Acquisition of Multi-Resistance of Campylobacter jejuni Isolates with Antimicrobial Usage in Poultry

Acquisition of Multi-Resistance of Campylobacter jejuni Isolates with Antimicrobial Usage in Poultry Loinda R. Baldrias 1 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 SUSPECTED TYPE NO. OF % NO. OF % (%) X 2 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 Kf = Cephalothin C = Chloramphenicol Cip = Ciprofloxacin Col = Colistin sulphate E = Erythromycin Gm = Gentamicin Na = Nalidixic acid Nor = Norfloxacin Spc = Spectinomycin Sul = Sulphamethazine Sm = Streptomycin Tet = Tetracycline Tm = Trimethoprim 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. ANTIBIOTIC TESTED C. jejuni isolates from commercial producers C. jejuni isolates from backyard 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 9 that 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 (X 2 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 foodproducing 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 ANTIBIOTIC TESTED % RESISTANCE of C. jejuni ISOLATES Penicillin X 2 VALUES FOR TYPE OF ANTIBIOTIC RESIDUE DETECTED Sulfas Tetracyclines Aminoglycosides Macrolides 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. References Alfredson DA and Korolik V. 2007. Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli. Federation of European Microbiological Societies (FEMS) Microbiol Lett 277: 123-132. 8

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