionic acid, and ethanol. Controls Of uninocculated media were used to determine baseline values. 4. BIOLOG TM Identification BIOLOG TM Identification followed the protocol for gram positive organisms in the BIOLOG TM manual. (Bochner 1996) The BIOLOGTM identification system tests the organisms carbon source utilization Of 95 substrates, with water as a control in the 96th well. The isolate BAW #1 was tested on BIOLOG TM gram positive plates, and the BIOLOG TM gram positive database was selected for data analysis. The redox based dye tetrazolium violet is used to detect metabolic activity colorimetrically. Positive utilization is observed as the dye is irreversibly reduced to a 35 purple formazon. Any chemical substrate oxidized will result in the production of NADH. The tetrazolium is reduced by electrons from the electron transport chain. The isolate BAW #1 was tested anaerobically and aerobically. The first test was anaerobic. The isolate was grown on GCS-RF plates and transferred by sterile swab into ADM to an OD of 0.25 using anaerobic technique. Two BIOLOG TM plates were inoculated with this solution and the plates were incubated under C02 at 39°C and read at 4 hours and 24 hours. After the isolate tested positive for growth under aerobic conditions, it was tested again on two BIOLOG TM plates using standard BIOLOG TM BUGM media and saline to prepare the inoculate. The aerobically incubated plates were incubated at 39°C for 24 hours. In both cases metabolic activity on 95 substrates was measured by the BIOLOG TM Plate Reader and the BIOLOG TM Gram Positive Database used tO identify the organism BIOLOG TM calculations and interpretations of BIOLOG TM data are as follows: The data produced by BIOLOG TM is presented on printed data sheets as: BIOLOG TM DATA FORM SUBSTRATE UTILIZATION positive {xxx} borderline {XXIH borderline {xxx- borderline xxx negative 36 Numerical values were assigned for the BIOLOG TM dataforms, 10 for positive, 5 for borderline and 0 for negative. The average value for each substrate was calculated and variability noted. This data manipulation serves to produce a set of numbers O comparable to other data in the microbiological literature, including Bergey’s Manual for Determinative Bacteriology (9th) (Holt et a1 1993) . Cluster Analysis was generated using the BIOLOG TM software by entering the BIOLOG TM Bionumber generated at the time of the plate reading. 5. API STREP Test The isolate BAW #1 was brought to the Animal Health Diagnostic Laboratory (AHDL) of the College of Veterinary Medicine at MSU for testing on API Rapid STREP system. The API STREP strip tests the isolate for acetoin production, B-glucosidase, pyrrolidonylaryl-amidase, B- galactosidase, leucine arylamidase, arginine hydrolysis, ribose, L- arabinose, mannitol, lactose, trehalose, raffinose, starch, hippurate hydrolysis, a-galactosidase, B-glucuronidase, alkaline phospatase, sorbitol, inulin, glycogen and B-hemolysis. Test results are presented as positive or negative for each test. From this a seven digit profile is produced which is used to determine species identification. This profile was called in on two separate occasions to the API STREP/ bioMerieux phone line. Operators used the most recent version 3 for data analysis. 37 The database includes E. gallinarum, but does not include E. saccharolyticus. An identification which is “good to the species level” indicates that the isolate BAW #1 matches at least 90% of what would be expected for the designated species. 6. Antibiotic resistance testing The isolate BAW #1 was sent to the Animal Health Diagnostic Laboratory (AHDL) of the College of Veterinary Medicine at MSU for analysis of antibiotic susceptibility. The isolate BAW #1 was tested for sensitivity to: ampicillin, cephalothin, cefotoxin, ciprofloxacin, clinamycin, erythromycin, gentamicin, nitrofurantoin, oxacillin, penicillin, tetracycline and trimethoprim —sulfa. 7. Analysis Of 16s rRNA sequence Of the isolate BAW#l The 16S rRNA sequence of the isolate BAW #1 has been determined (Lawes 1995) and was entered into the Ribosomal Database Project (RDP) in 1995 by Lawes . The RDP programs identified the isolate BAW #1 as a strain of E. saccharolyticus. In 1995, The RDP database only included six enterococcal species: B. cecorum, E. faecalis, E. hirae, E. sulfureus, E. columbae and E. saccharolyticus. Phenotypic data in this study did not support that identification, in fact the isolate was identified by API STREP and BIOLOG as E. casseliflavus. The sequences in the RDP database at the time of the 38 late“ lotes enter: were gallin 2375 I100 stIai inch SOC! SCC)‘ usi: anc Lawes analysis did not include members of the E. faecium species group. To test the identification of the isolate, a full species complement of the enterococcus was needed. Additional 16s rRNA enterococcal sequences were included in the analysis. These were E. casseliflavus NCDO2376, E. gallinarum NCDO 2313 (T), E. faecium NCDO 942 (T), E. Mundtii NCDO 2375, and E. durans, NCDO 596 (T). Partial sequences for two strains of E. casseliflavus, strain AFO 39903 and strain AF039989, and two strains of E. gallinarum, strains AF039900 and AFO 39898 were also included in the analysis (Patel et a1 1998). The type strain of E. saccharolyticus was also included in the analysis. The additional sequence data was entered into the ARB program Fast DNA ml. These sequences were examined for phylogenetic relationship using the least squares distance matrix program of ARB. An alignment and a phylogenetic tree were generated. 39 Results 1. Purification of the isolate BAW #1 The isolate BAW #1 readily grew overnight at 39 ° C under anaerobic conditions on the complex medium GCS-RF. The isolate is gram positive and occurs in chains of two up to 20 cocci in length. The cocci are slightly ovoid in shape. 2. Conventional Microbiological tests. Conventional test results include data generated when the isolate BAW #1 was tested on anaerobic and aerobic media, including several types Of selective media. Aerobic media supportiveof overnight growth at 39 ° C included TSA, TSA with 5% defibrinated sheep’s blood and BIOLOG TM BUGM. The isolate BAW #1 grew rapidly in TSB, reaching an ODaoo of 0.5 in 4-6 hours. The results of the growth curve study of the isolate BAW #1 in GCS-RF broth are presented in Table 3 and Figure 3. The isolate BAW #1 attained rapid growth rates in anaerobic media, with a peak doubling time of less than one hour. The isolate entered log phase growth approximately three hours after inoculation. Optical densities indicative of log phase growth are in the 0.2 to 0.6 range for this media (Figure 2). The stationary phase Of growth was entered when the colony forming units per milliliter reached 8 X 107 in GCS-RF broth. The isolate 4O BAW#l grows rapidly, even under anaerobic conditions. The growth rate of the isolate was determined by the formula (Meynell and Meynell 1965): “=(108NM‘108N) / t where )1: Specific growth rate N = Population at a given time N (0‘ Population after an elapsed time T= Time elapsed since last measurement The facultative nature of the isolate provided evidence that it could not be a member of the genus Ruminococcus. The isolate BAW #1 was tested on m-enterococcus agar, which contains 0.04% sodium azide and 2,3,5, triphenyl tetrazolium Chloride. Colonies were tiny, pink and visible after 24 hours. The isolate was tested on motility sulfide media which contains 0.2% ferric citrate and 0.2% L-cystine. After 48 hours, growth was positive, but not pigmented, indicating the isolate does not produce H2S from L-cystine. The colonies did not spread, indicating a lack of motility. The isolate BAW #1 tested positive for growth in TSA and Antibiotic Media with growth evident after 24 hours. The isolate BAW #1 was tested in TSB broth with 6.5% NaCl with growth evident after 24 hours. The isolate BAW #1 grew overnight on TSA slants with 15% glycerol. 41 Table 3. Growth characteristics of the isolate BAW #1 in GCS-RF broth: Optical Density and Colony Forming Units charted against time Optical Density Colony Specific Growth Forming Rate Units TIME Minutes Tube A Tube B average u=(log(Nt)-logN) / t 9:30 AM 0 0.04 0.05 12540000 1 1:15 AM 105 0.09 0.08 17995000 0.0015 11:55 AM 155 0.11 0.12 26962500 0.0035 12:30 PM 180 0.17 0.18 41050000 0.0073 12:55 PM 205 0.25 0.25 86000000 0.0128 1:40 AM 250 0.43 0.42 94075000 0.0009 Figure 2. Growth data for isolate BAW # 1 grown in GCS-RF anaerobically: Colony Forming Units and Time. ' 1.E+08 ae— .g . i D 1 E A E 1.E+07 L— w o t—— 4— -~- ll. _; >‘ I C 2 a O o .__ 1'E+06 iITITm fl—I VIIIIIIIIIIIIIIIIIITTTIIITTTTTTTITITI—IIll OOOOOOOOO (OQONV'CDQONV s—s-s-s-FNNN Time in minutes 42 The ability Of the isolate to tolerate high salt and azide concentrations confirmed its identification as a member Of the genus Enterococcus. Sterile cotton swabs used to pick up colonies Off of TSA and BIOLOG TM BUGM turned yellow. Control swabs rolled across TSA and BIOLOG TM BUGM with no inoculate picked up no pigmentation. Control swabs rolled across plates Of S. bovis grown on TSA and BIOLOG TM BUGM also picked up no pigmentation. The pigment production of the isolate contradicted its identification as E. saccharolyticus, since this species is not pigmented. Based on pigmentation, the isolate BAW#I could be E. mundtii, E. casseliflavus or E. sulfureus, however, the lack Of motility would exclude most strains of E. casseliflavus. 3. High-pressure liquid chromatography (HPLC) analysis of fermentation products Of the isolate BAW #1 . The end point fermentation products Of the isolate BAW #1 grown in TSB are 51% acetic acid, 34% lactic acid and 15% ethanol (Table 4). The end point fermentation products Of the isolate BAW #1 grown anaerobically in GCSX-RF are 95% lactic acid and 5% ethanol. The endpoint fermentation products of S. bovis are 89% lactic acid, 2% acetic acid and 9 % citric acid. (Table 4).The fermentation product analysis provided further evidence that isolate is not a strain of Ruminococcus, and supports the thesis that the isolate is an enterococci. 43 Table 4. Fermentation Products of the isolate BAW#l and Streptococcus bovis as determined by High Pressure Liquid Chromatography. I. Fermentation Products of the isolate BAW#I Cultured aerobically in Trypticase Soy Broth. Fermentation Product Percent of Total Lactic acid 0.34 Acetic acid 0.51 Ethanol 0. 15 II. Fermentation Products Of the isolate BAW#l Cultured Anaerobically in GCSX-RF Fermentation Product Percent of Total Lactic acid 0.95 Ethanol 0.05 III. Fermentation Products of Streptococcus bovis Cultured Aerobically in Trypticase Soy Broth Fermentation Product Percent of Total Citric acid 0.09 Lactic acid 0.89 Acetic acid 0.02 44 4. BIOLOG TM Identification The BIOLOG TM identification system tests the organisms carbon source utilization of 95 substrates, with water as a control in the 96th well. Thirty-nine of the substrates tested positive, fifteen showed an intermediate reaction and forty-one tested negative. A complete listing of substrate reactions is provided in Tables 5, 6, and 7. Some variability was found in substrate utilization on the four plates. Some substrates tested positive on one plate and borderline on another, as well as negative on one plate and borderline on another. None of the substrates tested positive on one plate and negative on another. An anaerobic environment was more conducive to the utilization of a-ketoglutaric acid, a -ketovaleric acid, L—rhamnose, L-lactic acid and pyruvic acid. The isolate demonstrated greater utilization of D-ribose and uridine 5- monophosphate under aerobic conditions. The BIOLOG TM software assigns a Bionumber to a reading which is a condensed version of the test results. The BIOLOG TM numbers for the four plates were: A: 3627-5656-7764-5623-3000-1300-000 1 -7600 B: 3607 -5656-77 64-5623-3000- 13 10-000 1 -7600 C: 3627-5656-7764-5663-3000-0300-OOO1-76OO D: 3607-5656—7764-5663-3200- 13 1 O-000 1-76OO 45 Based on this profile, the BIOLOG TM identification system identified the isolate BAW #1 as E. gallinarum on each plate using BIOLOG TM Gram Positive Release 3.5. The BIOLOGTM program rated the similarity and distance of the isolate BAW #1 to E. gallinarum for each plate read (Table 8.) BIOLOG TM Similarity is a calling criteria, not a probability, and is based on empirical results. It is considered a judge of the reliability and confidence of the identification. The value of distance in the BIOLOG TM system roughly approximates the number of mismatches, with allowances for substrates known to be variable for a given species. A similarity index of greater than 0.5 is considered an acceptable identification for bacterial species measured with the BIOLOG TM system. Therefor, the identification Of the isolate BAW #1 as E. gallinarum was considered greater than acceptable using the BIOLOG TM Gram Positive Release 3.5. This identification conflicted with the API STREP identification of i the isolate BAW #1 as E. casseliflavus and with the RDP identification of the isolate BAW #1 as E. saccharolyticus. The BIOLOG TM Software Release 3.5 includes E. casseliflavus , E. gallinarum and E. saccharolyticus in its database. 46 Table 5. BIOLOG TM SYSTEM Substrate utilization profile of the isolate BAW #1. PART 1. Substrates the isolate was unable to utilize. (mean utilization value less than 2). BIOLOG MEAN PLATE WELL A B C D 3-methyl glucose c1 1 1.25 0 0 0 5 a-hydroxybutyric acid e7 0 0 0 0 0 a-methyl D mannoside d2 0 0 0 0 0 a-methyl D-glucoside c12 0 0 0 0 0 acetic acid e6 0 0 0 0 0 adenosine-5'monophosphate h6 1 .25 0 0 0 5 alaninamide gl 0 0 0 0 0 asparagine g5 0 0 0 0 0 B-hydroxybutyric acid e8 0 0 0 0 0 D- arabitol b2 0 0 0 0 0 D-alanine g2 1 .25 5 0 0 0 D-galacturonic acid b8 0 0 0 0 0 D-lactic acid methyl ester f2 0 0 0 0 0 D-malic acid f4 1.25 5 0 0 0 D-tagatose e1 1.25 5 0 0 0 fructose-6-phosphate h9 0 0 0 0 0 y-hydroxybutyric acid 69 1.25 5 0 0 0 glycyl-L glutamic acid g7 0 0 0 0 0 glucose- 1-phosphate h 10 0 0 0 0 0 glucose-6-phosphate h1 1 0 0 0 0 0 inulin a6 0 0 0 0 0 Iralanine g3 1.25 5 0 0 0 L-alanyl-glycine g4 0 0 0 0 0 L-fucose b6 0 0 0 0 0 L-glutamic acid g6 0 0 0 0 0 L-pyrglutamic acid g8 0 0 0 0 0 L-serine g9 0 0 0 0 0 lactamide f1 0 0 0 0 0 m-inositol b12 1.25 0 5 0 0 mannan a7 0 0 0 0 0 methyl succinate f7 0 0 0 0 0 N-acetyl L-glutamic acid f12 1.25 0 5 0 0 p-hydroxybutyric acid e10 0 0 0 0 0 ropionic acid f8 1.25 5 0 0 0 putrescine g10 l .25 5 0 0 0 sedoheptulose d9 1 .25 0 0 0 5 succinamic acid HO 0 0 0 0 0 succinic acid f11 0 0 0 0 0 thymidine-5'monophosphate h7 0 0 0 0 0 tween 80 a9 0 0 0 0 0 xylitol e4 0 0 0 0 0 47 Table 6. BIOLOG TM SYSTEM Substrate utilization profile of the isolate BAW# 1. PART 2. Substrates the isolate was able to utilize weakly. (mean utilization value between 2 and 8). SUBS TRA TE BIOLOG MEAN PLATE WELL A B C D a-methyl D galactoside c9 2.5 0 5 0 5 2,3-butanediol gl 1 2.5 5 0 0 5 a—ketoglutaric acid e1 1 2.5 5 5 0 0 a-ketovaleric acid e12 2.5 5 5 0 0 p-cyclodextrin a3 7. 5 5 10 5 10 D-L-a glycerol phosphate h 12 2 . 5 0 5 0 5 D-melezitose c7 6.25 5 5 5 10 D-ribose d7 7.5 5 5 10 10 D-sorbitol d 10 5 5 5 5 5 L-lactic acid 13 7.5 10 10 5 5 L-rhamnose d6 2.5 5 5 0 0 Pyruvic acid f9 5 10 5 0 5 Tween 40 a8 7.5 5 10 10 5 Uridine-S'monophosphate h8 2.5 0 0 5 5 Xylose e5 3.75 5 0 5 5 48 Table 7. BIOLOG TM SYSTEM Substrate Utilization by the isolate BAW # 1 as tested in the BIOLOG'I‘M system. Part 3. Substrates the isolate was able to utilize in the BIOLOGTM system (mean utilization value greater than 8). BIOLOG MEAN PLATE SUBSTRATE WELL Amt-1L“- _ -_ _ b3 _ _1_-o___ __ - __ .10 -_--19____-- 19 -- -19 -- a-9 glucose 911 10 10 19 1019 .- Adenosine _- - - - - - 111-- -19- - -_.-19 -.-_10-____19_ -_19.____ .- p-methyl D galactosxde e. _,c10 -19 _.... -10-10-10 10 B-methyl D- -glucos-iC19--- -, _ 1.1-1 ..__ -1 Q.-. __ ..- _‘ -10---_.10.-.. 19 .- -19__ Cem_ e- - . --p4- ._ 19 e ._--_1o_.--19__-_--_19_- 19__--._ D- glucuronic acrd 910..‘.1010-10-_-1010- anlgctgsgmg b7 10 10 10W 10 W W10 ID-mannitol - ‘3 -. 3’ .. 7 _Hc5 _‘ - 5 10 10 10 10 10 D-melibiose ' 08“; 1010 10___ 10 I0 P-PiSCOSC -d419-1.0 10 10 10,-.- Qigiflngie- - - - - - - - d5 .- .-1Q __ -_Q1_0-_-__-1Q_ Q 10 - 10 _._.___ D-trehalose - .. -_ -€2-10--10-_--. 1.9 _. Q19 .-10--.- Dextrin - __.__ ___. Q _ ._ -_- . ail-Q- ___. .10-_ --- Q19 1919 -_ 19,.Q -_ (Glycerol _ _ grgmw 10 10 10 ‘10 10 Glycogen W ,_-.Q._.-. W W a5 19-10 1.0 10 1.0 Inosine W h3lO Q I I W. 10__ 10 10 10__ L- arabinose -13110-19 W19 19 10 W 1211-9119-9919- - .. - . 1’5 -. ,, 1.0 10 . 10 _- 10 - 10W -- Lactulose -0210 10 1.9. . 19 1.9 Q Maltose ._ 7“ - i - -_ c3 Q. “10% Q 10 -__- _1_0 _ 10 +7 10 _ Maltotriose __ - . -._. --,<-=.‘.1------1.9- 10 19 19 10 _-._--_- Methylpyruvate f6 10 A 10 10 __ 10 .- 10 Q. N- acetyl glucoseamme .. V.___._9_1910 10 10 IQ 10- N- acetyl mannoseamineflm all 10 10 10 10 10 Palitinose d3 10 _--_.19 10 10 10W Salacin Q. " d8 10 1'0_10___, 10 10 8180111086 C111 __ 10 10 10W 10 10.- - Sucrgseww -_ g _ f . -9-1_2Q-Q 10 10 19_--- Q10 10 _,-_. Thymidine h4 8 .75 - W19W 5 10 Q 10__- ’I‘uranose --_ Q. Q. _ --_- e3 ” 10 10 _-.19-__-.-. ”10' 3 10:“ 7; Uridine h5 10 ”‘ "107 10 10 10 49 Table 8. Similarity and Distance as determined by BIOLOG'I'M software for the isolate. The first data set is based on BIOLOG TM software release 3.5, the second is based on BIOLOG TM software release 3.7. Plate BIOLOG TM Similarity Distance RELEASE A 3.5 0.813 2.807 B 3.5 0.809 2.871 C 3.5 0.821 2.683 D 3.5 0.821 2.683 A 3.7 0.922 1.160 B 3.7 0.915 1.258 C 3.7 0.828 2.571 D 3.7 0.868 1.956 BIOLOG W was contacted regarding the conflicting identifications. BIOLOG TM had released an upgraded database, Release 3.7 with changes in the criteria for E. casseliflavus and E. gallinarum. The new database was installed into the BIOLOG TM program. The bionumbers of each of the four plates was entered into the new database. The resulting identification matched the API STREP identification; the isolate BAW #1 was identified as E. casseliflavus by BIOLOG TM software version 3.7. The BIOLOG TM system provides cluster analysis of isolates based on its metabolic data. The dendrograrns produced with the isolate BAW #1 using release 3.7 are shown in Figure 3. Each dendrogram shows the isolate BAW #1 as closest to E. casseliflavus. 50 Figure 3. BIOLOGTM generated dendrograms of the isolate BAW#] showing metabolic proximity to E. casseliflavus and E. gallinarum. A) dendrogram produced with BIOLOGTM number from Plate A: isolate BAW#l is designated A6 A . B) dendrogram produced with BIOLOGTM number from Plate B: isolate BAW#l is designated B8 B. C11 COCA-1 (CELSPP) C12 CELT-16 | ooooooooo ' ........ 0 ......... I ......... i . . . . 3 o . o .l A l LCC.LHC SS DInCETYLa 41: a 2 mme *—-—'—— a 3 ENT.SflC A r— F— H 1 ENT.GfiL a 5 ENT.CflS 8 7 (SUCH-1 (NCB.SPP) B 8 comaoun B . B_ 9 comnoun a L f": C10 CEL.CELU l....:....|....:....l ......... I... .:....l ......... l DENDPOGRFm DISTANCES COP.HOUfl A COPJ-IOUFD B COCA-1 (HCB.SPP) ENTIFDECL ENT. SHC ENT.GflL ENT.CflS B CEL.CELU CEL.HOH (CDC.fl-3) CDCJ‘l-fl (CELSPP) CEL.FL6 j GOOD wmmwm 33.79 N—‘OCD common-a QM.- b—o— DENDROGPHH DI STfiNCES 51 5. API STREP Test The isolate BAW #1 tested positive for acetoin production, B- glucosidase, pyrrolidonylaryl-amidase, B-galactosidase, leucine arylamidase, arginine hydrolysis, ribose, L-arabinose, mannitol, lactose, trehalose, raffinose, and starch. The isolate tested negative for hippurate hydrolysis, a-galactosidase, B-glucuronidase, alkaline phospatase, sorbitol, inulin, glycogen and B-hemolysis (See Table 9). The Animal Health Diagnostic Lab at Michigan State University determined the isolate BAW #1 was an E. faecium, type two or three. To check the database for further information, I called the API STREP/ bioMerieux phone line. Based on the utilization profile, the seven digit profile 5157551 was produced. This profile was called in on two separate occasions to the API STREP/ bioMerieux phone line. Operators used the most recent version 3 to give a “ good to the species” level identification of the isolate BAW #1 as E. faecium 3 or enterococcus faecium 2. A supplemental Note 60 was accessed which suggests that if the isolate BAW #1 has a yellow pigment, it is to be considered a “good to the species level” identification as E. casseliflavus . The database includes E. gallinarum, but does not include E. saccharolyticus. The “good to the species level” identification indicates that the isolate BAW #1 matches at least 90% of what would be expected for the designated species. The API 52 STREP data supplemented by my data concerning pigmentation, determined that the isolate BAW #1 is an E. casseliflavus strain. 6. Antibiotic resistance testing The isolate BAW #1 tested sensitive to the antibiotics cephalothin, gentamicin, tetracycline, ciprofloxacin and nitrofurantoin. The isolate BAW #1 tested moderately susceptible to the antibiotics ampicillin and penicillin. The isolate BAW #1 tested resistant to the antibiotics cefoxitin, clindamycin, erythromycin, oxacillin and tribissen (Table 10). 7. Analysis of 163 rRNA sequence of the isolate BAW#l Tree building using the ARB program Fast DNA ml for the enterococcal species show isolate BAW#l very close to both E. gallinarum and E. cassellflavus, slightly closer to the latter (Figure 4). Seven strains made up the E. gallinarum-E. casseliflavus group which clearly branched separate from the other enterococcal species. The proximity of this entire group precludes identification to the species level using this data. The 16s rRNA data shows the E. gallinarum-E. casseliflavus group distinct from the faecium-mundtii- hirae—durans group, as well as the other enterococcal species. E. saccharolyticus is quite far from BAW#l. The distance matrix shows the relationships between the strain BAW#l, and the E. saccharolyticus, E. casseliflavus and E. gallinarum strains. (Table 11). The isolate BAW#l is closest to the E. gallinarum strain AFC 53 39900. E. saccharolyticus is clearly not in the same phylogenetic group as the E. casseltflavus and E. gallinarum strains, with a distance of greater than 0.022 from all strains. The entire gallinarum- casseliflavus group has distances an order of magnitude less, with values less than 0.0022. Based on the distance matrix, the isolate is clearly not a strain of E. saccharolyticus. This data is insufficient to determine the identity at the species level, however it is clear that the strain is a part of the E. gallinarum-E. casseliflavus group. 54 Table 9. Metabolic profile of the isolate BAW #1 and related species as determined by API StrepTM. Numerical Data is the percent of strains testing positive for a given species. Data from the API STREP package insert. Isolate data is “+” or “-”, Reaction data for the isolate is from the Animal Health Diagnostic Lab at Michigan State University. acetoin production Hippurate hydrolysis B—glucosidase Pyrrolidonylaryl- Amidase a—galactosidase B—glucuronidase B—galactosidase alkaline phosphatase leucine arylamidase arginine hydrolysis Ribose L-arabinose Mannitol Sorbitol Lactose Trehalose Inulin Raffmose Starch Glycogen B—hemolytic Isolate BA W # 1 ++'++'+++ faecium 99 96 100 100 90 99 99 99 99 60 99 15 99 99 99 80 E. faecium 3 100 100 100 90 98 99 66 99 99 99 15 99 99 96 99 99 S. bovis 100 100 83 99 100 97 S. bovis H-1 99 93 OO‘HO 00 100 S. bovis II—2 100 100 78 41 38 100 \IOOOOHO E. gal- linarum 100 100 100 100 100 83 10 100 100 100 100 100 100 100 100 100 83 16 55 Table 10. Antibiotic Sensitivity of the isolate BAW #1. Antibiotic Class Mechanism Target organisms Isolate BAW# l Penicillin B-lactam inhibit cell wall streptococcal and Moderately synthesis staplylococcal susceptible Ampicillin B-lactam inhibit cell wall streptococci, moderately synthesis staphylococci, susceptible E.coli, proteus, haemophilus Cephalothin B-lactam: inhibit cell wall staphylococci, sensitive first generation synthesis streptococci cephalosporin Cefotoxin B-lactam inhibit cell wall gram-, some [3- resistant third synthesis lactamase generation producing gram + cephalosporin strains Oxacillin B-lactam- inhibit cell wall penicillinase resistant (methicillin) penicillinase synthesis producing resistant staphylococci penicillin Gentamicin amino- inhibit protein enterococci, gram- sensitive glycoside synthesis bacillus Tetracycline tetracyclines inhibit protein many gram- and sensitive synthesis gram + plasmid mediated resistance common Cipro- flouroquinone inhibits DNA enterobacteriaceae sensitive floxacin gyrase neisseria sp., some streptococcus and staphylococcus Nitro- inhibits DNA, enterococci and sensitive furantoin RNA and cell enterobacteriaceae wall synthesis urinary infections Clindamicin modified from inhibits most gram+ cocci resistant lincomycin protein and anaerobes. synthesis enterococci are resistant Erythromycin macrolide inhibits some resistant protein streptococcus, synthesis legionella, etc. Tribissen/ * Brand name inhibits steps enterobacteriaceae, resistant trimethoprim is Bactrim in microbial staphylococcus, sulfa folic acid etc. synthesis Data in Table 10 is drawn from Hindler et a1 1994 and Physicians Desk Reference 1997. 56 Figure 4. Phylogenetic relationship of the enterococci and the isolate BAW#l based on 16s rRNA sequence analysis by the ARB Fast DNA ml program. Vagococcus salmoninarum. (EBLDEW) NCFB 2777 Vagococcus fluvialis, [RDP] NCDO 2497; AT ____{ Enterococcus sulfureus, (EB!) NCDO 2379 (=- Enterococcus saccharolyticus, [RDP] NCDO 2594; AT Enterococcus pseudoavium, [DEM NCDO 2138 (T) Enterococcus malodoratus, (DEW) NCDO 846 (T) Enterococcus avium. (DEW) NCDO 2369 (7) Enterococcus raffinosus, (DEW) NCTC 12192 (T Enterococcus dispar, [DEW] E 18-1 (T) ”'— Enterococcus species, (DEW) LMG 12316 (EB L Enterococcus faecalis '—_ Enterococcus mundtii, (DEW) NCDO 2375 (T) ”' Enterococcus hirea. (DEW) NCDO 1258 (T) LL Enterococcus durans. [DEW] NCDO.596 (77 Enterococcus faecium, (DEW) NCDO 942 (T) Enterococcus casseliflavus, (DEW) NCDO 2376 Enterococcus casseliflavus , AF039903 Enterococcus casseliflavus, AF039899 isolate BA W#1 Enterococcus gallinamm, AF039900 Enterococcus gallinarum, AF039898 Enterococcus gallinarum, (DEW) NCDO 2313 (T) F_""" "" Enterococcus cecomm, (DEW) NCDO 2674 T ‘ Enterococcus columbae. momsmummm 0,”) 57 TABLE 11. Phylogenetic relationship of the enterococci and the isolate BAW#l based on 168 rRNA sequence analysis by the ARB least squares distance matrix program. E. Sac NCDO 2594 E. Gal 0.02446 NCDO 2313 Isolate 0.02596 BAW#l E. Cas 0.02446 NCDO 2376 E. gal 0.02667 AF039898 E.cas 0.02519 AF039899 E. gal 0.02593 AF039900 E. cas 0.02519 AF039903 E. Sac NCDO 2594 0.00287 0.00215 0.00143 0.00215 0.00215 0.00215 E. gal NCDO 2313 0.00430 0.00215 0.00072 0.00143 .00215 Isolate BAW# 1 58 0.00358 0.00358 0.00430 0.00215 E. cas NCDO 2376 0.00143 0.00072 0.00143 E. gal AFO 39898 0.00072 0.00143 E. cas AFO 39899 0.00214 E. cas AFO 39903 Discussion The isolate BAW #1 is a gram positive, facultatively anaerobic cocci which grows in chains of 2 to over 20 cocci. It is tolerant of high salt. The isolate grew on m-enterococcus media containing 0.04% sodium azide. Based on these tests, it is possible to identify the isolate as a member of the genus Enterococcus. The isolate is able to survive storage at 4 ° C for over 6 years. In rich media the isolate BAW #1 entered log phase within 2 to 4 hours, and attained a concentration of greater than 10‘8 colony forming units per milliliter even under anaerobic conditions. The organism is clearly a survivor. The isolate BAW #1produced a yellow pigment. Of the enterococci, only E. cassellflavus, E. mundtii and E. sulfureus produce a yellow pigment. E. casseliflavus is usually motile, E. mundtii and E. sulfitreus are not motile (Table 12). Phenotypic differentiation between E. casseliflavus and E. mundtii is generally based on motility. However, the type strain of E. casseliflavus is not motile. Recent research has determined that the motility test is not a reliable differentiator. The conventional tests suggested the identification of the isolate BAW #1 is E. casseliflavus, E. mundtii or E. sulfureus (Table 12). 59 Table 12. Phenotypic differentiation of E. casseliflavus from closely related enterococci with test results of the BAW#l isolate. BAW#l E. cas E. gal E. mun E. sul E. fcm E. sac Motility - +/(-) (2) - (2) - (2) - (4) - (2) "(6) +* (6) +* (6) Pigment Yellow Yellow None Yellow Yellow None None * (5‘16) (5) (5) (5) (5) Hippurate -API - (5) + (5) - (5) (+)(5) - (5) hydrolysis Tagatose - -/(+) +/(-) - (1) -/(+) BIOLOG (1) (1) (2) Arabinose + API + (1) + (1) + (1) - (4) + (1) Sorbitol - API - (5) - (5) d (5) - (4) - (5) + (5) Ribose + API + + (4) + (2) + (4) Raffmose + API + (3) + (3) - (3) + (4) - (3) Inulin - API + (3) + (3) - (3) - (3) B-gluco- No Data + (6) + (6) - (6) + (6) - (6) + (6) yranosidase - Efrotomycin No Data R (6) R (6) S (6) R (6) S (6) R (6) sensitivity 1) Cowan and Steele 1993 2) Bridge and Sneath 1982 3) Knudtston and Hartman 1993 4) Morrison et al 1997 5) Holt et al 1993 6) Carvalho et a1 1998 7) Martinez-Murcia and Collins 1991 * denotes variability of strains 60 Further data on the metabolic profile of the isolate BAW #1 obtained through the use of BIOLOGTM and API Strep. These tests demonstrated that the isolate is capable of utilizing a broad range of substrates under aerobic and anaerobic conditions. The metabolic based identification scheme BIOLOG TM names the organism E. casseliflavus. The earlier BIOLOG TM release 3.5 identified the organism as E. gallinarum. Release 3.7 changed the criteria for identification of E. gallinarum and E. casseliflavus , when the BIOLOG TM Bionumber produced by the BIOLOG TM plate reader was entered into the computer, the new software identified the isolate BAW #1 as E. casseliflavus. The distance criteria roughly approximates the number of mismatches, the isolate BAW #1 values were close to 1 for anaerobic conditions, and closer to two for aerobic conditions. The similarity index greatly exceeded the acceptable reading of 0.5. The API STREP test kit identified the organism as E. casseliflavus. The database also required further interrogation to produce accurate results. The API STREP kit identification as determined by code 5157551 named the organism E. faecium type 2 or 3, and considered the identification as “good to the species level”. This identification was questioned because it conflicted with identifications derived from other systems. This questioning pinpointed the significance of the pigment of the isolate BAW #1 , and based on this, the API STREP identification was 61 changed to E. casseliflavus. The API STREP package insert contains no reference to criteria for differentiating E. faecium from E. casselzflavus. E. casseliflavus and E. saccharolyticus are not mentioned on the package insert. The API Strep system is lacking in its ability to differentiate members of the faecium group of enterococcus. Fermentation product analysis produced results which fit the profile for a member of the genus Enterococcus, this test provided further evidence that isolate is not a member of the genus Ruminococcus. Anaerobically, in the nutrient rich rumen fluid based media GCSX-RF, the isolate BAW #1 produced 95% lactate and 5% ethanol. This is representative of the classic homofermentative metabolic pathway common to enterococci and streptococci (Garg and Mital 1991, Neijssel et al. 97). Grown in aerobic conditions, the isolate BAW #1 produced a broader complement of fermentation products: acetic acid, lactate and ethanol. Enterococci are known to produce these components under nutrient limited conditions. Streptococcal and enterococcal metabolic pathways continue to be the subject of research, it is known that the fermentation products of these genera are dependent on pH, p02, carbohydrate source and overall nutrient limitation (Neijssel et al 1997). Analysis of the 16s rRNA sequence data support the identification of isolate BAW#l as a member of the E. casseliflavus-E. gallinarum group. 62 This analysis is insufficient to differentiate these two species. The data clearly shows that the isolate is not a strain of E. saccharolyticus. This data supports the phenotypic identification of the organism as E. casseliflavus. The importance of a full set of data when conducting analysis using databases is underscored by this analysis. The identification of the isolate as E. casseliflavus by API Strep, BIOLOG TM , and ARB database sequence analysis contradicted the identification of the isolate as E saccharolyticus by Lawes and as Ruminococcus albus by Tadese. The characterization of the isolate in this study was multidimensional, including three databases in addition to the traditional approach using Bergey’s and phenotypic data. The identification of the isolate to the species level by LaWes as E saccharolyticus primarily rested upon the RDP analysis. However, the database used in that inquiry was strikingly incomplete. The RDP database only included six enterococcal species: E. cecorum, E. faecalis, E. hirae, E. sulfureus, E. columbae and E. saccharolyticus. None of these are of the E. faecium group of enterococci that the isolate clearly belongs to. However, despite this lack of depth of the initial analysis, the isolate BAW#] is listed in GENBANK as E. saccharolyticus, and has been given the accession number ESU30931 (Entrez Nucleotide Query). The organism is of a genus which is known to be pathogenic, usually only of serious concern in patients with a severely suppressed 63 immune system. The E. casseliflavus strain tested resistant to five of twelve antibiotics tested. It is only moderately susceptible to two more of these. It tested sensitive to penicillin and ampicillin. In each case, the isolate BAW #1 tested as would be expected of a member of the genus enterococcus. The isolate BAW #1 does not appear to have picked up resistance to any of the antibiotics for which it was tested. The isolate BAW #1 was not tested for vancomycin resistance. As a strain of E. casseliflavus, the isolate probably carries the VanC2 gene characteristic of that species (Coque et a1 1996). 64 Summary A bacterial isolate BAW #1 has been examined using BIOLOGTM, API StrepTM and conventional analysis. Examination and updating the databases of BIOLOGTM and API StrepTM produced a consensus that the isolate BAW #1 is a strain of E. casselzflavus. 1995 versions of the BIOLOGTM database named the isolate BAW #1 incorrectly. The API StrepTM diagnosis was also incorrect, until further tests were made and the company called for consultation. Analysis of 16s rRNA sequence data supports the identification of isolate BAW#I as a member of the E. casseliflavus-E. gallinarum group. The 16s rRNA data does not contain enough information to make a decisive identification at the species level. The data ”does not support the identification of the isolate as a member of the genus Rumincoccus or as E. saccharolyticus. The development of these databases has created a simple means of identifying commonly encountered bacteria. However, they may be deceptively simple and produce false results. At one point during this investigation, the well reputed databases API StrepTM, BIOLOGTM and RDP were identifying the isolate BAW #1 as E. faecium, E. gallinarum and E. saccharolyticus, respectively. Each had a limited database. Ironically, the simple testing of the color of a swab of the isolate BAW #1 could have greatly limited the search. However, useful phenotypic information has 65 been gathered through the use of BIOLOGTM and API StrepTM. Anaerobically, the major fermentation product of the isolate BAW #1 is lactic acid. Aerobically acetic acid, lactic acid and ethanol were produced. 66 CHAPTER TWO CHARACTERIZATION OF AN ENTEROCOCCAL BACTERIOPHAGE Phage Literature Review MORPHOLOGY The tailed phage include Siphoviridae, Myoviridae, and Podoviridae. While they show considerable difference in detail, they share important morphological features. The phage in these classes have single linear dsDNA contained in a capsid (Figure 6d). The capsid is built of Coat protein molecules arranged in icosahedrally symmetric arrays. All have a single host attachment apparatus (a tail) attached to one corner of the capsid. The tails may vary in contractibility and length but all are 67 thought to contain a threefold or sixfold rotational symmetry axis that projects through the center of the capsid shell of the virion (Casjens and Hendrix 1988). The capsid of the tailed phages may be isometric or elongated. The elongated capsid of the Myoviridae T4 phage has been studied in great detail. The T4 capsid is thought to be constructed from hemispherical caps having an icosahedral surface lattice and tubular sides with a matching helical surface lattice (Harrison et a1 1996). The chemical interactions between the subunits of the tube are likely to be similar to those of the cap; the surface lattice of the tubes is thought to be related to that of the icosahedral capsids. This theory is supported by the presence of tubular mutants of icosahedral viruses (Harrison et al 1996). DNA passes in and out of the capsid through the portal vertex during packaging and ejection. In phages where it has been studied, the portal vertex is composed of a dodecamer of a single polypeptide arranged in a ring with a 3 to 4 nm hole through its center. The hole is aligned with the long axis of the tail so that it acts as a pore (Casjens and Hendrix 1988). The tail is composed of helically arranged subunits. This structure is flexible, and of varying length based on strain. In lambda, it is composed of 32 rings (Casjens and Hendrix 1988). A “tape measure protein” controls tail length. At the distal end of the tail is a base plate, 68 which is frequently of 6 or 12 fold symmetry. This structure is frequently not seen in electron micrographs; its absence is most likely due to its destruction during preparation of specimens (Tikhonenko 1972). In some phages, the base plate has enzymatic activity, including lysozymes. Tail fibers attached to the end of the tail are the most important structural subunit for attachment to the host. The distal portion of the fibers is an important site of mutations determining host specificity. Fibers are also sometimes seen coming off of the sides of the tail (Casjens and Hendrix 1988) TAXONOMY Early taxonomic efforts by Bradley (1967) divided the tailed phages on the basis of capsid and tail morphology, as well as type of nucleic acid. According to this scheme, all phages with noncontractile tails were designated Group B. (Bradley 1967). Anna Tichonenko (1968) devised a similar scheme in which the same phages are considered group IV. In 1974, Ackermann and Eisensark (1974) subdivided the non- contractile tailed phages based on their capsid shape. Isometric phages in this scheme are in group Bl, phages with moderately elongated capsids are group B2, and phages with very long capsids (length to width ratio 2.7 to 5.5) are group B3. In 1991 the International Committee on the Taxonomy of Viruses (ICTV) moved to establish a universal database, the ICTVdB 69 (International Committee on the Taxonomy of Viruses Database). Its goal is to describe all viruses of animals, plants, bacteria, fungi and archaea from the family level down to strains and isolates. The database uses the DELTA system of programs for organization of virus. A decimal code hierarchy, similar to the system used for enzyme nomenclature, has been established. Families have been sorted and each assigned a number. The system can contain many more levels to accommodate strains and isolates. The decimal code 50.1.1. represents family 50, subfamily l, genus 1 (Murphy et a1 1995). In the ICTVdB the Siphoviridae is the family which contains the dsDNA, noncontractile tailed bacteriophages. The best known phage in this family is lambda. The decimal code for the Siphoviridae is 66; lambda is in the genus 66.0.1 “lambda-like phage”. INCIDENCE OF SIPHOVIRIDAE ASSOCIATED WITH STREPTOCOCCUS AND ENTEROCOCCUS. Ackermann (1996) has catalogued the descriptions of viable, negatively stained phages that have been included in periodicals, books and dissertations. Ninety—six percent of the over 4500 phages described are tailed phages. Of these 61.7% (2708) are Siphoviridae. Only 15% of the tailed phages have elongated capsids, of these 418 (B2) and 58 (B3) are Siphoviridae. Phage that can infect gram positive cocci account for 238 B2’s and 8 83’s. Eight of the most elongated B3 bacteriophages are infective of the bacterial genus enterococci. The genus streptococci are 70 listed as infected by 182 isometric Bl bacteriophages and no B2 or B3 bacteriophages. Phylogenetic relationships of the tailed phages to each other in unknown. However, protein and gene sequence data is beginning to accumulate. Tailed phage genomes apparently consist of gene blocks or modules that may be exchanged to produce new phage species (Ackermann et al 1995). Siphoviridae have been associated with S. bovis. Styriak et al (1991) isolated twenty strains of S. bovis from rumen contents. They tested filtered rumen fluid on the twenty strains and found two that produced plaques. The phage isolates were strain specific with the S. bovis isolates. Both phage strains had prolate capsids. Some strains previously identified as S. bovis are now classified as Enterococci (Chapter 1). A phage infective of S. faecalis (now E. faecalis) was also described as having an elongated “bacillus shaped” capsid and appears to be divided into three volumes. The tail had cross striations and ended in a baseplate with six-fold symmetry. The phage was designated 10C1 (Anderson 1973). Ackermann, Caprioli and Kasatiya (1975) describe an enterococcal phage VD 13. This phage was found while investigating urogenital group D Streptococci. It has a prolate capsid and a flexible noncontractile tail 7] terminated in a base plate. Capsids mostly appeared oval, flattened, and up to 65 nm wide. However, capsids deeply embedded in stain appeared to have hexagonal outlines. Tails showed cross-striations after staining with uranyl acetate (UA). Preparations contained spirals 10 nm wide, of variable length and with a l2-nm periodicity. The phage VD13 was tested against a battery of enterococci for plaque production. It lysed 37 out of 146 strains of S. (E. ) faecalis, S. (E. ) faecium, S. (E. ) durans and S. liquifaciens. It was inactive on 10 strains each of group A Streptococci, Bacillus, Staphylococcus, Micrococcus, Eschericia, Pseudomonas and Salmonella (Ackermann et al 197 5). The ICTVdB lists only five Siphoviridae infective of streptococcal species (A25, a25 PEI, A25 VD13, A25 omega8 anda25 24), and no phages infective of enterococci. METHODOLOGY OF PREPARATION OF PHAGE FOR TEM The preferred method for examination of phage is negative staining, followed by transmission electron microscopy. This method is quick and shows fine detail of phage ultrastructure. Several stains may be used depending on specific needs. The most common are uranyl acetate, and phosphotungstic acid. Staining with UA may produce negative and positive staining of adjacent areas, negative staining is considered superior in providing useful information. The capsid shape and tail striations are most easily 72 seen on phages negatively stained with UA. Positively stained capsids are blackened and shrunken, therefore size measurements of positively stained phages may be inaccurate. Positive staining with UA is known to produce shrinkage of from 11 to 31% of phage capsids (Ackermann 1987). Negative staining with UA may cause flattening and therefore an increase in the size of the phages (Hayat and Miller 1990). Phosphotungstic acid (PI‘A) produces only negative staining. It is known for producing rounded and flattened capsids, and is not the best for showing tail striations (Hayat and Miller 1990). PTA is not capable of contrasting specimen details smaller than 1 nm. UA has an ionic size of 0.4nm to 0.5nm, PTA has an ionic size of 0.9, and because of this UA can demonstrate finer details (Hayat and Miller 1990). I An alternative preparative technique is shadow casting. The Kleinschmidt technique (a variation of shadow casting) and its derivatives utilize cytochrome C in ammonium acetate to form a monolayer (Kleinschmidt 1968). This monolayer facilitates spreading of nucleic acids or particulate samples such as phages. The phage solution is deposited on a coated grid and shadowed with a thin film of platinum. Staining with a dilute UA solution to enhance contrast may proceed the shadowing. Resolution with shadow casting is limited by the granular structure of the shadowing film. Platinum granules deposited from an electron beam under optimum conditions may measure between one and 73 four nanometers. Specimen detail which is not twice the diameter of the grain must be interpreted carefully. Specimen dimensions may be exaggerated by the accumulation of shadowing materials, forming what is known as a cap (W illison et a1 1980). Shadow casting techniques are superior to negative staining for showing up fine fibrous structures such as the fiber of bacteriophage tails (Bradley 1967). Thin sectioning of plastic embedded phage samples is useful in the study of intracellular phage development. It is considered less prone to the flattening and emptying artifacts frequently associated with negative staining (Kellenberger et al 1995). Ultrathin sections are between 70 and 90 nm thick, or approximately twice as thick as a viral particle, thereby obscuring morphological details (Ackermann 1987). 74 Objectives: 1) To examine the structure of the phage isolated from the isolate BAW#I with transmission electron microsc0py. 2) To test the phage lysate for plaque production on Streptococcus bovis 3) To isolate the phage DNA harvested from an infection of the isolate BAW# 1 . 75 Materials and Methods 1. Purification of lysate Methods developed for isolation and purification of DNA from Eschericia coli and lambda phage were utilized in the study of the isolate and its bacteriophage. These standard methods were modified to accommodate the anaerobic metabolism of the isolate. Glucose- Cellobiose-Starch - Rumen Fluid media and TSA were used in these experiments. GCS-RF medium is the standard for the cultivation of anaerobic rumen bacteria. (See Appendix I). The media are prepared as broth. Soft agar GCS-RF contained 0.7% agar, and hard agar GCS-RF contained 2.0% agar in this research. For aerobic phage work, Trypticase Soy Broth (TSB), Trypticase Soy Agar (TSA) and TSA soft agar were used. Lysate was diluted and used to infect the isolate as follows. The lysate was used to prepare a dilution series in sterile dH20. Broth culture (0.3-ml) with an OD600 of 0.25 was added to the lysate and incubated 30 minutes. The solution was then added to 3 mls of soft agar, vortexed and poured onto a GCS-RF plate. The plates were incubated overnight. A well-isolated plaque was picked using a sterile pasteur pipette and deposited into 1 ml of dH20. One drop of chloroform was added. The phages were allowed to diffuse out of the agar for one hour, and centrifuged for 20 min. at 10,000g. The supernatant obtained was then 76 filter sterilized through a 0.45um Gelman filter and held at 4 0C. This phage picking procedure was repeated three times to ensure purification (Sambrook et al 1989). The phage was tested for plaque production under aerobic conditions using the soft agar overlay technique (Sambrook et a1 1989). TSA Plates were used with TSA soft agar. TSB was used as the broth to prepare a log phase culture of the isolate. Plates were incubated at 39° C. 2. Phage tested for plaque production on Streptococcus bovis S. bovis ATCC str 3317 91-08 was grown in TSB. The culture was tested for plaque production using the soft agar overlay technique. Media used were TSA (agar 2.0%) and soft agar (0.7 % agar) in TSB. Plates were incubated at 39 0C. A dilution series of the lysate collected from an infection of the isolate BAW#I was used for this experiment. The most concentrated in the dilution series produced 10'7 plaques per ml when plated on the Isolate BAW# lusing the soft agar overlay technique (Sambrook et a1 1989). 3. Purification and restriction digest of phage DNA The phage was plated, lysate collected, and phage DNA isolated using a Promega Lambda purification kit. Briefly, the protocol of the PROMEGA DNA isolation kit for Lambda was followed. (Promega Insert, Sambrook et a1 1989) 77 Lysate preparation: The isolate was used instead of E. coli in the protocol. The soft agar overlay method using GCS-RF media was used to produce plaques. After overnight incubations (anaerobic at 39° C) plaques were nearly confluent. 2 to 3 ml dH20 was overlaid on each plate. The top agarose was scraped off and transferred to a centrifuge tube to be incubated with intermittent shaking for 30 min. The tube was then centrifuged at 10,000g for 10 min. at 4°C. The supernatant was removed, 0.3% chloroform was added and the lysate was stored at 4°C. DNA extraction: 40 ul Nuclease was added to lO-ml lysate and incubated for 45 min. at 37°C. 4 ml Phage Precipitant was added, the tube was gently mixed and placed on ice for 30 min. The tube was centrifuged at 10,000g for 10 min. The supernatant was decanted and discarded. The pellet was resuspended in 500 111 of Phage Buffer and 3 mg Proteinase K was added. The resuspended phage were transferred to a 1.5-ml eppendorf tube and centrifuged for 10 sec. at 12,000g. The supernatant was transferred to a fresh tube. One ml of Purification Resin was added to the tube and mixed by gently inverting the tube. The solution was pipetted into a syringe barrel. The resin / lysate mixture was pushed into the Promega Minicolumn, then washed with 2 ml of 80% isopropanol. The Minicolumn was transferred to a fresh eppendorf tube in a microcentrifuge, 100 111 of 80°C double distilled water was added and 78 the column was then immediately centrifuged for 20 sec. at 12,000g. The eluted DNA in doubly distilled water was stored at 20° C till further use. Restriction digest: Phage DNA was digested with Hindlll and run on a gel with a Lambda Hindlll standard. Sambrook et al 1989) 4. Transmission electron microscopy of the phage Phage lysate produced from soft agar overlay plates was used to prepare samples. The phage was prepared for electron microscopy by two methods: A) Negative Staining with Uranyl Acetate (UA) B) Phage spread on a monolayer of Cytochrome C and shadow casted. Negative Staininuvith Uranyl Acetate (UA) The negative stain samples were processed as follows: A dr0p of lysate (@ 5 ul) was deposited on a formvar coated grid and allowed to stand for 1 min. Excess lysate was removed by draining from below with filter paper. A 5-ul drop of 2% UA was deposited on the grid, allowed to stand for one minute, and drained. Grids were examined with the Philips CM 10 or JEOL 100CX II and micrographs recorded. PhAage spread on a monolayer of Cytochrome C and shadow casted. The shadow cast samples were processed as follows: 40 ul 0.3M ammonium acetate was deposited on a sheet of parafilm. A 10-ul drop of 1 mg/ml 0.03% Cytochrome C and 20-ul lysate was added to the drop. 79 The drop was allowed to stand covered for 10 minutes. Coated grids were then touched to the drop, held for 30 seconds, held to a drop of 95% ethanol for 30 seconds and deposited face down on a filter paper. Grids were then removed from the filter paper and placed in the modified Balzer 510A freeze etch device. Grids were then shadow casted with 250 Hz of platinum evaporated in the electron beam gun. Platinum deposition was monitored with a Quartz Crystal Monitor, and controlled with a Control Unit EVM052. The angle of platinum deposition was 7 degrees. A TSR1000b rotary unit controlled the rotary stage. The stage was rotated at 80 rpm during platinum and carbon deposition. A thin layer of carbon was evaporated onto the specimens immediately after platinum deposition. Grids were examined with the Philips CM 10 and micrographs recorded. For the second Kleinschmidt shadowing experiment, the grids were touched to a drop of 0.5% UA immediately after the grid was picked up from the sample drop. The samples were washed as above in ethanol, dried, and mounted on the stage. The samples were again shadowed with platinum at 7 degrees. A thin layer of carbon was evaporated onto the specimens immediately after platinum deposition, with the stage rotating at 80 rpm. Grids were examined with the Philips CM 10 and micrographs recorded. 80 Bacteriophage measurement: Prior to photographing the phage for measurement, the Philips CM 10 was z-axis centered and aligned. Micrographs were taken of tobacco mosaic virus (TMV), known to have a width of 18 nm. The magnification of the phage micrographs was adjusted based on the TMV measurements. Micrographs were digitized using a Kodak MEGAPLUS Camera Control Unit Model 1.4 and Kodak MEGAPLUS Camera, Model 1.4. The digitized images were imported into Photoshop 3.5 on a PowerMac 7100. On screen magnification was increased at least five fold, and the “get info” tool used to measure the size of the phages and TMV. Using this methodology twenty phages were measured and sized averaged. Phage with obvious malformations were not used in the determinations of size. The size of the phages were determined by the formula: L0 = Length of object in nanometers M = magnification of micrograph after calibration S = Size in nanometers and Lo/ M = S Periodicity of the unwound tail was determined by counting 16 spirals on the computer screen, as above. 81 5. Phage infection of isolate BAW#l followed by electron microscopy. The phage and isolate BAW#l were prepared for electron microscopic examination as follows: 100 ul of phage lysate at a concentration of 10‘7 pfu/ ml was added to 1 milliliter of a log phase culture of isolate BAW#l. TSB was used as liquid media. The multiplicity of infection was assumed to be approximately 1 phage per 100 colony- forming units. The culture was sampled for electron microscopy by withdrawal of 2 ul at various time points. The 2-ul aliquot was deposited on a coated grid and allowed to stand for one minute. A 2-u1 drop of deO was added to the grid and removed from the side with filter paper after 15 seconds. A drop of uranyl acetate was immediately added to the grid and allowed to stain the phage / enterococcal mixture on the grid for 15 seconds. The stain was removed from the side with a filter paper and the grid allowed to dry. Grids were examined in a Philips CMIO transmission electron microscope. Grids were scanned at low magnification (about 5000X) for enterococcal chains and at a higher magnification of 25000X for phage. The time points sampled were: prior to addition of phages, 15 minutes after addition of phages, 2 hours after addition of phages, 3 hours after addition of phages, 4 hours after addition of phages, 4.5 hours after addition of phages and 5 hours after addition of phages 82 Results 1. Purification of lysate and testing of the phage under aerobic conditions. Methods developed for lambda phage and Eschericia coli were useful in the study of the isolate and its bacteriophage. Picking of a single plaque produced about 100,000 plaque-producing particles. When grown aerobically at 39° C, the phage was capable of producing plaques on a lawn of the isolate on TSA in less than five hours. 2. Phage tested for plaque production on Streptococcus bovis The phage did not produce plaques on S. bovis ATCC str 3317 91-08 when grown on TSA at 39°C or 35°C. Examination of the plates with a 10X ocular revealed no microscopic plaques. 3. Purification and restriction digest of phage DNA The Promega kit designed for lambda phage DNA extraction was used to extract DNA from the isolate BAW#l bacteriophage. This DNA was sufficiently pure for restriction digestion (Figure 4). The DNA was cut with Hindlll, producing a gel with nine clear bands. The size of the bands was determined by comparing them to a lambda DNA Hindlll digest standard. The bands lengths were 5950, 4000, 3800, 2800, 2100, 1650, 83 1300, 1050, and 680bp. The total length of phage DNA based on this is 23330bp. 4. Transmission electron microscopy of the phage prepared for electron microscopy. Negative Staining with Uranyl Acetate (UA) Staining with UA allowed visualization of fine structures of the phages (Figure 6). Figures 6b, 6n, and 60 show negatively stained phages with visible baseplates. Ridges were visible on the edges of the phage capsid (Figure 60) and in some micrographs, the top of the capsid was angular (Figure 6b). Note that the stain was pooled at the edges of the phage, and only lightly stained the phage itself. In contrast, positive staining of the phage capsid appeared dark. This demonstrates the affinity of UA for DNA (6a, 6g, 6m, and 6i). The positively stained capsids appeared angular, and have maintained their shape. Occasionally phages were seen with an intermediate staining reaction (Figures 6e and 6h). Two tail fibers and a base plate were visible on Figure 6i; this positively stained phage had an elongated head. Phage stained with UA were seen with capsids that were empty headed and swollen. (Figures 6f and 6k), and with a visible valve structure. A longitudinal ridge was sometimes visible in the tails of 84 Figure 5. Bacteriophage DNA extracted from the isolate BAW#l and cut with the restriction endonuclease Hindlll (4)) The DNA of the phage lambda is used as a standard for size determination (7.). 85 phages with the empty capsids. Some phages appeared to have lost their DNA (Figure 61). Figure 6c shows a phage and tobacco mosaic virus (TMV) side by side. TMV is known to be 18 nm wide, and is used as a standard for calibration of magnification in the transmission electron microscope. Figure 6j shows an unwound tail with a periodicity and width of 10 nm. A diagram summarizing the morphological features of the phage is presented in Figure 6d. The average size of the phages negatively stained with UA was: The capsid is 90 nm by 36 nm, the tail is 136 nm by 9 nm (Table 13). A higher magnification view of phages with empty heads is seen in Figures 7a and 7b. The valve structures that join the “capsid to the tail is highlighted in Figure 7a. The tails of the phages showed a longitudinal ridge. The perimeters of the phage capsid were balloon like and visible around a hollow core. A group of phages in Figure 7b shows one phage positioned approximately perpendicular to the image plane (see double arrow). Another phage in the group shows the empty head and valve structure (see single fat arrow). The tails appeared to be joined together in a knot (long arrow). The six pointed tailed structure in Figure 7c may be a phage capsid perpendicular to the image plane. 86 Table 13. Measurements of the phage infective of isolate BAW#l. Phage stained with uranyl acetate and examined in Philips CM 10 Transmission Electron Microscope. All measurements are in nanometers. Capsid Capsid Tail Tail Sides Height / length Width Width Length Width 86.6 38.5 9.6 125.0 62.5 2.3 91.6 38.8 7.4 133.7 70.9 2.4 93.3 38.5 11.4 134.3 2.4 91.0 38.3 8.0 140.4 73.6 2.4 86.3 35.3 2.5 90.5 36.2 8.7 140.4 76.9 2.5 88.3 35.4 8.7 140.4 66.9 2.5 91.7 36.9 2.5 86.8 34.7 2.5 89.5 35.8 2.5 88.2 34.7 8.7 134.3 2.5 92.2 36.4 2.5 91.7 35.8 2.6 90.6 35.3 2.6 90.9 35.4 8.7 140.4 76.2 2.6 89.5 34.2 9.5 134.0 2.6 89.5 34.2 9.5 137.0 2.6 93.3 35.5 2.6 88.2 33.1 8.7 135.7 2.7 91.4 32.8 2.8 Mean 90.06 35.79 135.97 8.98 2.52 Standard Deviation 2.12 1.72 4.63 1.04 0.11 Minimum 86.3 32.8 125.0 7.4 2.25 Maximum 93.3 38.79 140.4 11.4 2.78 Count 20 20 11 11 20 Confidence Level 1.00 .8 3.11 .70 .05 87 Phage spread on a monolayer of Cytochrome C and shadow casted. Micrographs of phages spread with cytochrome C and shadowed with platinum (the Kleinschmidt techniques) are shown in the Figure 8a through 8f. Micrographs of phages stained with UA prior to shadowing are shown in Figures 8a, 8d, and 8e. Angularity of the phage capsid was apparent, the angularity of the vertices of the phage was particularly evident in some cases (Figure 8a.) The apical vertex was 115 degrees. The base plate of the phage in these lightly shadowed micrographs was visible, and a tail fiber is seen extending from the thickened distal end of the tail to what appears to be a membranous particle (Figure 8d). Phage spread with cytochrome C, unstained, and shadowed with platinum were also examined (Figures 8b, 80, and 7 f). The thicker platinum shadow produced a different image than in the more lightly shadowed preparations (Figures 8a, 8d and 8e). Phage capsids and tails appeared thicker. Tail fiber were visible (Figure 8b) and an unusual triangular thickening at the distal end of the tail was noted (Figure 8c). The phages prepared by the shadow casting technique did not show valve structures or the longitudinal ridge of the tail. The end plate of the tail was exaggerated and appeared quite thick (Figures 8c and 8b). The image of the phage is longer and wider (120 nm by 57 nm) than other preparation methods. The tails in these micrographs were not longer, however they were wider. Background surface of the micrographs 88 was irregular and splotchy in appearance. Phage in these photos appeared to lack angularity and definition. Micrograph 8f shows a group of phage. The phage in the upper and lower right hand corners appeared to have a cap of platinum grain around a central core. The measurements of this central core (capsid: 100 nm by 33 nm) were in agreement with the size of the phage determined by the UA staining method. 5. Phage infection of the isolate BAW#] followed by electron microscopy. Micrographs were recorded of the isolate BAW#l prior to infection with the phage. Isolate BAW#l grows in chains, repeatedly dividing longitudinally to produce the characteristic chains. (Figures 9a and 9b). The separation of the chains was occasionally noted (Figure 9e). The size of individual cocci was approximately 0.9um by 1.1um. Most chains appeared elongated, with the maximum chain length over 20 cocci. The phage was very difficult to find in samples taken during the first 3 hours of the experiment. After 4 hours, the phage was occasionally seen, but not in high concentrations. At 5 hours, the phage was seen near most enterococcal chains. The chains frequently appeared to be lined with phage (Figure 8b). In this image, the phage were noted externally all over the four cocci enterococcus. The enterococcal chains were examined to determine if certain positions within the chain were 89 more frequently attacked, however, no differences were noted. The individual infected cocci were examined to determine if the phage was attacking a certain part of the cocci (i.e. new membrane near the septum or older membrane at the polar regions), again, no differences were noted. Phages were seen that appeared to attach to the outside of the bacterium (Figures 9f and 9c). An unwound tail was observed close to a bacterium under multiple attack, (Figure 9c). At five hours, bacteria were found which appeared to have burst, (Figure 9g) and in one image several phages were attached to the debris. 90 Figure 6. Phage harvested from the isolate BAW#l and stained with uranyl acetate prior to examination in the transmission electron microscope. The phages in these micrographs show positive and negative staining reactions. Micrographs are lettered from top left to bottom right. a) b) g) h) j) k) 1) Positive staining of the phage capsid due to the affinity of UA for DNA. The positively stained capsid is angular. Negatively stained phage with a visible baseplate. The top of the capsid is angular. Note that the stain is pooled at the edges of the phage, and only lightly stains the phage itself. Phage and tobacco mosaic virus (TMV) side by side. TMV is known to be 18 nm wide, and is used as a standard for calibration of magnification in the transmission electron microscope. A diagram summarizing the morphological features of the phage is presented. A phage with an intermediate staining reaction Phages stained with UA appears empty headed and swollen, the valve structure is visible. A longitudinal ridge is visible in the tails of these phages with empty capsids. Positive staining of the phage capsid due to the affinity of UA for DNA. The positively stained capsid is angular. A phage with an intermediate staining reaction. A negatively stained phage with two tail fibers. An unwound tail. The periodicity and width is 10 nm, A phage with an empty and swollen head. Several phages are visible in this micrograph. Note that two capsids are empty and swollen, the others are negatively stained. m) Positive staining of the phage capsid due to the affinity n) 0) of UA for DNA. The positively stained capsid is angular. A negatively stained phage. A negatively stained phage with ridges visible along the sides of the capsid. 91 92 b) Figure 7. Phages harvested from the isolate BAW# land stained with uranyl acetate prior to examination in the transmission electron microscope. Micrographs are lettered from top left to bottom right. Phages have tangled tails. In the phage with an empty capsid (see arrow), the valve is visible joining the capsid to the tail. The tail of this phage is thickened and has a lengthwise ridge. The other phages have capsids full of DNA, the capsids are angular. Phages with tangled tails (see long arrow). Three phages have full capsids, one has an empty capsid with a visible valve (see medium arrow). Small arrows point to what may be a top view of another phage. A long fine structured tail protrudes from what may be a phage perpendicular to the plane of view. 93 94 Figure 8. Phages harvested from the isolate BAW# land shadow cast with platinum prior to examination in the transmission electron microscope. The phages in Figures a, d, and e were lightly stained with uranyl acetate prior to shadowing, The phages in Figures b, c, and f were shadow cast with an excessive deposition of platinum. Micrographs are lettered from top left to bottom right. a) b) d) 6) Edges of the phage are clearly visible and have been measured. The top apex of the phage has an angle of 115 degrees. Heavy shadow on phage, tail width approaches 25 nm, tail fiber is visible. Phage is very heavily shadowed, it appears round and fat. The tail appears to end in an angular structure not characteristic of tail fiber or baseplate. Phage with tail fiber attached to membranous particle (see arrow). Phage is evenly and lightly shadowed, tail baseplate is visible, capsid is angular and full. Phage capsid shows significant staining due to affinity of DNA for uranyl acetate. 1) A large group of phages is joined together in a snarl of their tails 95 stti J./.. , u rope aCt‘i- ) lm 96 Figure 9. The micrographs show the bacterium prior to, during and after attack by the bacteriophage. All grids were stained with UA for 15 seconds. Micrographs are lettered from top left to bottom right. a) Isolate BAW#] prior to infection with the phage. The arrows point to developing septa in dividing cells. b) A large number of phages (see short arrows) are attached to this chain of four. c) High magnification allows visualization of the phage ultrastructure on the surface of isolate BAW#I. Short arrows point to an unwound tail. The inset magnification is 286,000,and contrast has been inverted. d) ’I‘wo long chains of isolate BAW#I show the usual elongation of cocci. e) Two cocci separating. 1) High magnification shows the surface of isolate BAW#l densely populated by phages. g) A lysed isolate BAW#I. Arrows point to several phages attached to the bacterial debris. 97 100 nm 98 Discussion Purification of the phage. The phage was successfully purified after infection of the isolate BAW#l. A single plaque produced about one hundred thousand plaque forming units. Inoculation with phage lysate always produced plaques on a lawn of the isolate BAW#I isolate. Colonies appearing within the margins of plaques were picked. These phage resistant strains were spotted on to a lawn of the isolate BAW#I isolate, however, no plaques were produced. No evidence was found of lysogeny. The phage demonstrated insignificant reduction in plaque forming ability after treatment with 0.3% chloroform. The most rapid plaque production recorded was a log phase isolate BAW#l culture inoculated into TSA supplemented with glucose, in this case, plaques were visible after 5 hours. The restriction digest of the isolated phage nucleic acid demonstrated that the phage was a double stranded DNA phage. This is to be expected given the morphology of the phage. The length of the phage DNA by this analysis was 23330 bp, short in comparison with the tailed phage lambda. The phage did not produce plaques on S. bovis ATCC str. 3317 91-08. This is not a surprise, considering that isolate BAW#l and S. 99 Bovis are not in the same genus, and phage which are capable of infecting more than one species are rare (Ackermann 1987). Electron microscopic examination of the phage. The examination of the phage with the TEM showed the following features. The capsid was prolate, the tail was non-contractile. An "8 endplate was visible on the tail in several micrographs (Figures 51, 5k, 51, -- f“ _“.. a a . 5n, 50 7b, 7c and 7d). A fiber was seen extruding from the tip of the tail (Figures 5I, 7b and 7d). The shadow cast micrographs, while obscuring fine structure of the capsid, most clearly demonstrated the tail fiber. The tail fiber of the phage was visible in micrographs prepared with the Kleinschmidt technique and where the platinum shadow was light. The fiber was only seen once in a non-shadowed micrograph. Frequently, phages were noted in groups that appear to be connected (Figures 6a, 6b and 7f). The phages in these groups may have been attacking a common bacterial receptor site, or this may be an artifact of specimen preparation. Negative staining is a very useful method for providing detail of the capsid and valve structure, tail structure and baseplate of the phage. The tail fiber was only seen once using this technique. Phages which were negatively stained may have undergone osmotic shock, resulting in the loss of the capsid content, DNA. Phages with empty capsids were clearly 100 misshapen and were not used in data collection for size determination. These shocked phages did, however, provide key information on the portal valve and tail structure. Determination of the size of the phage after negative staining requires close attention to the osmotic state of the phage being examined. Negatively stained phage capsids appeared angular. The area surrounding the phages was more electron dense than the capsid or tail. These phages were used in size determination. Positively stained phages appeared angular, with the capsid core darkened. Several of the phages with positively stained capsids measured larger than the negatively stained phages, data from these phages-were not used for size determination. Phages in all three forms, positively Stained, negatively stained and osmotically shocked appeared on the same grid. Differences may have been due to differential concentrations of stain versus buffer or partial drying of grids. Shadow casting of the phage produced variable results. The first of the variations was the apparent thickness of the shadow itself. Two runs of the shadow cast produced two distinctly different results. One of the runs laid a very heavy shadow on the specimen and support structure, the shadow here appears piled on itself and knobby (Figures 7b, 7c and 7 f). In Figure 7f, the heavy coat of platinum created a cap on either side of two of the phages. The phage capsid measured 30 nm wider than in 101 other preparation techniques. The fine structure of the phage was buried under a drift of platinum shadow. Contrast in these images was due solely to the platinum. On the second shadow cast run, the phages were stained with uranyl acetate and rinsed in ethanol. The shadow layer was much lighter and finer, the outlines of the phage capsid were clearly visible. The uranyl acetate added contrast to the image. Measurements of the phages ~.-_.1 .: i I in this treatment were close to the measurement of negatively stained phages. In the Balzer freeze fracture apparatus deposition of platinum was by electron beam gun, which can short out and frequently needs to be reset. This may have been one factor that may have “caused a differing total deposition. Exact placement of the specimen within the gun was also a variable, as was the condition of the electron beam gun itself. It was also possible that the pre-shadowing UA treatment of the grids in the second run altered the dynamics of the shadowing process in an unknown manner. The second anomaly of the shadow casting was that the rotary shadow casting of both runs produced some phages that appeared to be unidirectionally shadowed. Figure 8b is from the first, heavily shadowed run, and the phage appears to be unidirectionally shadowed. Phages in figures 8c and 8f are from the same run and appeared rotary shadowed. 102 This phenomenon was also seen on the second shadow cast (not shown). Because the angle of platinum shadow deposition was only seven degrees, a minor bend in a grid or its support structure on the rotary stage may have blocked the platinum from one direction. The repetition of the shadow cast provided an important reality check for the size of the phage. The lightly shadowed phages resembled (‘2 the phages stained with UA in size and shape. The heavily shadowed L: phage preparations provided insight into the tail fiber and base plate, for these structures, exaggeration of size provides useful information. The Kleinschmidt technique of spreading macromolecules with cytochrome C and subsequent shadow casting is frequently used to illuminate DNA structure. The DNA strand itself was only 2nm wide, it appeared about 20 nm wide after the Kleinschmidt process. It is likely that the tail fiber of the phages underwent a similar size enhancement in these experiments. 103 Summary A phage infective of the isolate BAW#l was examined by transmission electron microscopy (TEM). The phage has a long non- contractile tail and an elongated capsid. The valve joining the capsid to the tail was visible in preparations where the phage had lost its DNA. Preparation of the phage for TEM using shadowcasting and negative staining provided information on the morphology of tail fibers. The phage capsid is 90 nm by 36 nm, the tail is 136 nm by 9 nm. The phage did not look like the phage described by Tadese, however, it did have a similar morphology to one of the phage reported by Lawes. The phage has double stranded DNA. It is a member of the Siphoviridae family of phage, a B2 in Bradley’s scheme (Bradely 1967). 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Agents Chemother.32(5):768-9. 116 Appendix I AEROBIC MEDIA ATCC MEDIA 260: TRYPTICASE SOY AGAR (BBL 11043) WITH 5% DEFIBRINATED SHEEP BLOOD Pancreatic digest of casein 15.0 g Agar 15.0 g Papaic digest of soybean meal 5.0 g NaCl 5.0 g Sheep Blood defibrinated 50.0 ml dH20 950.0 ml pH: 7.3+/- 0.2 at 25C, makes 1000ml. TRYPTICASE SOY BROTH WITH 5% DEFIBRINATED SHEEPS BLOOD ‘ Bacto Tryptone 3.75 g Bacto Soytone 1.25 g NaCl 1.25 g dH20 225 ml Defibrinated sheep blood 25 ml pH: 7.3+/- 0.2 at 25C, makes 250 ml ANTIBIOTIC MEDIA (DIFCO) is 26.5 g/L Bacto Beef Extract 1.5 g Bacto-Yeast Extract 3.0 g Bacto- Peptone 6.0 g Bacto-Dextrose 1.0 g Bacto Agar 15.0 g 117 MOTILITY SULFIDE MEDIA 104.4 g/ L Bacto beef extract 3.0 g Protease peptone No 3 10.0 g L-cystine 0.2 g Ferric Ammonium Citrate 0.2 g Sodium Citrate 2.0 g Sodium Chloride 5.0 g Bacto gelatin 80.0 g Bacto agar 4.0 g KF STREPTOCOCCUS AGAR 76.05 g/ L Bacto-Protease Peptone No 3 10.0 g Bacto- Yeast Extract 10.0 g Sodium Chloride 5.0 g Sodium Glycerophosphate 10.0 g Maltose 20.0 g Lactose 1.0 g Sodium Azide 0.04 g Bacto Brom Cresol Purple 0.015 g Bacto-agar 20.0 g 118 m- Enterococcus Agar 41.5 g/ L Bacto- Tryptone 20.0 g Bacto—yeast extract 5.0 g Bacto dextrose 2.0 g Dipotassium Phosphate 4.0 g Sodium Azide 0.4 g Bacto Agar 10.0 g 2,3,5, Triphenyl Tetrazolium 0.1 g Chloride ANAEROBIC MEDIA RUMEN FLUID Rumen fluid is passed through two layers of cheesecloth into large flasks. After overnight incubation at 39C, fluid is centrifuged at 15000rpm for 30 min. The supernatant is then autoclaved at 15psi for 20 min. After cooling the bottles are stores at 4C. Immediately prior to use in media, the fluid is re-centrifuged for 30 minutes at 15000rpm. SODIUM CARBONATE SOLUTION 8% I NazCO3 24-0 8 I dH20 300 ml Boil water under CO2 to push 02 out. Add Na2 C03 and mix thoroughly. Gas with CO2 for 15 min. Tube up under CO2, and clamp in press. Autoclave at 248°C, 15psi for 20min. 119 MINERAL 1 SOLUTION I K2HPO4 6.0 g I dH20 1000 mls Place solution in 1.5 liter bottle. Autoclave at 248°C, 15psi for 20min. When solution is cool, tighten lid and store at 4C. MINERAL 2 SOLUTION KH2PO4 6.0 g (NH4)2SO4 6.0 g NaCl 12.0 g MgSO4:7H20 2.45 g CaC12*2H20 1.59 g dH20 to 1000 mls Place solution in 1.5 liter bottle. Autoclave at 248°C, 15psi for 20min. When solution is cool, tighten lid and store at 4C. BRANCHED CHAIN FATTY ACID SOLUTION Isobutyric acid 30 ul Isovaleric acid 30 ul 2-methylbutyric acid 30 ul n-valeric 30 ul Branched Chain Fatty Acids are filter sterilized through a 0.22um sterile Acrodisk and stored at 4C. 120 CYSTEINE SULFIDE SOLUTION ( 2.5% ) REDUCING AGENT cysteine HCl 7.5 g Na2S*9l-120 5.0 g dH20 100 ml 10 N NaOH to pH 10.0 dH20 to 300 mls Weigh out and dissolve cysteine-HCL and Na2S*9H20 in small amount of dH20. Adjust pH to 10.0 with 10 NaOH. Boil under C02 and wire down in a boiling flask. Autoclave at 248°C, 15psi for 20min. Pipette under C02 and incubate for 24 H to check sterility. ANAEROBIC DILUTION MEDIA Mineral solution 1 1 1.2 ml Mineral solution 2 11.2 ml Resazurin 0.3 ml dH20 256.3 ml Cysteine Sulfide 6.0 ml Sodium Carbonate ‘ 15.0 ml Combine first 4 components, adjust pH to 6.7-6.8 range. Boil under CO2 and wire down in a boiling flask. Autoclave at 248°C 15psi for 20min. When solution has cooled add sodium carbonate and cysteine sulfide solutions. Pipette under C02 and incubate for 24 H to check sterility. 121 GCS-RF BROTH, SOFT AND HARD AGAR Glucose 0.2 g Cellobiose 0.2 g Starch 0.2 g Yeast Extract 0.6 g Trypticase 1.5 g Rumen Fluid 90 ml Mineral 1 11.2 ml Mineral 2 11.2 ml Resazurin 0.3 ml dH20 163.6ml HCI to adjust pH as necessary Cysteine Sulfide 6.0 ml Sodium Carbonate 15.0 ml Agar: for soft agar 2.05 g Agar: for plates and slants 6.0 g Net Volume to: 300 mls GCS-RF BROTH: Weigh out and dissolve solids in small amount of H20. Add rumen fluid and resazurin. Adjust pH to 6.7 to 6.8 range with HCl. Boil under C02 and wire down in a boiling flask and autoclave. ' GCS-RF Soft Agar: Soft agar is 0.65% agar added to pH adjusted solution prior to boiling down. Cysteine sulfide and sodium carbonate are added to solution at the end of the boiling down process. 2.5 m1 tubes are then prepared under C02, and put into a press to autoclave. GCS-RF Hard Agar: For plates add 2.0% agar to pH adjusted solution. Boil under CO2 and wire down in a boiling flask and autoclave. GSCX-RF: As GCS-RF Hard Agar, add 15ml glycerol ( 5% ) as substitute for 15ml of water. All GCS-RF formulations are autoclaved at 248°C 15psi for 20 min. All component addition and pipetting is conducted under C02, All media is incubated for 24 H to insure sterility. 122 Appendix H Dendrogram showing phylogenetic relationship of some of the most similar organisms to isolate BAW#I (str. 1 BAW) based on 16s rRNA sequence comparison. From Angela Lawes 1993. This tree was built with the sequences from the following enterococcal species: E. columbae, E. saccharolyticus, E. sulfureus, E. faecium, E. cecorum and E. hirae. Note that the RDP did not contain E. casseliflavus, E. gallinarum, E. mundtii, E. durans, E. malodoratus, E. pseudoavium, E. raffinosus or E. faecium. E. colum bae S. cecorum str.) BAW E. saccharolyticus E. sulfureus S. salivarius l L——-——- S. pnem oniae _.__.___ E. faecalis C. divergent B. SLDtiliS 596 123 Alignment of 16s rRNA sequence of isolate BAW#I with closely related Appendix III sequences of E. casseliflavus and E. gallinarum strains. Name: name: Name: name: Name: Name: Name: Eanalli IslBAW#1 NDCO 2313 Eanasse NCDO 2376 AF039898 AF039900 AF039903 .......... ACAUGCAAGU ACAUGCAAGU ACAUGCAAGU acatgcaagt acatgcaagt acatgcaagt acatgcaagt 101 AAAAAGAGUG AAAAAGAGUG AAAAAGAGUG aaaaagagtg aaaaagagtg aaaaagagtg aaaaagagtg 151 AGGGGAUAAC AGGGGAUAAC AGGGGAUAAC aggggataac aggggataac aggggataac aggggataac 201 GCAUGGAAGA GCAUGGAAGA GCAUGGAAGA gcatggaaga gcatggaaga gcatggaaga gcatggaaga 251 E. gall AF039899 E. E. gall E. C358 C383 CGAACGCUNN CGAACGCUUU CGAACGCUNN cgaacgcttt cgaacgcttt cgaacgcttt cgaacgcttt 111 GCGAACGGGU GCGAACGGGU GCGAACGGGU gcgaacgggt gcgaacgggt gcgaacgggt gcgaacg99t 161 ACUUGGAAAC ACUUGGAAAC ACUUGGAAAC acttggaaac acttggaaac acttggaaac acttggaaac 211 AAGUUGAAAG AAGUUGAAAG AAGUUGAAAG aagttgaaag aagttgaaag aagttgaaag aagttgaaag 261 Len: Len: Len: Len: Len: Len: Len: oooooooooo I UUCUNUCACC UUCUUUCACC NNCUNUCACC ttctttcacc ttctttcacc ttctttcacc ttctttcacc 121 | . GAGUAACNCG GAGUAACACG GAGUAACNCG gagtaacacg gagtaacacg gagtaacacg gagtaacacg 171 AGGUGCUAAU AGGUGCUAAU AGGUGCUNAU aggtgctaat aggtgctaat aggtgctaat aggtgctaat 221 l GCGCUNUUGC GCGCUUUUGC GCGCUNUUGC gcgcttttgc gcgcttttgc gcgcttttgc gcgcttttgc 271 124 1554 Check: D6735E1F 1554 Check: ECBSE486 1554 Check: 384A3986 1554 Check: IEO4DSC6 1554 Check: BEDSDDOS 1554 Check: SZBCBSOA 1554 Check: D34FC5E0 31 71 ISO ACGCUGGCGG CGUGCCUAAU 50 ACGCUGGCGG CGUGCCUAAU 43 ACGCUGGCGG CGUGCCUNAU 50 acgctggcgg chgcctaat 22 acgctggcgg cgtgcctaat 26 ................ taat 4 acgctggcgg cgtgcctaat 25 T1 91 I100 GGAGCUNGCU CCACCGAAAG 100 GGAGCUUGCU CCACCGAAAG 93 GGAGCUNGCU CCACCGAAAG 100 ggagcttgct ccaccgaaag 72 ggagcttgct ccaccgaaag 76 ggagcttgct ccaccgaaag 54 ggagcttgct ccaccgaaag 75 131 T41 l150 UGGGUNACCU NCCCAUCAGA 150 UGGGUAACCU GCCCAUCAGA 143 UGGGNNACCU NCCCAUCAGA 150 tgggtaacct gcccatcaga 122 tgggtaacct gcccatcaga 126 tgggtaacct gcccaccaga 104 tgggtaacct gcccatcaga 125 181 191 I200 ACCGUAUAAC ACUAUNNUCC 200 ACCGUAUAAC ACUAUUUUCC 193 ACCGUAUAAC ACUNUNNUCC 200 accgtataac actattttcc 172 accgtataac actattttcc 176 accgtataac actacettcc 154 accgtataac actattttcc 175 231 241 I250 GUNACUNAUG GAUGGACCCG 250 GUCACUGAUG GAUGGACCCG 243 GUNACUNAUG GAUGGACCCG 250 gtcactgatg gatggacccg 222 gtcactgatg gatggacccg 226 gtcactgatg gatggacccg 204 gtcactgatg gatggacccg 225 281 291 300 Enr 131 Sn: APO AFO AFO Appendix III-cont’d CGGUGCAUUA' CGGUGCAUUA CGGUGCAUUA cggtgcatta cggtgcatta cggtgcatta cggtgcatta 301 I UAGCCGACCU UAGCCGACCU UAGCCGACCU tagccgacct tagccgacct tagccgacct tagccgacct 351 GACUCCUACG GACUCCUACG GACUCCUACG gactcctacg gactcctacg gactcctacg gactcctacg 401 I UGACCGAGCA UGACCGAGCA UGACCGAGCA tgaccgagca tgaccgagca tgaccgagca tgaccgagca tgttgttaga tgttgttaga tgttgttaga tgttgttaga 501 I UAUCUAACCA UAUCUAACCA unucunAcca tat: ctaacca tatctaacca tatctaacca tacctaacca 551 I GCUNGUUGGU GCUAGUUGGU GCUGGUUGGU gctagttggt gctagttggt gctagttggt gctagttggt 311 l GAGAGGGUNA GAGAGGGUGA GAGAGGGUNA gagagggtga gagagggtga gagagggtga gagagggtga 361 GGAGGCAGCC GGAGGCAGCA GGAGGCAGCA ggaggcagca ggaggcagca ggaggcagca agaggcagca 411 l ACGCCGCGUG ACGCCGCGUG ACGCCGCGUG acgccgcgtg acgccgcgtg acgccgcgtg acgccgcgtg 461 l GAAGAACAAG GAAGAACAAG GAAGAACAAG gaagaacaag gaagaacaag gaagaacaag gaagaacaag 511 I GAAAGCCACG GAAAGCCACG GAAAGCCACG gaaagccacg gaaagccacg gaaagccacg gaaagccacg 561 GAGGUAACGG GAGGUAACGG GAGGUAACGG gaggtaacgg gaggtaacgg gaggtaacgg gaggtaacgg 321 | UCGGCCACAN UCGGCCACAC UCGGCCACAC tcggccacac tcggccacac tcggccacac tcggccacac 371 GUAGGGAAUC GUAGGGAKUC GUAGGGAAUC gtagggaatc gtagggaatc gtagggaatc gtagggaatc 421 l AGUGAAGAAG AGUGAAGAAG AGUGAAGAAG agtgaagaag agtgaagaag agtgaagaag agtgaagaag 471 GAUGAGAGUN GAUGAGAGUA GAUGAGAGUN gatgagagta gatgagagta gatgagagta gatgagagta 521 GCUAACUACG GCUAACUACG GCUNACUACG gctaactacg gctaactacg gctaactacg gctaactacg 571 125 CUCACCAAGG CUCACCAAGG CUNACCAAGG ctcaccaagg ctcaccaagg ctcaccaagg ctcaccaagg 331 UGGGACUGAG UGGGACUGAG UGGGACUGAG tgggactgag tgggactgag tgggactgag tgggactgag 381 UUCGGCAAUG UUCGGCAAUG UUCGGCAAUG ttcggcaatg ttcggcaatg ttcggcaatg ttcggcaatg 431 GUNNUCGGAU GUUUUCGGAU GUNNUCGGAU gttttcggat gttttcggat gttttcggat gttttcggat 481 AAACGUNCAU GAACGUUCAU AAAUNUNNKU aaacgttcat gaacgttcat gaacgttcat aaatgttcat 531 l UGCCAGCAGC UGCCAGCAGC UGCCAGCAGC tgccagcagc tgccagcagc tgccagcagc tgccagcagc 581 CNACGAUGCA CAACGAUGCA CAACGAUGCA ccacgatgca caacgatgca ccacgatgca caacgatgca l“ I ACACGGCCNN ACACGGCCCA ACACGGCCCN acacggccca acacggccca acacggccca acacggccca 391 GACGAAAGUC GACGAAAGUC GACGAAAGUC gacgaaagtc gacgaaagtc gacgaaagtc gacgaaagtc 441 l I CGUAAAACUC CGUAAAACUC CGUNAAACUC cgtaaaactc cgtaaaactc cgtaaaactc cgtaaaactc 491 I CCCUNNACGG CCCUUGACGG CCCUNNACGG cccttgacgg cccttgacgg cccttgacgg cccttgacgg 5:“ I CGCGGUNAUA CGCGGUAAUA CGCGGUNAUA cgcggtaata cgcggtaata cgcggtaata cgcggtaata 591 350 400 450 300 293 300 272 276 254 275 350 343 350 322 326 304 325 400 400 372 376 354 375 450 443 450 422 426 404 425 500 493 500 472 476 454 475 550 543 550 522 526 504 525 600 Isl APO APO AFO AFO Appendix III-cont’d CGUAGGUGGC CGUAGGUGGC CGUAGGUGGC cataggtggc cgtaggtggc cgtaggtggc cgtagstgsc I01 CGGUUUCUUA CGGUUUCUUA CGGUUUCUUA cggtttctta cggtttctta cggtttctta cggtttctta 651 UUGGAAACUG UUGGAAACUG UUGGAAACUG ttggaaactg ttggaaactg ttggaaactg ttggaaactg T01 UAGCGGUGAA URGCGGUGAA UAGCGGUGAA tagcggtgaa tagcggtgaa tagcggtgaa tagcggtgaa 751 I UCUCUNGUCU UCUCUGGUCU UCUCUNGUCU tctctggtct tctctggtct tctctggtct tctctggtct 801 I GGAUUAGAUA GGAUUAGAUA GGAUUAGAUA ggattagata ggattagata ggattagata ggattagata 851 NRGCGUNNUC AAGCGUUGUC NAGCGUUGUC aagcgttgtc aagcgttgtc aagcgttgtc aagcgttgtc 611 I AGUCUNAUGU AGUCUGAUGU AGUCUNAUGU agtctgatgt agtctgatgt agtctgatgt agtctgatgt 661 I GGAGACUNGA GGAGACUUGA GGAGACUNGA ggagacttga ggagacttga ggagacttga ggagacttga 711 I AUGCGUAGAU AUGCGUAGAU AUGCGUAGAU atgcgtagat atgcgtagat atgcgtagac atgcgtagat 761 I GUNACUGACG GUAACUGACG GUAACUGACG gtaactgacg gtaactgacg gtaactgacg gtaactgacg 811 l CCCUNGUAGU CCCUGGUAGU CCCUNGUAGU ccctggtagt ccctggtagt ccctggtagc ccctggtagt 861 CGGADNNAUU CGGAUUUAUU CGGAUUUAUU cggatttatt cggatttatt cggatttatt cggatttatt 621 I GAAAGCCCCC GAAAGCCCCC GAAAGCCCCC gaaagccccc gaaagccccc gaaagccccc gaaagccccc $71 GUGCAGAAGA GUGCAGAAGA GUGCAGAAGA gtgcagaaga gtgcagaaga gtgcagaaga gtgcagaaga 721 I AUAUGGAGGA AUAUGGAGGA AUAUGGAGGA atatggagga atatggagga atatggagga atatggagga le CUNAGGCUCG CUGAGGCUCG CUNAGGCUCG ctgaggctcg ctgaggctcg ctgaggctcg ctgaggctcg 821 CCACGCCGUA CCACGCCGUA CCACGCCGUA ccacgccgta ccacgccgta ccacgccgta ccacgccgta 871 126 GGGCGUNAAG GGGCGUAAAG GGGCGUAAAG gggcgtaaag gggcgtaaag 999C9t3339 gggcgtaaag 631 I GGCUCAACCG GGCUCAACCG GGCUCAACCG ggctcaaccg ggctcaaccg ggctcaaccg ggctcaaccg 781 GGAGAGUGGA GGAGAGUGGA GGAGAGUGGA ggagagtgga ggagagtgga ggagagcgga ggagagtgga 731 I ACACCAGUGG ACACCAGU.G ACACCAGUGG acaccagtgg acaccagtgg acaccagtgg acaccagtgg 781 I AAAGC-GUGG AAAGCCGUGG AAAGC-GUGG aaagc-gtgg aaagc-gtgg aaagc-gtgg aaagc—gtgg 831 AACGAUGAGU AACGAUGAGU AACGAUGAGU aacgatgagt aacgatgagt aacgatgagt aacgatgagt 881 I CGAGCGCAGG CGAGCGCAGG CGAGCGCAGG cgagcgcagg cgagcgcagg cgagcgcagg cgagcgcagg I41 I GGNNGGGUCA GGGAGGGUCA GGNNGGGUCA gggagggtca gggagggtca gggagggtca gggagggtca 691 I I AUUCCAUGUG AUUCCAUGUG AUUCCAUGUG attccacgtg attccatgtg attccatgtg attccatgtg 741 CGAAGGCGGC CGAAGGCGGC CGAAGGCGGC cgaaggcggc cgaaggcggc cgaaggcggc cgaaggcggc I” I GGAGCGAACA GGAGCGAACA GGAGCGAACA ggagcgaaca ggagcgaaca ggagcgaaca ggagcgaaca I“ I GCUNAGUGUU GCUAAGUGUU GCUAAGUGUU gctaagtgtt gctaagtgtt gctaagtgtt gctaagtgtt 891 700 750 600 593 600 S72 576 554 575 650 643 650 622 626 604 625 700 693 700 672 676 654 675 750 742 750 722 726 704 725 799 792 799 771 775 753 774 849 842 849 821 825 803 824 Appendix III-cont’d ggagggtttc agagggtttc ggagggtttc ggagggtttc 901 tggggagtac ngggagtac tggggagtac tggggagtac 951 CACAAGCGGU CACAAGCG.U CACAAGCGGU cacaagcggt cacaagcggt cacaagcggt cacaagcggt 1001 l CCAGGUCUUG CCAGGUCUUG CCAGGUCUUG ccaggtcttg ccaggtcttg ccaggtcttg ccaggtcttg 1051 GGGCAAAGUG GGGCAAAGUG GGGCAAAGUG gggcaaagtg gggcaaagtg gggcaaagtg gggcaaagtg 1101 I GUUGGGUNAA GUUGGGUUAA GUUGGGUNAA gttgggttaa gttgggttaa gttgggttaa gttgggttaa 1151 CGCCCUUCAG CGCCCUUCAG CGCCCUNCAG cgcccttcag cgcccttcag cgcccttcag cgcccttcag 911 I GACCGCAAGG GACCGCAAGG GACCGCAAGG gaccgcaagg gaccgcaagg gaccgcaagg gaccgcaagg 961 GGAGCAUGUN GGAGCAUGUG GGAGCAUGUN ggagcatgtg ggagcatgtg ggagcatgtg ggagcatgtg 1011 I ACAUCCUNUG ACAUCCUUUG ACAUCCUNUG acatcctttg acatcctttg acatcctttg acatcctttg 1061 ACAGGUGGNG ACAGGUGGUG ACAGGUGGNG acaggtggtg acaggtggtg acaggtggtg acagstgstg 1111 GUCCCGCAAC GUCCCGCAAC GUCCCGCAAC gtcccgcaac gtcccgcaac gtcccgcaac gtcccgcaac 1161 UGCUGCAGCN UGCUGCAGCA UGCUGCAGCA tgctgcagca tgctgcagca tgctgcagca tgctgcagca 921 UUNAAACUCA UUGAAACUCA UUGAAACUCA ttgaaactca ttgaaactca ttgaaactca ttgaaactca 971 GUUUAAUUCG GUUUAAUUCG GUUUAAUUCG gtttaattcg gtttaattcg gtttaattcg gtttaattcg 1021 ACCACUCUAG ACCACUCUAG ACCACUCUAG accactctag accactctag accactctag accactctag 1071 CAUNGUUGUC CAUGGUUGUC CAUNGUUGUC catggttgtc catggttgtc catggttgtc catggttgtc 1121 GAGCGCAACC GAGCGCAACC GAGCGCAACC gagcgcaacc gagcgcaacc gagcgcaacc gagcgcaacc 1171 127 AACGCAUUAA AACGCAUUAA AACGCAUUAA aacgcattaa aacgcattaa aacgcattaa aacgcattaa 931 AAGGAAUUGA AAGGAAUUGA AAGGAAUUGA aaggaattga aaggaattga aaggaattga aaggaattga 981 AAGNAACGCG AAGCAACGCG AAGNRACGCG aagcaacgcg aagcaacgcg aagcaacgcg aagcaacgcg 1031 l AGAUAGAGCU AGAUAGAGCU AGAUAGAGCU agatagagct agatagagct agatagagct agatagagct 1081 GUCAGCUCGU GUCAGCUCGU GUCAGCUCGU gtcagctcgt gtcagctcgt gtcagctcgt gtcagctcgt 1131 cttattgtta cttattgtta cttattgtta cttattgtta 1181 I GCACUCCGCC 899 GCACUCCGCC 892 GCACUCCGCC 899 gcactccgcc 871 gcactccgcc 87S gcactccgcc 853 gcactccgcc 874 941 I950 CGGGGGC.CG 948 CGGGGGCCCG 942 CGGGGGC.CG 948 cgggggcccg 921 cgggggcccg 925 cgggggcccg 903 cgggggcccg 924 991 1000 AAGAACCUNA 998 AAGAACCUUA 991 AAGAACCUNA 998 aagaacctta 971 aagaacctta 975 aagaacctta 953 aagaacctta 974 T041 l1050 UN.CCUUCGG 1047 UCCCCUUCGG 1041 UU.CCUUCGG 1047 tccccttcgg 1021 tccccttcgg 1025 tccccttcgg 1003 tccccttcgg 1024 1091 I1100 GUCGUGAGAU 1097 GUCGUGAGAU 1091 GUCGUGAGAU 1097 gtcgtgagat 1071 gtcgtgagat 1075 gtcgtgagat 1053 gtcgtgagat 1074 T141 1150 GUUGCCAUCA 1147 GUUGCCAUCA 1141 GUUGCCAUCA 1147 gttgccatca 1121 gttgccatca 1125 gttgccatca 1103 gttgccatca 1124 1191 1200 En: Isl Enr APO 131 Eur APO AFO AFO APO Appendix III-cont’d I UUUAGUUGGG UUUAGUUGGG UUUAGUUGGG tttagttggg tttagttggg tttagttggg tttagttggg 1201 I GGGAUGACGU GGGAUGACGU GGGAUGACGU gggatgacgt eggatgacgt Sggatgacgt gggatgacgt 1251 UACAAUGGGA UACAAUGGGA UACAAUGGGA tacaatggga tacaatggga tacaatggga tacaatggga 1301‘ UNAAAGCUGC UUAAAGCUUC UNNAAGCUNC ttaaagcttc ttaaagcttc ttaaagcttc ttaaagcttc 1351 I CCGGAAUCGC CCGGAAU CCGGAAUCGC ccggaaccgc ccggaatcgc ccggaatcgc ccggaatcgc 1401 GGGCNUNGUA GGGCCUUGUA GGGCCNNGUA gggccttgta gggccttgta gggccttgta gggccttgta 1451 CACUCUAGCG CACUCUAGCG CACUCUAGCG cactctagcg cactctagcg cactctagcg cactctagcg 1211 CAAAUCAUCA CAAAUCAUCA CAAAUCAUCA caaatcatca caaatcatca caaatcatca caaatcatca 1261 I AGUACAACGA AGUACAACGA AGUACAACGA agtacaacga agtacaacga agtacaacga agtacaacga 1311 UCUCAGUUCG UCUCAGUUCG UCUCAGUUCG tctcagttcg tctcagttcg tctcagttcg tctcagttcg 1361 | UNGUAAUCGC 98C UAGUAAUCGC UAGUAAUCGC tagtaatcgc tagtaatcgc tagtaatcgc tagtaatcgc 1411 CACACCGCCC CACACCUCCC CACACCGCCC cacaccgccc cacaccgccc cacaccgccc cacaccgccc 1461 AGACUGCCGG AGACUGCCGG AGACUGCCGG agactgccgg agactgccgg agactgccgg agactgccgg 1221 UGCCCCUUAU UGCCCCUUAU UGCCCCUUAU tgccccttat tgccccttat tgccccttat tgccccttat 1271 GUUGCGAAGU GUUGCGAAGU GUUGCGAAGU gttgcgaagt gttgcgaagt gttgcgaagt gttgcgaagt 1321 GAUUGUAGGC GAUUGUAGGC GAUNGUAGGC gattgtaggc gattgtaggc gattgtaggc gattgtaggc 1371 I GGAUCAGCAC UCAGCAC UCAGCAC ggatcagcac ggatcagcac ggatcagcac ggatcagcac 1421 GUCACACCA- GUCACACCAC GUCACACCA- gtcacacca- gtcacacca- gtcacacca- gtcacacca- 1471 128 UGACAAACCG UGACAAACCG UGACAAACCG tgacaaaccg tgacaaaccg tgacaaaccg tgacaaaccg 1231 GACCUGGGCU GACCUGGGCU GACCUGGGCU gacctgggct gacctgggct gacctgggct gacctgggct 1281 CGCGAGGCUN CGCGAGGCUA CGCGAGGCUN cgcgaggcta cgcgaggcta cgcgaggcta cgcgaggcta 1331 UGCAACUCGC UGCAACUCGC UGCAACUCGC tgcaactcgc tgcaactcgc tgcaactcgc tgcaactcgc 1381 I GCCGCGGUNA GCCGCGGUGA GCCGCGGUNA gccgcggtga gccgcggtga gccgcggtga gccgcggtga 1431 CGAGAGUUUG CGAGAGUUUG CGAGAGUUUG cgagagtttg cgagagtttg cgagagtttg cgagagtttg 1481 GAGGAAGGUG 1197 GAGGAAGGUG 1191 GAGGAAGGUG 1197 gaggaaggtg 1171 gaggaaggtg 1175 gaggaaggtg 1153 gaggaaggtg 1174 1241 1250 ACACACGUGC 1247 ACACACGUGC 1241 ACACACGUGC 1247 acacacgtgc 1221 acacacgtgc 1225 acacacgtgc 1203 acacacgtgc 1224 1291 1300 AGCUAAUCUC 1297 AGCUAAUCUC 1291 AGCUNAUCUC 1297 agctaacctc 1271 agctaatctc 127$ agctaatctc 1253 agctaatctc 1274 1341 1350 CUNCAUGAAG 1347 CUACAUGAAG 1341 CUNCAUGAAG 1347 ctacatgaag 1321 ctacatgaag 1325 ctacatgaag 1303 ctacatgaag 1324 1391 I1400 AUACGUUCCC 1397 AUACGUUCCC 1391 AGACGUUCCC 1397 atacgttccc 1371 atacgttccc 1375 atacgttccc 1353 atacgttccc 1374 1441 l1450 UAACACCCGA 1446 UAACACCCGA 1441 UAACACCCGA 1446 taacaccc.. 1418 taacacccga 1424 caacacccga 1402 taacacccga 1423 1491 1500 Eur Isl Enr AFO APO AFC Appendix III-cont’d agtcggtgag agtcggtgag agtcggtgag 1501 .......... attggggtga attggggtga attggggtga 1551 cg.. cacc cgga OOOOOOOOOO gtaacctttt gtaacctttt gtaacctttt 1511 agtcgtaaca agtcgtaaca agtcgtaaca teeieeeaée tggagccagc tggagccagc 1521 aggtigeee: aggtagccgt aggtagccgt 129 .......... 0000000000 cgcctaaggt gggatagatg cgcctaaggt gggatagatg cgcctaaggt gggatagatg -_-------- 1541 ------- tat accggaaggt gcggctggat -------- at 1489 1521 1446 1418 1508 1506 1509 Appendix IV. Endnotes While my Master's Thesis research did not land on the cover of Science magazine, my graduate research was very fruitful. Entering graduate school after almost a lO-year absence from science was a serious challenge. Sixty hours a week reviewing undergraduate algebra and chemistry ended with 4.03 in Biochemistry. Coursework in molecular biology, rumen microbiology and microbial ecology provided a foundation for my research. I had the opportunity to use a variety of methods to investigate the bacterium and phage. I learned and applied a variety of late twentieth century microbiological methods. I used BIOLOG; API Strep and 168 rRNA based databases to identify a bacterial isolate. I explored the strengths and weaknesses of these databases. HPLC analysis of the chemical byproducts of the bacterium provided important supporting data and an introduction to a powerful analytical tool. To study the genetic make up of the phage, I applied molecular biology techniques, and used the data to interrogate earlier data on the phage and its hosts. To study the ultrastructure of the phage, I was introduced to the electron microscope, which led directly to a position as an electron microscopist at the Center for Electron Optics at MSU. I learned a fundamental caution for scientific work. Decisions are made about what receives the attention of the scientists eye. High hopes regarding a phage capable of infecting important rumen species led to dismissal of data that didn’t "fit". The three different microbial databases produced an analysis based on partial data for the genus Enterococcus. The most important point, however, is the confirmation of the 60's axiom to "Question Authority". I found this crucial in determining validity of results from both world-renowned databases and laboratory cohort. Science can't move forward without a rigorous interrogation of data, data 130 analysis methods and data collectors. "Objective science" only approximates objectivity if the subjective factor is made conscious. Future research Changes in medicine and the efficacy of antibiotics are bringing the microbiologists eye back to phage therapy. Phage of the species group E. casseliflavus and E. gallinarum are worthy of investigation for use in phage therapy. Multiply drug resistant enterococci are increasingly becoming feared pathogens. Phage therapy was attempted and discarded earlier this century in the US. The rapid development of bacterial resistance to phage makes the development of phage therapy complex. In the former Soviet satellite of Georgia, phage therapy was developed and successfully used. The methods involved using many strains of phage infective of a pathogen. An interesting discussion of these efforts and phage therapy in general was published on the web by Elizabeth Kutter, at Evergreen State College. see http: / / www.cvergreen.edu / user / T4 / PhageTherapy/ Phagethea.html Collection of a battery of strains in the E. gallinarum-E. casseliflavus and a battery of phage infectious to them could be the beginning of an interesting research initiative. An initial investigation would be to test pathogen survival when challenged with single and multi-phage batteries. Phage receptor sites on enterococci have not been investigated. A bacterial strain that has mutations for resistance may have altered viability and pathogenecity. A thorough investigation into these questions could lead to important medical breakthroughs. 131 "Illll‘lllllllllllll