Acquisition of Multi-Resistance of Campylobacter jejuni

Isolates with Antimicrobial Usage in Poultry

Loinda R. Baldrias1
Professor
College of Veterinary Medicine
University of the Philippines Los Banos

Abstract
The antimicrobial profiles of 12 Campylobacter jejuni isolates recovered from poultry ceca
were determined using the Kirby Bauer method. The isolates came from chickens detected
to be positive for antibiotic residues. These showed multi-resistance, being resistant to more
than 7 out of 14 different antibiotics tested. Statistical analysis showed a significant
relationship between antibiotic usage in poultry production and the development of
microbial resistance. Observed multi-resistance among C. jejuni isolates adds to the evidence
on emergence of resistant bacteria in animals following administration with antibiotics,
either prophylactically or therapeutically. The growing resistance to different antibiotics,
which is being observed to be higher in developing countries, appears to be a similar trend in
the Philippines, where the use of antimicrobial drugs in humans and animals is relatively
unrestricted. This is of public health relevance as antibiotic resistance among bacteria from
foods of animal origin may have an impact on antibiotic–associated bacterial infection of
humans.

Introduction
Development and use of antimicrobial agents were considered among the most important
measures leading to the control of bacterial diseases in the 20 th century (Cohen, 1992).
Antibiotics have greatly enhanced human life expectancy, reduced mortality, improved the
quality of life and almost won the war against many infectious diseases. However, reports of
antibiotic-resistant bacteria isolated from farms and animal carcasses are raising concerns
that antibiotic use in agriculture may play a role in selecting for antibiotic resistance among
foodborne bacteria (Nawaz et al., 2001; Alfredson and Korolick, 2007). The emergence of
antimicrobial resistance is a very controversial issue. Some claim that indiscriminate use of
antibiotics in agriculture, either as growth promotants or for performance enhancement, has
created a reservoir of resistant organisms in the environment that could infect humans
through the food chain. On the other hand, it is also possible that abuse of antibiotics in
human medicine may be largely responsible for the increase in antibiotic resistance.

1 Research supported by the Department of Agriculture Bureau of Agricultural Research (DA-BAR), Philippine
Council of Health Research and Development (PCHRD), Philippine Council of Advanced Research in Science and
Technology Development (PCASTRD) and Commission on Higher Education (CHED).

1
Among food-borne illnesses from foods of animal origin, campylobacteriosis is now
considered the leading gastrointestinal infection throughout the world, even exceeding
salmonellosis (Rosef and Kapperud, 1983 and Walker et al., 1986; Steel et al., 1998;
Altekruse et al., 1999; Alfredson and Korolick, 2007). Their significance, as causative agents
of foodborne diseases in man, is well documented by various food-borne outbreak reports
(Engberg et al., 2001). Most cases of Campylobacter enteritis do not require antimicrobial
treatment, but a substantial portion of these infections does require treatment, particularly
if it is severe and prolonged. There are, however, reports on the emergence and spread of
resistance spectra among Campylobacter strains, particularly C. jejuni, which is recognized as
the most important etiologic agent of acute diarrheal Campylobacteriosis in humans
worldwide (Gibreel et al., 1998; Smith et al., 1999; Saenz et al., 2000; Engberg et al., 2001;
Alfredson and Korolick, 2007). This study investigates a possible link between the use of
antibiotics in poultry production and the development of antibiotic resistance using local C.
jejuni isolates from chickens from commercial and backyard raisers that were detected to be
positive for antibiotic residues.

Materials and methods

Antimicrobial sensitivity testing of Campylobacter Isolates. Local putative Campylobacter
isolates, recovered from the ceca of randomly selected freshly dressed chicken at dressing
plants of commercial and backyard chicken producers, were confirmed to be C. jejuni by
polymerase chain reaction using specific primers CL2 and CR3 (Ng et al., 1997; Magistrado et
al., 2001). Pure cultures of twelve revived C. jejuni isolates were subjected to antimicrobial
susceptibility testing using the Kirby Bauer method (Atlas et al., 1988; NCCLS, 1981 and
1998; Lucey et al. 2000). The 14 antimicrobial substances used for sensitivity/resistance
testing were: ampicillin (10 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin
(5 μg), colistin sulphate (10 μg), erythromycin (15 μg), gentamicin (10 μg), nalidixic acid (30
μg), norfloxacin (10 μg), spectinomycin (10 μg), streptomycin (25 μg), and tetracycline (30
μg) from MAST Diagnostics, U.K.; trimethoprim (1.25 μg) and sulfamethazine (25 μg) from
SIGMA, Italy. Their patterns of antibiotic resistance were analyzed (Baldrias and Raymundo,
2009).

Antibiotic residue detection using the Four Plate Test (FPT). Data from Four Plate testing of
liver samples from the same study population of freshly dressed chickens at dressing plants
of commercial and backyard raisers were analyzed for type of antibiotic residues (Baldrias et
al., 2008).

Statistical Analysis. Chi-square and Pearson correlation (Noether, 1991) was used to
determine if a relationship existed between the percent antibiotic resistance of the C. jejuni
isolates (Baldrias and Raymundo, 2009) and the type of antibiotic residue as inferred by Four
Plate Test (Baldrias et al., 2008). This was to examine an association between the
antimicrobial profile of isolates with the antibiotic usage (by type of antibiotic residue
detected) of the same chicken population from commercial and backyard raisers, computed
using Chi-square with continuity correction and linear regression as used by Schmidt et al
(2001).

2
Results and discussion
On determining the frequency of detection by type of antimicrobials (Table 1), as inferred by
the Four Plate Test, it was found that penicillin type of antibiotic ranked highest in
occurrence at 53.1% for both backyard (62.8%, 49/78) and commercial (44%, 37/84),
followed by aminoglycosides. Both producers, then varied on the frequency for tetracyclines
and sulfonamides. However, the least frequent type of antibiotic detected for both
producers were the macrolides at 21.6%. The number of chickens positive for penicillin,
tetracycline, sulfonamide and aminoglycoside antibiotics was significantly higher at 5% level
of significance for backyard raisers than for commercial producers. In contrast, no significant
difference was found between both producers for number of chickens positive for
macrolides. It was observed, however, that in many instances, more than one type of
antibiotic residue was detected in the liver samples, possibly indicating simultaneous dosing
of a combination of two or more antibiotics.

Table 1. Suspected type of antibiotic detected in fresh chilled chicken by producer using
Four Plate Test

COMMERCIAL (n=84) BACKYARD (n =78) (N=162)

PERCENT
2
SUSPECTED TYPE NO. OF % NO. OF % (%) X VALUE
OF ANTIBIOTIC POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE
Penicillin 37 44.05 49 62.82 53.09 0.0167421*
Tetracycline 27 32.14 43 55.13 43.21 0.0031686*
Sulfonamide 22 26.19 47 60.26 42.59 1.1BE-05*
Aminoglycoside 25 29.76 47 60.26 44.44 9.50BE-05*
Macrolide 23 27.38 12 15.38 21.60 0.1289373

Legend: * = significant at p < .05 level of significance

Resistance profiles (along with the measured zones of inhibition) of the 12 C. jejuni from
commercial (C. jejuni isolates V5, V7, V19 and V26) and backyard producers (52c, 53m, 55c,
57b, 60c, 64c, 65b and 68c) are presented in Table 2. The 14 antimicrobial agents chosen for
the test are those being used for treating clinical cases of gastroenteritis in both man and
animals, had a broad spectrum of activity, and routinely used for sensitivity testing in
diagnostic laboratories (National Committee for Clinical Laboratory Standards, 1981 and
1998). They are also similar to those used by Lucey et al. (2000). All of these isolates showed
multi-resistance (being resistant to more than 7 different antibiotics tested). For commercial
producers, two (V5 and V26) of the four (50%) isolates came from chickens detected to be
positive for antibiotic residues. These two isolates showed resistance to all (100%)
antibiotics tested. For backyard raisers, all isolates came from chickens positive for antibiotic
residues by FPT (Baldrias et al, 2008).

On the specific percentage of resistance to the different antibiotics (Figure 1 and Table 2), all
12 C. jejuni isolates were resistant to trimethoprim (100%); 91.7% were resistant to
cephalothin, ciprofloxacin, colistin, gentamycin, nalidixic acid, sulphamethazine,
streptomycin, and tetracycline; 83.3% to ampicillin; 75% to chloramphenicol and norfloxacin;
and 66.7% to spectinomycin. In contrast, only 33.3% of the isolates were resistant to
erythromycin. The 100% resistance to trimethoprim among all C. jejuni isolates is an

3
important finding, as resistance to trimethoprim had been increasingly observed for C. jejuni
by Gibreel and Skold (2000) and Lucey et al. (2000). Gibreel and Skold (2000) related this to
the acquisition of foreign resistance genes (dfr1, dfr9 or both) that code for resistant variants
of the enzyme dihydrofolate reductase — the target of trimethoprim (Alfredson and
Korolick, 2007). These dfr genes were found to occur as integron cassettes inserted in the
chromosome of the clinical isolates examined by these workers.

Legend:
Amp = Ampicillin Col = Colistin sulphate Nor = Norfloxacin Tet = Tetracycline
Kf = Cephalothin E = Erythromycin Spc = Spectinomycin Tm = Trimethoprim
C = Chloramphenicol Gm = Gentamicin Sul = Sulphamethazine
Cip = Ciprofloxacin Na = Nalidixic acid Sm = Streptomycin

Figure 1. Percent total antimicrobial resistance of 12 Campylobacter jejuni isolates

from poultry of commercial and backyard producers to different types of antibiotics

4
 Table 2. Antibiotic resistance profiles and zones of inhibition (mm) of Campylobacter jejuni
isolates from poultry of commercial and backyard producers.
C. jejuni isolates from commercial C. jejuni isolates from backyard producers
ANTIBIOTIC TESTED producers
V5 V7 V19 V26 52c 53m 55c 57b 60c 64c 65b 68c
ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP ZI RP
Ampicillin, 10 μg 7 R 15 S 0 R 5 R 0 R 0 R 0 R 0 R 0 R 13 I 0 R 1 R
Cephalothin, 30 μg 6 R 15 I 0 R 1 R 2 R 0 R 1 R 0 R 0 R 12 R 3 R 1 R
Chloramphenicol, 30 μg 5 R 1 R 0 R 4 R 3 R 0 R 18 S 3 R 5 R 5 R 15 I 17 I
Ciprofloxacin, 5 μg 7 R 7 R 12 R 11 R 0 R 15 R 0 R 0 R 1 R 15 R 18 I 0 R
Colistin sulphate,10 μg 1 R 1 R 4 R 4 R 0 R 15 S 9 R 0 R 4 R 5 R 3 R 7 R
Erythromycin,15 μg 7 R 10 R 0 R 10 R 19 S 17 I 20 S 17 S 19 S 14 S 18 S 20 S
Gentamicin,10 μg 6 R 6 R 12 R 8 R 0 R 17 S 7 R 0 R 2 R 10 R 5 R 9 R
Nalidixic Acid, 30 μg 0 R 0 R 7 R 7 R 3 R 0 R 2 R 0 R 2 R 6 R 0 R 3 R
Norfloxacin, 10 μg 6 R 7 R 15 I 12 R 2 R 16 I 2 R 0 R 2 R 17 I 15 I 2 R
Spectinomycin, 100 μg 5 R 5 R 12 R 10 R 4 R 16 S 18 S 1 R 5 R 0 R 12 S 20 S
Suphamethazine, 25 μg 0 R 0 R 0 R 12 R 0 R 17 S 0 R 10 R 12 R 0 R 2 R 5 R
Streptomycin,10 μg 4 R 5 R 15 S 7 R 1 R 0 R 1 R 0 R 1 R 7 R 0 R 1 R
Tetracycline, 30 μg 4 R 10 R 8 R 9 R 5 R 0 R 0 R 0 R 1 R 16 S 0 R 1 R
Trimethoprim, 2.5 μg 5 R 5 R 0 R 5 R 2 R 0 R 0 R 0 R 1 R 0 R 0 R 2 R
No. of antibiotics resistant
to 14 12 12 14 13 8 11 13 13 10 9 11
% AMR 100.0 85.7 85.7 100.0 92.9 57.1 78.6 92.9 92.9 71.4 64.3 78.6

Legend: ZI = zone of inhibition (mm); RP = resistance profile; Interpretation: R= resistant; S = susceptible;

I = intermediate (based on NCCLS, 1981 and 1998; Atlas et al., 1988; and Quinn et al., 1994)

The multi-resistance of C. jejuni isolates from chickens in this study reflects a similar trend
for increasing frequency of resistance being observed by other investigators from other
countries (Alfredson and Korolick, 2007). For example, Saenz et al. (2000) in Spain found that
on 1997-1998, C. jejuni strains recovered from broilers were resistant to cephalothin,
ampicillin, gentamicin, and tetracycline. Resistance to ciprofloxacin (a quinolone antibiotic)
in the present study is also reflective of the high frequency of resistance being observed
among Campylobacter strains in other countries. Altekruse et al. (1999) in Minnesota on
1997 also cited C. jejuni resistance to ciprofloxacin in his locality. The licensing of
fluoroquinolone for use in poultry in 1995 was observed to coincide with reports on
emergence of quinolone-resistant strains of C. jejuni (Khachatorians, 1998; Altekruse et al.,
1999; Alfredson and Korolick, 2007; Kabir, 2010).

Among the features used for identification of C. jejuni in diagnostic laboratories are its
resistance to cepthalothin and susceptibility to nalidixic acid (Carter and Cole, 1990). As
such, the observed high level of resistance to cephalothin was an expected finding. However,
the high level of resistance to nalidixic acid vouches for the increasing occurrence of nalidixic
acid-resistant strains of C. jejuni, as reported by Saenz et al., (2000). The growing resistance
to different antibiotics, which is being observed to be higher in developing countries,
appears to be a similar trend in the Philippines, where the use of antimicrobial drugs in
humans and animals is relatively unrestricted. Likewise in Malaysia, Selaha (2002) found 9that

5
76 C. jejuni isolates, recovered by cloacal swabs of chickens from 10 broiler farms, were
resistant to at least one of the seven antibiotics tested. Multiple resistance was also
observed in 58 (76.3%) isolates with ten (13.1%) resistant to all antibiotics tested.
Comparably similar results were observed for resistance to tetracycline; 91.7% in this study
and 100% in Selaha‘s study (2002). Least resistance was also observed for erythromycin;
33.7% in this study and 23.7% in Selaha’s (2002), among the antibiotics tested.

The low resistance (33%) to erythromycin (a macrolide) by C. jejuni isolates validates the
observation that among the antibiotic residues (Baldrias et al, 2008), macrolides were the
least frequent type of antibiotic (21.6%) detected for both commercial and backyard
producers. This may provide another good association between antibiotic usage in poultry
production and development of microbial resistance. Similarly, Hernandez and Raymundo
(1988) also observed a clear association between higher level of resistance of Escherichia coli
isolates and feeding pigs diets supplemented with antibiotics, as compared to pigs not given
antibiotics in their feeds. Likewise, Bradbury and Munroe (1985) raised the concern that a
relationship might be present between antibiotic use in feeds and in the development and
presence of antibiotic resistance among bacteria of food-producing animals. This is of public
health relevance as antibiotic resistance among bacteria from foods of animal origin may
have an impact on antibiotic–associated bacterial infection of humans (Kabir, 2010).

Observed multi-resistance among C. jejuni isolates adds to the evidence on emergence of

resistant bacteria in animals following the administration of antibiotics, either
prophylactically or therapeutically. For instance, in the survey conducted by Evangelista
(1994), all of the participating poultry farms in the survey indicated using antibacterials in
their operations. Use of antibiotics may have created a heavy selective pressure causing
microorganisms, like C. jejuni, to undergo needed changes to adapt to the new antibiotic
environment (Rowe-Magnus and Mazel, 2002; Alfredson and Korolik, 2007; Kabir, 2010).

Using Chi-square test, an analysis was conducted to determine if a relationship does exist
between the type of antibiotic residue (Baldrias et al, 2008) and percent antibiotic resistance
of the C. jejuni isolates (Baldrias and Raymundo, 2009). Pearson correlation (X2 values in
Table 3) showed that a statistically significant relationship (p < .05) exists between the
occurrence of penicillin type of residues in chickens sampled with the existence of antibiotic
resistance of C. jejuni isolates, particularly for cephalothin (a beta-lactam antibiotic) with a P-
value = 0.02 and for erythromycin (a macrolide) with a P-value = 0.028.

Similarly, a significant relationship was also present between the detection of tetracycline
type of antibiotic residue and with resistance to erythromycin of C. jejuni isolates (P-value =
.001). Likewise, for the aminoglycoside residue with erythromycin (P-value = .030).

The P-value or probability values are herein specified to provide a better appreciation on the
observation of associations between antibiotic residue and resistance of C. jejuni isolates, in
the light of the 0.05 level of significance cut-off. This provides an objective measure of the
strength of evidence, such that if the P-value is smaller than the level of significance, then
the result is highly significant (Ott and Mendenhall, 1990; Noether, 1991). When reporting
results of a statistical investigation (rather than stating whether or not a given hypothesis is
rejected), it is currently much preferred to report the actual P-value associated with the

6
experimental evidence. This makes the report more informative so that the reader can
decide whether to accept or reject the hypothesis being tested. For trimethoprim, no
statistics can be computed as all the isolates showed 100% resistance to this antibiotic. A
100% level of resistance is, without a doubt, biologically significant.

The general guideline observed by the Philippine Generics law of 1988 on antibiotic
supplementation of animal feeds follows the recommendation of the Swann Report of 1969
that antibiotics used to treat infections in humans are not to be used as animal-food
additives. This is based on the concept that structurally similar drugs would have the same
target of action and is, therefore, subject to cross-resistance within the same class of related
antibiotics. For instance, the initial expectation was that penicillin residue will be related to
the occurrence of ampicillin resistance; or aminoglycoside residue with resistance to
aminoglycoside antibiotics, like streptomycin, kanamycin or gentamicin. However, the
relationship revealed between the occurrence of antibacterial residues in the liver samples
(Baldrias et al, 2008) and multi-resistance of C. jejuni isolates (Baldrias and Raymundo, 2009)
to chemically unrelated antibiotics raises a very important concern of cross-resistance that
antibiotics can confer to other classes of antibiotics. As presented in Table 3, statistically
significant relationships were established between detection of penicillin type residue with
resistance to cephalothin (a cephalosporin) and erythromycin (a macrolide); between
tetracycline type residue with erythromycin resistance, and between aminoglycoside residue
and erythromycin resistance. The relevance of these observed relationships might be related
to the example cited by Courvalin (2001) on apramycin, an antibiotic used exclusively in
animals because it had an unusual structure. It was not expected to be recognized by
aminoglycoside-modifying enzymes in bacteria. However, enterobacteria of animal origin
became resistant to apramycin by synthesis of a plasmid-mediated 3-N aminoglycoside
acetyltransferase type IV, which confers resistance to gentamicin (Chalus-Dancia et al.,
1986). Following spread in animal strains, the plasmid was later found in clinical isolates
from hospitalized patients (Chalus-Dancia et al.1991). As such, use of antibacterials in food-
producing animals may, in turn, result to spread of resistant bacteria from animals to
humans. Spread of resistance may involve transfer of antibiotic-resistant genes from
bacteria in animals to human pathogens, and even strains of resistant bacteria that are
zoonotic that can cause disease in man (Alfredson and Korolick, 2007). The occurrence of
multi-resistant pathogens is currently creating a dilemma in the treatment of infections, not
only in animals, but also in human medicine.

7
 Table 3. Pearson Chi-square values for type of antibiotic residue detected in relation to
percentage of resistance to antibiotic tested
2
% X VALUES FOR TYPE OF ANTIBIOTIC RESIDUE DETECTED
RESISTANCE of
C. jejuni Tetracy- Aminogly-
ANTIBIOTIC TESTED Penicillin Sulfas Macrolides
ISOLATES clines cosides
Ampicillin 83.3 .166 .584 .584 .584 .640
Cephalothin 91.7 .020 * .140 .460 .140 .753
Chloramphenicol 75.0 .371 .157 .157 .157 .546
Ciprofloxacin 91.7 .640 .460 .140 .460 .753
Colistin sulphate 91.7 .640 .460 .460 .460 .753
Erythromycin 33.3 .028* .001* .083 .030 * .140
Gentamicin 91.7 .640 .460 .460 .460 .753
Nalidixic Acid 91.7 .640 .460 .140 .460 .753
Norfloxacin 75.0 .371 1.00 1.00 .157 .546
Spectinomycin 66.7 .273 .083 .386 .083 .460
Suphamethazine 91.7 .640 .460 .460 .460 .753
Streptomycin 91.7 .640 .140 .460 .460 .753
Tetracycline 91.7 .640 .460 .140 .460 .753
Trimethoprim** 100
Legend:
* Significant relationship exists at p < .05 level of significance between type of antibiotic residue and percent
antibiotic resistance of the Campylobacter jejuni isolates
**No statistic computed because trimethoprim is a constant with all isolates showing resistance.

Significant findings gained from occurrence of multi-resistance among the C. jejuni isolates
(Baldrias and Raymundo, 2009) and their relationship with detection of antibiotic residues
(Baldrias et al., 2008), indicating antibiotic exposure of the sampled chicken population,
provides proof that development of multi-resistance among the isolates may be a response
to selective pressure or stresses created by exposure to antimicrobials. Thus, this study
shows concrete evidence of a definite association between the development of
antimicrobial resistance and usage of antibiotics in poultry production.

Acknowledgements
Dr. Loinda R. Baldrias would like to express her deepest gratitude to the Department of
Agriculture Bureau of Agricultural Research (DA-BAR), the Philippine Council of Health
Research and Development (PCHRD), the Philippine Council of Advanced Research in Science
and Technology Development (PCASTRD) and the Commission on Higher Education (CHED)
for providing the funds needed for the research.

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