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THE DEVELOPMENT OF A STANDARDIZED IN VITRO
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CAMPYLOBACTER SPECIES

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ROBERT ANDREW HANSON

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6/01 cJCIRC/DateDue.p65-p.15

 

THE DEVELOPMENT OF A STANDARDIZED IN VITRO ANTIMICROBIAL
SUSCEPTIBILITY TESTING METHOD FOR CAMPYLOBACTER SPECIES

By

Robert Andrew Hanson

A THESIS
Submitted to Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Medical Technology Program

2002

ABSTRACT

THE DEVELOPMENT OF A STANDARDIZED IN VITRO ANTIMICROBIAL
SUSCEPTIBILITY TESTING METHOD FOR CAMPYLOBAC T ER SPECIES

By

Robert Andrew Hanson

Campylobacterjejuni is the most commonly isolated sporadic foodborne
pathogen in the United States. Efforts to monitor the prevalence and antimicrobial

resistance patterns of Campylobacterjejum‘ require the development of a standardized in
vitro antimicrobial susceptibility testing method to ensure accurate, reproducible and
reliable reporting data. There are currently several published antimicrobial susceptibility
testing methods for Campylobacters; however, there is no National Committee for
Clinical Laboratory Standards (NCCLS) method available.

In order to develop a standardized method for the antimicrobial susceptibility
testing of Campylobacter spp. three specific objectives were accomplished. First, define
the optimal environmental growth conditions on artificial media allowing robust growth
of Campylobacter isolates. Second, identify a Quality Control (QC) organism which
would survive several passages on artificial media and exhibit a reproducible in vitro
antimicrobial susceptibility profile. And third, define a testing method with preliminary
QC ranges for each antimicrobial evaluated. Mueller-Hinton agar with 5% defibrinated
sheep’s blood provided optimal growth in a 10% CO2 atmosphere at 42'C . ATCC 33560
Campylobacterjejuni produced the most reliable and reproducible MIC data points using

the broth microdiluton method and has been endorsed by the NCCLS as a QC organism.

To my parents, Ron and Kim Hanson
thank you for all the love and support throughout the years

iii

ACKNOWLEDGMENTS

I would like to thank my research professor, Dr. Robert D. Walker for his
everlasting sense of duty, honor, kindness, and wisdom. His personal and
professional work ethic is rare and inspirational.

“ If you can fix the problem, then fix the problem and don’t worry about
If you can’t fix the problem, then don’t worry about it”

Robert D. Walker
July 18, 1998

I would like to thank Dr. John B. Kaneene, for his perpetual sense of
humor, warmth, compassion and wisdom. His omnipresent support for
his students, to achieve and succeed, is in one word....”inspirational.”

I would like to thank Dr. Doug Estry for his sense of leadership and
integrity he cultivated and fostered as the director of the Medical
Technology program, which directly affected my opportunities as an
undergraduate, graduate and professional student.

I would like to thank Dr. Leo Mendoza for his kindness, dedication,
enthusiasm, and wisdom. His willingness to provide opportunities and
support for students is admirable.

I would like to thank Dr. Edward Maher for his ongoing career advice and
financial support via the Kellogg Biological Station Grant, MSU.

I would like to thank Nino Soave and Roger Herr for their perpetual
friendship, loyalty and joyfitl times throughout the years.

I would also like to thank RoseAnn Miller and Sonya Bodeis for the
technical support and enormous contributions to this study.

Lastly, thank you Drs Walker, Kaneene, Estry and Mendoza for
participating on my graduate school committee.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ vi
INTRODUCTION ................................................................................................. 1
LITERATURE REVIEW ....................................................................................... 14
MATERIALS AND METHODS:GENERAL ......................................................... 17
MATERIALS AND METHODS:DETAILED ........................................................ 22
RESULTS ................................................................................................................ 25
DISCUSSION .......................................................................................................... 30
SUMMARY AND CONCLUSIONS ...................................................................... 32
CURRENT AND FUTURE RESEARCH ............................................................... 33
APPENDICES (A-D) ............................................................................................... 34
BIBLIOGRAPHY ................................................................................................... 39

List of Tables

Table 1. BMD (Nalidixic Acid) .................................................... Appendix A
Table 2. BMD (Tetracycline) ....................................................... Appendix A
Table 3. BMD (Gentamicin) ......................................................... Appendix B
Table 4. BMD (Amoxicillin) ......................................................... Appendix B
Table 5. BMD (Ciprofloxacin) ...................................................... Appendix C
Table 6. BMD (Erythromycin) ...................................................... Appendix C
Table 7. BMD (Doxycycline) ........................................................ Appendix D
Table 8. BMD (Trimethoprim/Sulfamethoxazole) .......................... Appendix D

vi

INTRODUCTION

During the latter part of the 20th century, Campylobacterjejuni has become
recognized as the most common isolated sporadic foodbome pathogen and the leading
cause of bacterial gastroenteritis reported in the United States and has achieved
worldwide attention among the international medical communities(1,2). Campylobacter
organisms have long been recognized as a cause of diarrhea in cattle and sheep, but have
only been recognized as an important cause of human illness for the last 25 years (2).
Campylobacter was thought to be an opportunistic human pathogen when it was isolated
from human blood cultures in the 1950s (3).

The most commonly identified species, Campylobacterjejuni, was first isolated
from human stools in 1972 by a filtration technique developed for veterinary research (4).
Campylobacterjejuni is commonly found as commensal of the gastrointestinal tract of
wild and domesticated cattle, sheep, swine, goats, dogs, cats, and most varieties of fowl.
Some of these animals, including cattle, pigs and poultry, are part of the human food
chain (5). C. jejuni is not considered to be part of the normal intestinal flora in humans,
thus when ingested via contaminated animal food products it can cause severe
gastroenteritis. Meats originating from infected animals fi'equently become contaminated

with intestinal contents during the slaughtering process.

The consumption of contaminated poultry is estimated to be responsible for 50 to
70 percent of sporadic bacterial gastroenteritis infections within the United States.
It has been estimated that 70 to 90 percent of poultry products in the United States
destined for human consumption are contaminated with Campylobacterjejuni (6). Other
vehicles of infection include raw clams, unpasteurized goat’s milk and cheeses and
contaminated vegetables. Investigations of more than 50 outbreaks have also indicated
that unpasteurized cow’s milk can lead to bacterial enteritis by Campylobacter jej uni (6).

In 1997, laboratory ~confirmed human bacterial gastroenteritis cases were made
up as follows: 46% were campylobacteriosis, 26% were salmonellosis, 15% were
shigellosis, 4.0% were E. coli 0157:H7 infections, 1.6% were yersiniosis, and
approximately 1% each were listeriosis, and vibrio infections. The aforementioned
laboratory data was reported by the Centers for Disease Control and Prevention (CDC),
Food and Drug Administration (FDA), and the Food Safety and Inspection Service(FSIS)
F cod-Borne Disease Active Surveillance Network (FoodNet)(7). Data provided by the
1996 FoodNet surveillance program established Campylobacrer as the most commonly
isolated sporadic foodbome pathogen in the United States (1) and this trend continued in
1997 and 1998 (7,8)

Today, Campylobacters, particularly, C. jejuni, are known to be a leading cause of
human gastroenteritis worldwide(1,7,8). Campylobacter jej uni infections occur
throughout the year in the United States and other developed countries, showing sharp

peaks in the summer and early fall.

As a result of incomplete national surveillance, the actual incidence of
Campylobacter infections in the US. is not known. However, Passive reporting by
laboratories across the country have indicated that C. jejuni is usually the more commonly
isolated from fecal specimens provided by patients with diarrhea] syndromes than
Salmonella or Shigella (6).

The effect of campylobacteriosis is usually self-limiting in humans; however, in
some cases re-infection may occur. It is estimated that annually there are between two and
eight million cases of enteric campylobacteriosis in the US. with approximately 200-
1000 deaths (9). The current treatment for campylobacteriosis includes re-hydration
therapy with isotonic saline and antimicrobial agents, such as the flouroquinolones (10).
Chronic sequelae of C. jejuni includes reactive arthritis, hemolytic uremic syndrome, and
other gastrointestinal disorders.

One of the most important sequelae associated with Campylobacterjejuni is
Guillian-Barre syndrome. Guillian-Barre syndrome is a subacute, acquired, demyelinating
neuro-perpherial disorder. This condition is rare in the United States, but may be fatal to
those suffering from the disease. Severe damage to the perpherial nervous system can
result if the patient is left untreated (11). It is estimated that there are 5,000 cases of
Guillian-Barre syndrome each year in the United States. Studies indicate that 36-40% of
these cases follow a Campylobacter infection (12).

Disease Prevalence

In the United States, an estimated 2.1 to 2.4 million cases of human
campylobacteriosis occur each year(2). Common symptoms that patients experience with
laboratory-confirmed infections include fever, diarrhea, bloody stools and abdominal
cramping(13). Bacteremia, septic arthritis, and extraintestinal symptoms are far less

common (14).

The incidence of campylobacteriosis in HIV positive patients is much higher than
in the general population. For instance, in Los Angeles County between 1983 and 1987,
the reported incidence of campylobacteriosis in patients living with AIDS was
approximately 519 cases per 100,000 population, which was 39 times higher than the rate
in the general population (15). Recurrent infections and infection with antimicrobial
resistant strains of C. jejuni are major complications faced by patients living with AIDS
(16).

In the United States, infants have the highest age-specific Campylobacter isolation
rate, approximately 14 per 100,000 person years. In young adolescents the isolation rates
decline to approximately 4 per 100,000 person years. The peak isolation rate in neonates
and infants has been attributed in part to susceptibility upon first exposure and to the low
threshold for seeking medical care for infants. An interesting feature of the epidemiology
of human campylobacteriosis is the high isolation rate among young adults,
approximately 8 per 100,000 person years. The high rate of infection during early
adulthood, which is pronounced in men, is thought to reflect poor food-handling practices
and lack of proper cooking times, especially when cooking poultry. Among middle-aged
and older adults, the isolation rate is < 3 per 100,000 person years (2).

Animal Reservoirs

Campylobacterjejuni is commonly found as commensal of the gastrointestinal
tract of domesticated beef cattle, poultry, sheep, swine, goats, dogs, cats and rodents.

Animals and animal products have been identified as sources of infection in
several outbreaks, and many of the Campylobacter serotypes that cause disease in

humans have been isolated from animals.

C. jejuni is not considered to be part of the normal intestinal flora in humans, thus
when ingested via contaminated animal food products it can cause severe gastroenteritis
(17). C. jejuni appears to be normal commensal of all classes of bovines. Carcasses may
become contaminated with intestinal contents during slaughter (18). One study
conducted in Sweden showed the presence of C. jejuni in the feces of 17 out of 90 (19%)
cattle, and another study conducted in Finland showed a C. jejuni incidence of 5.5%
from fecal samples taken from 200 cattle (46).

A community outbreak of gastroenteritis in Vermont was traced to the
consumption of unpasteurized milk produced at a commercial dairy farm. Two different
testing methods showed a C. jejuni isolate from a sick patient and an isolate from a
diseased cow to be the same serotype (47). Carcass contamination by Campylobacter
species is more common in swine carcasses than in sheep, cattle, or goats. The danger of
intestinal spillage is greater with the procedures used in dressing swine carcasses than
with those used in dressing the carcasses ruminants (48).

Campylobacter species are frequently found in the intestinal flora of commercially
raised birds and such wild birds as pigeons, crows, ravens, and seagulls (49). A Dutch
study of broiler chickens found the highest rate of Campylobacter contamination from the
months of June to September and the lowest in March.

Potential routes of entry of organisms into a flock include infection of newborn
chicks from older birds, contamination of feed and water, and wild or game birds.
Infection of poultry is often without clinical symptoms (50). Reservoirs in the poultry
environment include beetles (51), unchlorinated drinking water (32), and farm workers
(29,30,31). Vertical transmission (i.e. from breeder flocks to progeny), as seen in
Salmonella species, has been hypothesized, but not widely accepted.

5

Because of its association with animals and their feces, it has been found in
surface water and parts of the human food chain, resulting in its being classified as a
zoonotic foodbome pathogen (52)

Fecal-oral person-to-person infection has been documented for C. jejuni. As with
other enteric pathogens, those in contact with excreta of infected people are at risk.(19).
Human-to-human transmission has been reported from food handlers that are infected and
carry the organism (20).

A food handler was the most probable source of an outbreak of acute
gastroenteritis due to Campylobacterjejuni that occurred at a military base in Israel.
Stool cultures were taken from 17 clinically affected as well as 23 asymptomatic soldiers.

In 6 of the 17 patients with enteritis (35%), Campylobacterjejuni serotype (ii)
was isolated, while the stool cultures of all the asymptomatic soldiers were negative. The
food handler had suffered from acute gastroenteritis before the outbreak but had not
reported the illness. He was found to harbor the same serotype as the affected patients
(21).

Transmission: Animal to Human

A majority of human Campylobacter infections are sporadic in nature. Sporadic
Campylobacter infections usually occur from June to early August. A number of case-
control studies have identified one of the most important risk factors associated with
sporadic campylobacteriosis, specifically, handling raw and ingesting undercooked

poultry, which eventually caused campylobacteriosis in humans.

Outbreaks usually occur during the spring and fall months in the US. Another
outbreak vehicle, the consumption of unpasteurized milk, has been responsible for 30 of
the 80 reported outbreaks of campylobacteriosis to the CDC between 1973 and 1992.
Outbreaks caused by drinking unpasteurized milk often involve visits to farms (e.g.,
school field trips) during the temperate seasons (22).

Less frequent risk factors associated with campylobacteriosis include drinking
milk from bird-peeked bottles, handling young companion pets like dogs and cats,
especially pets with chronic and persistent diarrhea. Reports of serotype overlaps exists
between clinical C. jejuni isolates recovered from humans, poultry, cattle, and swine
indicating that foods of animal origin can contribute to a major pathway in the
transmission of C. jejuni to humans(23).

Camgzlobacters md the Food Supply

Epidemiological evidence supports the hypothesis that raw agricultural products
such as poultry, beef, raw milk are sources of human Campylobacter infections. Most
chickens destined for human consumption is contaminated with C. jejuni, one study
reported an isolation rate of 98% for retail chickens. The bacterial counts on the carcasses
can often exceed 103 per 100 grams.

Skin and giblets have particularly high levels of contamination(24). In another
study, raw milk samples from dairy farms in Tennessee yielded a contamination rate of
12% and all the isolates were identified as C. jejuni(25). Raw milk is thought to be
contaminated with bovine feces; however, direct contamination of milk can also occur as
a result of mastitis(26). Campylobacters can also be found in beef and other red meats. In
one study, C. jejuni was recovered in approximately 5% of raw ground beef and in 40%

of veal samples(27).

The Farm Environment

The control of Campylobacter contamination of farms has been a major topic of
discussion between farm owners and the Food and Drug Administration. The goal
remains to reduce the risk of contamination of food animal carcasses, such as poultry and
beef products bound to enter retail venues(28). Epidemiological studies indicate that strict
hygiene reduces intestinal carriage in food-producing animals(29,30,31). In field studies,
poultry flocks that drank chlorinated water had lower intestinal colonization rates than
poultry that drank unchlorinated water(30,32).

Meats originating from infected animals frequently become contaminated with
intestinal contents during the slaughtering process. Bacterial counts on carcasses usually
increase during the slaughtering process 13). In one study, up to a 1000 fold increase in
bacterial counts on carcasses was reported during transportation to slaughter(33).

In another study, the defeathering and evisceration of chickens(3 4) and turkeys at
slaughter increased the bacterial counts by approximately 10 to 100 fold during those
processes (35).

Treatment of Campylobacteriosis

The primary treatment for most campylobacteriosis patients include fluid and
electrolyte replacement. Severely dehydrated patients should receive rapid intravenous
infusion of normal saline (0.9%) which will expand the patients blood volume(36).
Campylobacter infections are usually self limiting, antibiotic therapy may be indicated for
patients who have a high fever, bloody diarrhea with more that 8 stools in a 24 hour
period, immunocompromised patients suffering from HIV or chemotherapy, and patients

with bacteremia or septicemia (3 6).

The enteric disease caused by C. jejuni has responded to several different
antimicrobial agents, including the flouroquinolones. Unfortunately, the enteric disease
caused by this organism may be clinically indistinguishable from those caused by other
intestinal pathogens of humans, such as Salmonella, Shigella and Vibrio cholera.

The recommended antimicrobial therapy for gastroenteritis caused by these
organisms is ciprofloxacin, a flouroquinolone (10); however, the plasma concentration of
ciprofloxacin required to inhibit the growth of C. jejuni may be higher than the
concentration required to inhibit growth of Salmonella, Shigella and Vibrio cholera
which may enhance the selection for resistant isolates of C. jejuni.(37).

Antimicrobial Resistance

Prior to 1989, there were no reported incidences of drug resistance to
fluoroquinolones from any laboratory isolated and confirmed Campylobacter organisms.
Since 1989, fluoroquinolone resistant Campylobacter isolates have been reported by
several European countries and more recently, in the United States (38). The incidence of
fluoroquinolone-resistant C. jejuni in Europe has been associated with an increase in the
use of fluoroquinolones in human and veterinary medicine.(10,38).

The lack of prudent use of antimicrobial agents, namely flouroquinolones, in both
human and veterinary medicine may contribute the current problem of antimicrobial
resistance among enteric pathogens(3 9). The rate of antimicrobial resistant enteric
infections is highest in the developing world, where the use of antimicrobial drugs in
humans and animals is relatively unrestricted. A study conducted in 1994 reported that
most of the clinical isolates of C. jejuni fiom US. army troops in Bangkok, Thailand

were resistant to ciprofloxacin(40).

The first fluoroquinolone approved in the United States for use in human
medicine was norfloxacin in 1986, and shortly thereafier in 1988, ciprofloxacin was
approved for use in human medicine. Since 1988, there has been several other
fluoroquinolones approved in the US. for use in human medicine such as levofloxacin,
ofloxacin, lomofloxacin, fleroxacin and trovafloxacin. Enrofloxacin was approved for use
in dogs and cats in 1989. Currently, enrofloxacin is being used to treat dogs, cats,
chickens, turkeys, and beef cattle. Furthermore, difloxacin, orbifloxacin, and
marbofloxacin are being used to treat dogs and/or cats.

Sarafloxacin, which is no longer available, was used on poultry farming units to
promote better health and growth. After flouroquinolone use in poultry was approved in
Europe, resistant C. jejuni strains emerged rapidly in humans during the early 19905.
Such widespread use of fluoroquinolones in human and veterinary medicine within the
United States could result in an increased incidence of antimicrobial drug resistance
similar to the incidence rates of antimicrobial drug resistance evident in some European
countries(39).

Concern for the development of bacterial resistance to the fluoroquinolones
resulted in a meeting between the Infectious Disease Society of America (IDSA), the
Centers for Disease Control and Prevention (CDC), and both the human and the
veterinary components of the Food and Drug Administration’s (FDA), specifically the
Center for Drug Evaluation and Research (CDER) and the Center for Veterinary

Medicine (CVM), respectively.

10

A recommendation that resulted from this meeting was that when a
pharmaceutical company has a fluoroquinolone approved for use in food animal species,
the company is responsible for establishing a nationwide resistance monitoring system to
monitor the possible variation in susceptibility patterns of the target pathogen in relation
to time. Since this recommendation, enrofloxacin, sarafloxacin, orbifloxacin, difloxacin
and marbofloxacin have been approved for use in veterinary medicine with the approval
of other fluoroquinolones pending. The USDA/CDC/F DA National Antimicrobial
Resistance Monitoring System was established to monitor drug resistance in Salmonella
isolates from animals and humans. Antimicrobial testing of Campylobacter spp. was
added to the program in 1998.

In 1988 the Food Safety and Inspection Service (F SIS), a department of the
United States Department of Agriculture (USDA), initiated a scientific collaboration with
the Agriculture Research Service (ARS) to strengthen and establish an official
interrelationship between the two agencies to cooperate indefinitely on food safety
research efforts(41). In 1995 an important and unique public health initiative was
established known as the CDC/F DA/F SIS Foodbome Diseases Active Network called
FOODNET (1,7,8).

The justification for the Development of an In Vitro Sweptibilig Testing Memod for
Campylobacter spp.

In order to address and implement the recommendations for drug resistance
monitoring set forth by the aforementioned agencies, the development of a standardized
in vitro antimicrobial susceptibility testing method was necessary to ensure accurate,
reproducible and reliable in vitro antimicrobial susceptibility testing of Campylobacter
spp.

11

The National Committee for Clinical Laboratory Standards (N CCLS) has defined
the criteria for developing such a standard in their M23-T3 and M37-A documents. For
the in vitro antimicrobial susceptibility testing of almost all other bacterial pathogens,
laboratories doing such tests follow the procedures recommended by the NCCLS.

Tests results that are generated without NCCLS standardization may not be
reproducible and thus, may not be verifiable. While there are several published
procedures for the in vitro antimicrobial susceptibility testing of C. jejuni (42,43,44,45),
there is no NCCLS approved standards for performing these tests.

The NCCLS testing procedure required the standardization of a quality control
organism, including defining the Minimal Inhibitory Concentration (MIC) ranges for each
antimicrobial agent tested, defining the artificial media required for optimal bacterial
growth, optimal atmospheric conditions and interpretive criteria for antimicrobial
susceptibility ranges.

The selection of quality control organism is based on its phenotypic stability as
determined by its colony characteristics, the survival of the organism following numerous
passages on artificial media, and genotypic stability as determined by its reproducible in
vitro antimicrobial susceptibility profile. In order for C. jejuni to be included in a national
resistance monitoring system, as recommended by the aforementioned agencies, a
standardized method for performing in vitro antimicrobial susceptibility testing on
clinical Campylobacter isolates was required.

Testing Objectives

There were three specific objectives to accomplish in order to develop a
standardized method for the in vitro antimicrobial susceptibility testing of Campylobacter
species.

12

First, define the optimal growth conditions on artificial media capable of
producing luxuriant growth of Campylobacter isolates recovered from biological and/or
environmental sources (i.e. feces, blood, carcasses, water).

Second, identify a Quality Control (QC) organism that possess genotypic and
phenotypic stability based on the QC organism’s capacity to survive numerous passages
on artificial media and the reproducibility of the QC organism’s in vitro antimicrobial
susceptibility profile. And third, define a testing method and define the preliminary QC

ranges for each antimicrobial agent evaluated.

13

LITERATURE REVIEW

Published, non standardized antimicrobial testing methods for Campylobacter z’ez’uni

A study conducted by Huang et al., utilized heart infusion agar media .
supplemented with 5% rabbit blood (HIAB) to recover frozen Campylobacter isolates and
then incubated them for 48 h at 35°C, and subsequently restreaked the recovered isolates
onto a flesh HIAB plate and incubated the isolates for an additional 24 h at 42°C to
capture the thermophilic species. The suspensions of the organisms used for the broth
microdilution assay were prepared in 5 ml of Mueller-Hinton broth (Difco Laboratories,
Detroit, Mich.) and adjusted to equal the turbidity of a 0.5 McFarland standard (1 x 108
CFU/ml). When performing antimicrobial susceptibility testing using the broth
microdilution method the Huang Group used the guidelines established by the NCCLS
Document M7-A2 for bacteria that grow aerobically.

A study conducted by Aarestrup et al., used a selective enrichment phase with
Preston broth from isolates collected from animal fecal samples. Afier the Preston broth
was inocculated, the suspension was incubated for 18 to 24 h at 42'C, to enhance the
growth of thermophilic Campylobacter spp., in a microaerobic atmosphere
(approximately 6% 02, 7% C02, 7% H2, and 80% N2) created by a gas generation
system. One loop of the broth was then transferred to mCCDA (Oxoid CM739 plus
selective supplement SR 155E) selective media plate for performing a Tenover et al.

modified agar dilution method.

14

A study conducted by Van Leoveren et al., used different minced farm animal
meats to recover Campylobacter organisms. The meat samples were collected with a
brucella broth (Oxoid, Basingstoke, UK) moistened sterile cotton swab. In the lab the
swabs were homogenized into Preston selective broth (Oxoid) and incubated in a micro-
aerophilic atmosphere at 42°C for 48 h. The enrichment suspension was then streaked on
to modified charcoal cefoperazone deoxycholate agar (mCCDA, Oxoid) and incubated at
42°C for 24-120 h. The susceptibility testing was conducted using the agar dilution
method based on guidelines established by the NCCLS Document M7-A4.

A study conducted by Gaudreau et al., used Campylobacter isolates recovered
from human samples. Upon collection the samples were frozen in Trypticase soy broth
(BBL Microbiology Systems, Cockeysville, Md) supplemented with 15% (v/v) glycerol.
Inocula suspension was prepared in Mueller-Hinton broth (BBL Microbiology Systems)
at a density adjusted to a 0.5 McFarland turbidity standard for the disc diffusion and
diluted 1:10 for the agar dilution. Both testing methods used the same inoculum
suspension to reduce intra-laboratory variation. The inoculated plates were incubated at
35°C under a microaerophilic atmosphere obtained with a glass generator envelope
(Difco), for 48 h. Susceptibility testing criteria for the agar dilution and the drug
concentrations in the discs were those of the NCCLS.

A study conducted by Huysmans et al., used 100 strains of thermophilic
Campylobacters isolated from stools of patients suffering from acute diarrhea. The Mle
of the 100 clinical isolates were determined by agar dilution using methods similar to
those recommended by the NCCLS Document M7-A3, using Mueller-Hinton agar

containing 5% lysed horse blood.

15

Zones diameters were determined on the same medium using NCCLS strength
antimicrobial discs and similar methodology. All results were read after 48 h incubation
at 37°C in a microaerophilic atmosphere (5% 02, 10 C02, 10% H2, and 75% N2).

Agar Dilution and broth microdilution remain the standard methods of
determining the susceptibility of Campylobacters to antimicrobial agents. However, this
review of literature reveals that there is no consensus about the optimal medium, the
requirement of blood supplementation, the temperature of incubation, or the time of
incubation. Temperature and time of incubation , for instance, vary from 42°C for 24 h to
37°C for 48 h. For agar dilution testing, Mueller-Hinton agar containing 5% sheep blood,
incubated in anaerobic jars with a gas mixture containing 5% 02, 10% C02 and 85% N2
for 48 h at 37°C, is the method most frequently used at the Centers for Disease Control
and Prevention in the United States, whereas cation-adj usted Mueller-Hinton broth
containing 5% lysed horse blood, incubated as described above, is used by the CDC for
broth microdilution assays. Other methods will produce acceptable results, but it is
critical that quality control strains be tested in parallel to ensure the accuracy of the
method.

The choice of method depends on several factors, including cost, ease of
performance, personal preference and experience and the availability of methods in each
laboratory. Susceptibility tests for Campylobacter are not yet standardized, and
consequently the literature contains some variability in susceptibility data reported. For
the interpretation of the results, breakpoints recommended by the NCCLS for aerobic
bacteria have been used in most cases. However, national and international breakpoints or

breakpoints established through population distribution studies have also been utilized.

16

MATERIAL AND METHODS: GENERAL

Materials
Bacterial Strains

A total of 71 Campylobacter isolates collected from human stool cultures and
farming animals have been screened for use as a potential QC organism. Four of these
isolates were submitted by Dr. Frank Aarestrup from the Danish Veterinary Laboratory in
Denmark. Four were submitted by Dr. Laura Piddock from the University of
Birmingham, United Kingdom. Six isolates were submitted by Dr.Konkel from the
University of Washington and the remaining isolates were submitted by Dr. Walker from
Michigan State University, College of Veterinary Medicine, who is currently at the CVM-
FDA. Three type strains fi'om the American Type Culture Collection (ATCC) were also
included in the study: C. jejuni ATCC 33560, C. jejuni ATCC 43430, and C. jejuni
43470.

Of the 71 isolates, 21 were identified as Campylobacterjejuni. Of these 21
isolates, three isolates, C. jejuni ATCC 33560, C. jejuni E97-2805 from Dr. Konkel’s
laboratory and C. jejuni 4239-928 from Dr. Walker’s laboratory were selected for
subsequent testing in five laboratories. The five participating laboratories included the
Clinical Microbiology Institute (CMI) in Wilsonville, OR., MRL: Pharmaceutical
Services (MRL) in Hemdon, VA., USDA-ARS (USDA) in Athens, GA., Duke University
Medical Center (Duke) in Durham, NC., and Michigan State University (MSU) in East

Lansing, MI.

17

To enhance and better visualize the cell walls of the Campylobacter isolates a
modified Gram staining method was employed using carbofusion stain instead of the
standard saffarin counterstain. Hippurate hydrolysis and oxidase testing was also
performed to identify the isolates. Criteria for subsequent testing included survival of
numerous passages on artificial media, growth characteristics, and susceptibility profile.
One isolate, 4239-928 died after being shipped to participating laboratories and was not

tested.

Antrmrcrobial agents

Eight different antimicrobial agents were used in this preliminary study. These
included, amoxicillin, gentamicin, doxycycline, ciprofloxacin, tetracycline, erythromycin,
trimethoprim/sulfamethoxazole and nalidixic acid for the broth microdilution method. All
the antirnicrobials were supplied by pharmaceutical companies as standard powders with
known potencies. The compounds were prepared according to the recommendations by
the manufacturers and NCCLS guidelines.
Antimicrobial disks

Zones of inhibition were determined using commercially available disks. The
concentrations of the antimicrobial disks were; trimethoprim/sulfamethoxazole 15ug,
erythromycin 15ug, nalidixic acid 30ug, ampicillin IOug, gentamicin 10ug, ciprofloxacin
Sug, doxycycline 30ug, and tetracycline 30ug.

Two lots of disks were used for each antimicrobial agent. The disks were supplied
by Becton Dickinson and shipped by Michigan State University to the participating

laboratories.

18

Environmental Materials
WWW

Growth characteristics and antimicrobial MIC profiles of clinical Campylobacter
isolates were investigated using commercially available Campy-pouches, which provides
a microaerophilic environment, and a C02 incubator set at 10% concentrations . The
preliminary disk diffusion and broth microdilution testing that was conducted using a
C02 incubator was set to deliver a 10% CO2 concentration. Based on a decision by the
National Committee for Clinical Laboratory Standards-Veterinary Antimicrobial
Susceptibility Testing (N CCLS-VAST) subcommittee, any subsequent Campylobacter
antimicrobial susceptibility testing will be conducted using incubator set to deliver a 5%
CO2 concentration.
Zimloc bag system

Grth characteristics and antimicrobial MIC profiles of clinical Campylobacter
isolates were also investigated using commercially acquired plastic Zip-10c bags filled
with a gas mixture (85% N2, 5% 02, and 10%C02). It was difficult to standardize the
amount of gas entering the bags and that may contribute a source of error and lack of
accuracy and precision to the testing methods.
Methfls: Preparation of Campylobacter susmnsions

A standard stock suspension of C. jejuni was prepared, using cation adjusted
Mueller-Hinton broth, to achieve a final concentration of 0.5 McFarland, which is

approximately 108 colony forming units.

19

When applicable, the broth microdilution and disk diffusion testing methods used
the same standard stock suspension in order to minimize inoculum variations and other
sources of error that may result from preparing two separate standard stock suspensions.
ELEM

Disk diffusion testing was performed as described in the NCCLS document M2-
A6. Mueller-Hinton blood agar plates (150mm), supplemented with 5% sheep’s blood.
Inocula were prepared using cation adjusted Mueller-Hinton broth (Difco Laboratories,
Detroit, MI, USA) to match an optical density equal with a 0.5 McFarland Standard. A
sterile cotton swab was used to inoculate the 150 mm blood agar plates by rotating the
plates three consecutive times, 60 degrees each time, to ensure a uniform distribution of
inocula across the surface of the plates. The disk diffusion plates were incubated at 37°C
in a 10% CO2 atmosphere for 48 hours prior to measuring the zone of inhibition
diameters.

Broth Microdilution

Broth microdilution MIC testing was performed according to NCCLS document
M7-4A. During preliminary testing both Brucella and Mueller-Hinton broth was utilized
and the growth patterns of the Campylobacter isolates were very reproducible and there
seemed to be no microbiological growth advantage of either broths. A decision was made
to use Cation adjusted Mueller-Hinton broth because it was more cost effective and more
readily available. The inocula was prepared by suspending the Campylobacter isolates in
cation adjusted Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) to achieve a

turbidity of 0.5 McFarland standard.

20

The suspension was further diluted with sterile water to provide a final inoculum
density of 5.0 x 10’ CPU/ml in the wells of the broth microdilution trays. Colony counts
were preformed on each inocula to ensure appropriate inocula concentrations. Following
the inoculation of the antimicrobial trays, the trays were incubated at 37°C for 48 hours in

a 5% and 10% CO2 environments.

21

METHODS AND MATERIALS: DETAILED

Objective one: Define the optimal growth conditions, on artificial media, capable of
producing luxuriant growth of Campylobacter isolates recovered from biological
and/or environmental sources (i.e. feces, blood, carcasses, water)

Midi;

Various media formulations were investigated to determine the optimal and most
reliable artificial growth media for in-vitro cultivation of clinical and laboratory
attenuated strains of Campylobacter.

Both agar and broth media was investigated. The criteria for the selection of a
suitable growth and antimicrobial susceptibility testing medium was based on its ability
to provide optimal growth of Campylobacter isolates under varying atmospheric
conditions. The mediums tested included cation-adjusted Mueller-Hinton broth, Brucella
broth, Mueller Hinton agar, and Mueller-Hinton agar supplemented with 5% defibrinated
sheep blood.

Temgrature

Most Campylobacter organisms are thermophilic and thrive within a temperature
range of 37°C to 42°C., these two temperature ranges were investigated in this study.
Incubation Period

The incubation period is directly correlates with the Incubation temperature. The

incubation times tested were 24 and 48 hours.

22

Moisture content of the Incubator or Campy-Gas Generation Systems

The clinical Campylobacter isolates that were cultivated in a moist environment
flourished and exhibited watery colony characteristics as compared with isolates that
were cultivated within an environment lacking moisture. An incubator with a water pan
was used to achieve a moisture-rich environment, along with moisture producing Campy-

pouches and jars for isolate cultivation and testing.

Objective two: Identify a Quality Control (QC) organism that possess genotypic and
phenotypic stability based on the QC organism’s capacity to survive numerous
passages on artificial media, and the reproducibility of the QC organism’s in vitro
antimicrobial susceptibility profile.

The second objective involved the identification of a quality control isolate that
could represent the Campylobacter genus for in vitro antimicrobial susceptibility testing
purposes. By using broth microdilution testing method, the MIC profiles of several
Campylobacter isolates were obtained and evaluated based on genetic and phenotypic
stability and growth survival characteristics on numerous passages on artificial media.

Initially 71 Carnpylobacter isolates were under investigation, these included 3
ATCC strain and 68 clinical isolates. Of the original 71 strains tested, 3 strains were
selected for interlaboratory testing. The criteria for selecting the 3 strains were based on

their stable growth patterns over numerous passages on artificial media and consistent

and reproducible MIC data values.

23

Objective three: Define the preliminary Quality Control ranges for the in-vitro
antimicrobial susceptibility testing of 8 antimicrobial agents against the selected QC
organisms.

The third objective involved testing 3 potential quality control Campylobacter
isolates in 5 laboratories. The 5 participating laboratories performed disk diffusion and
broth microdilution testing methods on 3 potential quality control isolates. The broth
microdilution trays and the disk diffusion plates were placed in an incubator set at 37°C in
10% CO2 for 48 hours.

Based on the reproducibility of the broth microdilution MIC profiles and disk
diffusion zone diameters reported by all 5 laboratories, one isolate was chosen to be
tested in the final phase of the study and the dilution range relative to the MICs of the
clinical isolates. During preliminary testing at Michigan State University, one
manufacturer of MH agar (BBL) was used for making disk diffusion plates and one
manufacturer of MH broth (Difco) was used for preparing broth microdilution trays. The
Mueller-Hinton broth (Difco) used in the antimicrobial dilution trays were prepared by

PML in Portland, Oregon.

24

RESULTS

Objective one
Ma

There was no significant difference in the optical density readings after the
incubation of Campylobacter suspensions for either Mueller-Hinton or Brucella broths
under investigation. Mueller-Hinton broth (Difco Laboratories, Detroit, MI, USA) has the
advantage of being more widely available and is currently being used as the broth of
choice for the in-vitro antimicrobial susceptibility testing of numerous other organisms
and for those two reasons, Mueller-Hinton is the broth of choice for Campylobacter
studies.

Optimal growth on agar surface studies were also conducted. Campylobacter
isolates were streaked on MH agar and MH agar supplemented with fresh and old sheep’s
blood. The addition of sheep’s blood resulted in a final Mueller-Hinton Blood (MHB)
agar concentration of 5%. A non-selective Enriched Blood Agar (EBA) was also
investigated. No growth was exhibited by all isolates tested using the non-supplemented
MH agar.

Abundant growth was observed when testing the MHB agar and the EBA plates.
The growth patterns between the MHB and EBA are indistinguishable and both provide
for abundant growth of the organism; however, selection of MHB agar for future
susceptibility testing of Campylobacter organisms is based on its wide distribution among
laboratories and it has been commonly used for susceptibility testing of other clinical

isolates.

25

11mm

Campylobacter isolates will grow at 37°C and 42°C with optimal growth at 42°C
occurring in 24 hours.

At a previous NCCLS-VAST subcommittee meeting, the working group selected
35-37’C for in vitro susceptibility testing since they felt time was not a factor and all

laboratories have 37°C incubators. Based on that recommendation, any subsequent
studies will be performed at 35-37'C.
Incubation mriod

The incubation period is related to the temperature of the testing environment. At
35-37’C the incubation period is 48 hours, whereas at 42°C the incubation period is 22-24
hours. An incubation period of 48 hours was used for all test conditions.
M_<_)i_sture Content of the Incubator or Campy-Gas Generation systems

Placing water in the bottom of the incubators or using the Campy-bag or Campy-
jar system will increase the humidity and produce abundant growth exhibiting watery
Campylobacter colony characteristics. Optimal growth can be achieved by addition of
water to an incubator or using Campy-pouches or Campy-jars which allows the organism
to flourish.
Objective two

A total of 71 clinical Campylobacter isolates were tested for their ability survival
numerous passages on artificial media and growth characteristics. Those isolates who
died after only a few passages were deemed not suitable and would not represent a viable
quality control organism. Of the 71 isolates observed, 21 isolates were selected for broth

microdilution testing to narrow the field to 3 potential quality control organisms.

26

Most clinical isolates clustered around the QC ranges for each of the antimicrobial
agents tested. Three isolates generated the most accurate and reproducible MIC data
points among the 15 originally selected. The 3 selected for further testing in phase three
were Campylobacterjejuni ATCC 33560, Campylobacterjejuni E97-2805 and
Campylobacterjejuni 4239-928.

Objective three

Three potential quality control isolates were investigated. Campylobacterjejuni
ATCC 33560, Campylobacterjejuni E97-2805, and Campylobacter jejuni 4239-928. C.
jejuni 4239-928 died out shortly after the 3 isolates were shipped, consequently it was
dropped from the study. For the two remaining candidates, the broth microdilution MIC
data values reported exhibited accurate and reproducibly MIC data points among all 5
laboratories. Preliminary testing was done in five laboratories using the broth
microdilution and the disk diffusion methods with eight antimicrobial agents.

Broth microdilution and disk diffusion data for isolate ATCC 33560 are shown in
appendices A-D. Broth microdilution data values for isolate E97-2805 and disk diffusion
data for ATCC 33 560 and E97-2805 are not included in this thesis paper. While both
isolates performed reasonably well against most drugs, there were some decrepencies.

The MIC ranges for isolate E97-2805 were off scale for both tetracycline
and doxycycline. However, this isolate was slightly more active than isolate ATCC 33560
when tested against ciprofloxacin and nalidixic acid. Both isolates performed poorly
when tested against trimethoprim/sulfarnethoxazole (table 8), suggesting that this

antimicrobial may not be an appropriate drug to test.

27

Overall, C. jejuni ATCC 33560 generated more reproducible data points with less
variability between participating labs than C. jejuni E97-2805. When testing the ATCC
33560 isolate, using the broth microdilution method, all antimicrobial agents tested
produced NCCLS acceptable MIC data values, which were between 2 to 3 dilutions away
from the mean MIC values, except for Trimethoprim/Sulfamethoxazole.

The agents that performed very well overall include nalidixic Acid (Table l),
tetracycline (Table 2), gentamicin (Table 3), amoxicillin (Table 4), ciprofloxacin (Table
5), erythromycin (Table 6), doxycycline (Table 7). trimethoprim/sulfamethoxazole (Table
8) should not be used for antimicrobial susceptibility testing of organisms in this genus.
Data collected from the disk diffusion testing exhibited zone diameters with excessive
variability and poor reproducibility among the 5 laboratories. Preliminary testing was
performed using the disk diffusion testing method with eight antimicrobial agents.

The resulting zones of inhibition varied considerably between the five laboratories
and even within a laboratory. Interlaboratory variation was seen when ampicillin was
tested against isolate E97-2805. Examples of intralaboratory variation with isolate ATCC
33560 may be seen with numerous isolate/drug combinations.

We hypothesized that this variation was due to the growth patterns on the plates
and how the plates were read in relation to the light source. In other words, the zone of
inhibition could appear as a hologram with the size of the zone of inhibition changing
with respect to how the light reflected off the surface of the medium. To investigate the
possible cause of this phenomena we looked at the effect variations in organism

concentrations had on the size of the zone of inhibition.

28

We tested isolate 33560 at concentrations of 107, 108, 109 CF U/ml. There was no
difference found in regards to the zone sizes. We also looked at the effect of varying
concentrations of CO2 had on the size of the zone of inhibition. Carbon dioxide
concentrations of 5%, 8%, and 10% were investigated.

We found that this had no effect on the variation seen in the zone diameters. Disk
difiusion should not be utilized for antimicrobial susceptibility testing of Campylobacters
until precise growth characteristics can be identified that would eliminate variations in

zone size and end point determinations.

29

DISCUSSION

The optimal environmental conditions, as endorsed by the NCCLS-VAST
subcommittee, to support luxurious growth of Campylobacter organisms include using a
Campy-Gas Generation System or a mechanical incubator with a C02 concentration
setting of 10% along with a temperature setting of 37°C. The incubation period should be
48 hours. However, since there was only a slight difference in growth patterns between
Mueller-Hinton broth tested at 5% and 10%, the NCCLS-VAST subcommittee
recommended that 5% CO2 be adopted as the standard CO2 concentration since most
clinical laboratories have access to a 5% CO2 incubator.

The moisture content of the Campy-Gas Generation Systems allow the
Campylobacter organisms to flourish and grow in abundance. The Campylobacter
isolates grew better in an environment with a high moisture content when compared with
the same environment lacking moisture. When using a mechanical incubator, water in a
tray with a large surface area should be placed on the bottom of the incubator to enhance
the growth of Campylobacter organisms. A Zip-10c bag system was investigated during
phase one of the study.

A gas mixture containing 85% N2, 5% O2 and 10% CO2 was injected into a
commercially acquired zip-10c bag. The Campylobacter isolates exhibited good growth
with this system; however, it was difficult to standardize the amount of gas that was
injected into the bag and the added expense of a special gas cylinder proved to be

unpractical as a standardized testing condition.

30

There are several different types of media that will support the growth of
Campylobacter organisms; however, both Mueller-Hinton broth(Difco) and agar (BBL)
preformed the best, supporting consistent and rapid growth of all clinical isolates tested.

Mueller-Hinton broth and agar are readily available and commonly used for the
in-vitro antimicrobial susceptibility testing of numerous other bacteria species. One of the
methods suitable for antimicrobial susceptibility testing of the Campylobacter genus is
broth microdilution. This method generated reproducible and verifiable MIC data values.
A recommendation to the NCCLS-VAST subcommittee was made to discontinue the use
of disk diffusion for the antimicrobial susceptibility testing of the Campylobacter genus.

Based on data points collected and evaluated, disk diffusion is not a valid method
of choice for antimicrobial susceptibility testing of the Campylobacter genus at this time,
due to its excessive variability in zone diameter interpretations, which makes the
interpretation of precise zone size difficult. Data tables for Campylobacterjejunr' ATCC
33560 using the broth microdilution method can came be found in the appendices. Of all
the antimicrobial agents tested in this study, only one agent,
Trimethoprim/Sulfamethoxazole, is not recommended for susceptibility testing of the
Campylobacter genus .

The overall performance of Campylobacterjejuni ATCC 33560 isolate during the
broth microdilution testing method and concurrent data analysis has reinforced our
decision to recommend that the Campylobacterjejuni ATCC 33560 isolate be used as a
quality control isolate for the in vitro antimicrobial susceptibility of the Campylobacter

genus.

31

SUMMARY and CONCLUSIONS

The recommended optimal environmental conditions for the antimicrobial
susceptibility testing of Campylobacter species includes using a Campy-Gas Generation
System or a 10% C02 incubator set at 37C with an incubation period of 48 hours.
Campylobacter isolates should be streaked onto Mueller-Hinton agar supplemented with
5% sheep’s blood and Mueller-Hinton broth (Difco) should be used for inocula
suspension preparation and antimicrobial susceptibility testing.

The broth microdilution method using Mueller-Hinton (Difco) broth produces
reproducible and reliable antimicrobial susceptibility results. The disk diffusion method
should not be used for the antimicrobial susceptibility testing of Campylobacters since
this testing method produced excessive variability, poor reproducibility and inconsistent
holographic zone sizes among the 5 laboratories, which makes interpertation challenging.

The QC organism for the antimicrobial susceptibility testing of the
Campylobacter genus should be Campylobacterjejuni ATCC 33560. This specific isolate
produced the most reproducible and reliable MIC data points among the 5 laboratories
and has exhibited a robust growth characteristic throughout all testing objectives in this

preliminary study.

32

CURRENT AND FUTURE RESEARCH

Based on the data collected from this preliminary broth microdilution study, the
NCCLS-VAST subcommittee has endorsed the aforementioned environmental testing
conditions for the Campylobacter genus. Meanwhile, testing of C. jejuni ATCC 33560 by
the “gold standar ” agar dilution method has been conducted in 10 laboratories
worldwide. Based on the data collected from this agar dilution study of C. jejuni ATCC
33560, the NCCLS-VAST and AST has endorsed the agar dilution method as one of the
standard antimicrobial susceptibility testing method for the Campylobacter genus and has
also endorsed C. jejuni ATCC 33560 as the QC organism.

MIC data and antimicrobial breakpoints from the agar dilution study will be
published in upcoming NCCLS M31 and M7 Documents. Further investigation of the
broth microdilution testing method using 3 different manufacturers of Mueller-Hinton
broth in 10 different laboratories worldwide will be conducted to validate and this testing
method as a standard antimicrobial susceptibility testing method for the genus

Campylobacter.

33

APPENDICES (A-D)

34

APPENDIX A

 

1

Table 1. BMD (Nalidixic Acid)

IdJ1 I232 I333 I834

10 10 10

1 0.8 0.45 1.1
1 1 0.5 1
1 0.5 0.25 1
1 1 0.5 2
1 2 2 2

Table 2. BMD (Tetracycline)

 

35

APPENDIX B

 

Table 3. BMD (Gentamicin)

W4 Id) 5 All lws

 

Table 4. BMD (Amoxicillin)

36

APPENDIX C

1
Table 5. BMD (Ciprofloxacin)

 

 

Table 6. BMD (Erythromycin)

37

APPENDIX D

0.5

 

Table 7. BMD (Doxycycline)

I831 Id)2 Ida3 Iw4 IwS
4

0.25
0.125
0.06
0.03
N 10 10 10 10 8 48
Geomem 24 528 4.05 10
Mode 2 64 2 256 16 2
Min 2 0.5 >256 4 0.5
Max 4 64 16 > 16 >256
2 3 6 2 10

Table 8. BMD (Trimethoprim/Sulfamethoxazole)

 

38

10.

11.

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