- Email: [email protected]
Antimicrobial Resistance in Salmonella and Escherichia coli Isolated from Commercial Shell Eggs
Antimicrobial Resistance in Salmonella and Escherichia coli Isolated from Commercial Shell Eggs M. T. Musgrove,*1 D. R. Jones,* J. K. Northcutt,† N. A. Cox,‡ M. A. Harrison,§ P. J. Fedorka-Cray,储 and S. R. Ladely储 *Egg Safety and Quality Research Unit, †Poultry Processing Research Unit, ‡Poultry Microbiological Safety Research Unit, and 储Bacteriology, Epidemiology, and Antimicrobial Resistance Research Unit, United States Department of Agriculture, Agricultural Research Service, Athens, GA 30604; and §Department of Food Safety and Technology, University of Georgia, Athens 30602 Salmonella isolates (n = 41) than in the E. coli isolates (n = 194). Salmonella Typhimurium was the most prevalent (69.0%) serotype and demonstrated the greatest multiple resistance. Salmonella Kentucky, the least prevalent (5.0%) serotype recovered, was the most susceptible. Although 34.1% of the Salmonella serotypes were susceptible to all antimicrobial agents, 60.1% were resistant to 11 or more compounds. Many Salmonella isolates exhibited resistance to tetracycline (63.4%), nalidixic acid (63.4%), and streptomycin (61.0%). Most E. coli isolates (73.2%) were susceptible to all antimicrobial drugs. Many E. coli isolates exhibited resistance to tetracycline (29.9%), streptomycin (6.2%), and gentamicin (3.1%). Only 1% of the E. coli isolates were resistant to 4 antimicrobial agents. These data indicate that shell eggs can harbor resistant foodborne and commensal bacteria; among Salmonella isolates, resistance was serotype-dependent.
Key words: Salmonella, Escherichia coli, egg, antimicrobial resistance 2006 Poultry Science 85:1665–1669
INTRODUCTION Antimicrobial resistance is an increasingly global problem, and emerging antimicrobial resistance has become a public health issue worldwide (Kaye et al., 2004). A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in human or veterinary medicine and in food-animal production (Bager and Helmuth, 2001; Anderson et al., 2003; Schroeder et al., 2004). Though many bacteria recovered from poultry or poultry-related samples have been monitored, few published studies have reported on antimicrobial resistance in bacteria, particularly Salmonella and Escherichia coli recovered from shell eggs (Yang et al., 2002; Antunes et al., 2003; Bajaj et al., 2003; Dargatz et
©2006 Poultry Science Association Inc. Received January 5, 2006. Accepted April 6, 2006. 1 Corresponding author: [email protected]
al., 2003; Zhao et al., 2003; Busani et al., 2004; Chung et al., 2004; Del Cerro et al., 2003; Dias de Oliveira et al., 2005). Studies were conducted in 2003 to monitor microbial populations, including Salmonella and other Enterobacteriaceae, along the shell egg-processing chain (Musgrove, 2004; Musgrove et al., 2005a,b). This paper reports on the antimicrobial susceptibility profiles of E. coli and Salmonella recovered in that study.
MATERIALS AND METHODS Description of Shell Egg Processing Plants A survey of in-line egg-processing facilities in the southeastern United States was conducted. Three plants were selected for sampling on 3 separate processing days. These plants were designated as X, Y, and Z to protect the anonymity of the participating companies. A more detailed description of the plants has been previously published (Musgrove et al., 2005b).
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ABSTRACT The development of antimicrobial resistance in bacteria has become a global problem. Isolates of Salmonella and Escherichia coli recovered from shell egg samples, collected at 3 commercial plants, were analyzed for resistance to 16 antimicrobial agents (n = 990). Eggs were sampled by rinsing in a saline solution. Pooled samples were preenriched in buffered peptone water and then selectively isolated using standard broths and agars. Salmonella-positive isolates were serogrouped immunologically before being serotyped. Enterobacteriaceae were enumerated from individual samples using violet red bile glucose agar plates. Escherichia coli were identified biochemically from presumptive Enterobacteriaceae isolates. Salmonella and generic E. coli antimicrobial-susceptibility testing was conducted using a semiautomated broth microdilution system. More resistance was observed in the
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Shell Egg-Sample Collection Eggs were collected from commercial plants at the following points of processing: at the accumulator, at prewash wetting, after the first washer, after the second washer, at the sanitizing rinse, at drying, at oiling, at check detection and weighing, at packaging (at 2 different packer head belts), at the entrance of the rewash belt, and at the exit of the rewash belt. Eggs were collected after the line had been operating for at least 2 h but during the midmorning break so as not to interfere with processing. This also allowed samples to be taken simultaneously from all sampling sites. Twelve eggs from each collection site were aseptically placed into clean foam cartons, packed into half-cases, and transported back to the laboratory.
As described in Musgrove et al. (2005b), 10 of the 12 eggs collected at each site were sampled using a shell rinse technique. Each egg was placed into a sterile WhirlPak bag (Nasco Modesto, Modesto, CA) with 10 mL of sterile PBS and rinsed by shaking for 1 min. Rinsate from the rinse and the crush method for every egg was then subjected to microbiological analyses as previously described (Musgrove et al., 2005b).
Water-Sampling Methodology Water samples were collected from the washer tanks in each of the 3 shell egg-processing plants and analyzed for pH, temperature, chlorine, protein, solids, and minerals, including iron, using procedures described previously (Northcutt et al., 2005). Samples were enriched for Salmonella. Collection procedures and other details are described in a previously published article (Northcutt et al., 2005).
Direct Plating Microbiology Microbial populations from the individual samples described above were enumerated for Enterobacteriaceae on violet red bile glucose agar (Becton, Dickinson and Co., Franklin Lakes, NJ) plates with overlay (purple-red colonies) as previously described. Presumptive colonies were counted and reported as log cfu/mL of egg rinsate. Up to 5 isolates for each positive sample were randomly selected for further analysis. An isolate from the third streak plate was saved on brain heart infusion agar slants at 37°C and Protect beads (Technical Service Consultants Ltd., Heywood, Lancashire, UK) at −20°C until further analyses for identification could be performed. Identification of Isolates A more detailed description of sampling methodology has been published previously (Musgrove, 2004). Each stored isolate was streaked onto plate count agar and incubated overnight at 37°C. A cultural suspension using 5 mL of physiological saline was prepared from each isolate. BioMe´rieux API 20 E strips (bioMe´r-
Salmonella Enrichment For each of the 12 collection sites, 2 pooled samples were formed by combining shell egg rinses or crushed shells and membranes from 5 eggs. Samples were preenriched in buffered peptone water at 35°C for 18 to 24 h, followed by enrichment in TT broth (Becton, Dickinson and Co.) and Rappaport-Vassiliadis broth (Becton, Dickinson and Co.) overnight at 42°C. Enriched samples were plated onto BG Sulfa (Becton, Dickinson and Co.) and XLT-4 (Becton, Dickinson and Co.) agar plates and incubated at 37°C for 24 h. Presumptive positive colonies were inoculated into lysine iron agar (Becton, Dickinson and Co.) and triple sugar iron slants (Becton, Dickinson and Co.) and incubated at 35°C for 18 to 24 h. Those samples giving presumptive results on each of these media were confirmed using serogrouping antisera (Becton, Dickinson and Co.). Confirmed isolates were then streaked for purity and stocked onto agar slants and ceramic beads in cryogenic protective media. A copy of each isolate was provided to the National Veterinary Services Laboratories (Ames, IA) for serotyping. A sample was recorded as positive if it was confirmed and serotyped from either the shell rinse or crushed shell and membrane composite samples.
Antibiogram Methodology Salmonella and generic E. coli were tested for antimicrobial susceptibility using a semiautomated broth microdilution system (Sensititre, TREK Diagnostic Systems Inc., Cleveland, OH). Custom-made panels of 16 antimicrobial drugs were configured in 96-well plates (National Antimicrobial Resistance Monitoring System, 2005). The minimum inhibitory concentration for each isolate was determined according to Clinical and Laboratory Standards Institute (National Committee for Clinical Laboratory Standards, 2003, 2004a,b). Breakpoint interpretations used in the National Antimicrobial Resistance Monitoring System (NARMS) were used when Clinical and Laboratory Standards Institute guidelines were not available (NARMS, 2005).
RESULTS AND DISCUSSION Since their discovery in the 1940s, antibiotics have been widely used in both human and veterinary medical practice (Salyers and Whitt, 2005). However, many important human and animal pathogens have developed resistance to these compounds (Walsh, 2003). Resistant bacteria are routinely isolated from a variety of foods, including poultry meat and eggs (NARMS, 2005). Since 1997, pathogenic and commensal organisms, such as Salmonella and E. coli,
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 30, 2015
Shell Egg-Sampling Methodologies
ieux, Marcy l’Etoile, France) were inoculated, incubated, handled, and analyzed according to the manufacturer’s instructions. Reactions were recorded, and identifications were determined using Apilab Plus software (bioMerieux).
ANTIMICROBIAL RESISTANCE IN SALMONELLA AND ESCHERICHIA COLI
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Table 1. Antimicrobial resistance patterns for Escherichia coli isolated from shell eggs at commercial shell egg processing plants (n = 194)
Antimicrobial agent None Cephalothin Cephalothin-tetracycline Gentamicin-streptomycin-sulfamethoxazole-tetracycline Gentamicin-tetracycline Streptomycin Streptomycin-tetracycline Tetracycline
No. of resistant isolates (%) 142 (73.2) 2 (1.0) 1 (0.5) 2 (1.0) 4 (2.1) 5 (2.6) 5 (2.6) 33 (17.0)
which many Salmonella isolates exhibited resistance were tetracycline (63.4%), nalidixic acid (63.4%), and streptomycin (61.0%). Few studies have reported on antimicrobial resistance of Salmonella isolates collected from eggs or the egg-processing environment. In a study conducted in India, all of the 66 Salmonella isolates were susceptible to at least one of the compounds tested (Bajaj et al., 2003). Highest resistance was observed for penicillin (96.9%), vancomycin (83.3%), and erythromycin (81.8%). Resistance was also noted for trimethoprim (42.4%), streptomycin (24.2%), tetracycline (9.0%), and gentamycin (3.0%). Bajaj et al. (2003) did not report on the serotype(s) of Salmonella isolated in their study. In the NARMS summary report for 2003, when considering all Salmonella serotypes, 27.1% were resistant to tetracycline, 0.4% were resistant to nalidixic acid, and 19.5% were resistant to streptomycin (NARMS, 2005). Figure 1 depicts the distribution of resistance in Salmonella by serotype. In a study conducted in India of 38 salmonellae isolated from eggs and egg packing materials, 95% were Salmonella Enteritidis (Suresh et al., 2005). All of the isolates were resistant to tetracycline, and 40% were resistant to nalidixic acid. There were 5,353 Salmonella isolates in the 2003 NARMS report (2005). The most frequently isolated serotypes were Kentucky (n = 555, 10.4%), Typhimurium-Copenhagen (n = 533, 10.0%), Newport (n = 483, 9.0%), Heidelberg (n = 439, 8.2%), and Typhimurium (n = 411, 7.7%). Serotypes Typhimurium, Heidelberg, and Kentucky are 3 of the most commonly isolated serotypes collected from poultry clinical and slaughter samples (NARMS, 2005), and they were the most commonly identified isolates in our study. No S. Enteritidis were recovered in the current study, whereas only 2.5% of S. Enteritidis (n = 139) was reported to NARMS in 2003 (NARMS, 2005). An Italian survey recovered antimicrobial-resistant Salmonella Typhimurium, Enteritidis, and Infantis from humans, food, and animal samples (Busani et al., 2004). In the Italian study, the highest rates of multiple resistances were detected for S. Typhimurium, as was the case with the current study. In a Turkish study involving S. Enteritidis isolated from chicken, eggs, and humans, 58 of 82 isolates (71%) were
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are monitored by the NARMS. However, little attention has been given to antimicrobial resistance of bacteria isolated from commercial shell eggs or the egg-processing environment. Antibiogram patterns for E. coli isolates are summarized in Table 1. Most of the 194 isolates in this study (73.2%) were pan susceptible. Antimicrobial agents for which many E. coli isolates exhibited resistance were tetracycline (29.9%), streptomycin (6.2%), and gentamicin (3.1%). Only 1% (n = 2) of the E. coli isolates were resistant to 4 compounds. Many E. coli isolated from meat and poultry have demonstrated resistance to at least one antimicrobial drug (Schroeder et al., 2004; NARMS, 2005). Lanz et al. (2003) analyzed E. coli isolates from veterinary clinical sources in Switzerland, including from laying hens. Of the 16 antimicrobial drugs tested, the isolates in the Swiss study were most resistant to sulfonamides, tetracycline, and streptomycin. Sulfonamides, tetracycline, and streptomycin are the oldest drugs used in infectious disease, and it is not surprising that some level of resistance would have emerged over time (Salyers and Whitt, 2005). In a NARMS summary of antimicrobial resistance in E. coli collected from a variety of species from 1998 to 2003, gentamicin resistance ranged from 14.7 to 26.5%, streptomycin resistance ranged from 35.4 to 62.2%, and tetracycline resistance ranged from 36 to 79.9% (NARMS, 2005). Levels of resistance for these compounds were considerably lower in the E. coli isolates analyzed in the present study compared with the published reports. In our study, prevalence of multiple resistances was 20.6% for 1 compound, 5.2% for 2 compounds, and 1.0% for 4 compounds. Salmonella antibiogram results are summarized in Table 2. From the perspective of total numbers, more resistance was observed in the Salmonella isolates than in the E. coli isolates. However, resistance in Salmonella is largely serotype-dependent (NARMS, 2005). Salmonella Typhimurium was the most prevalent (69.0%) serotype and demonstrated the greatest multiple resistance. Salmonella Kentucky, the least prevalent (5.0%) serotype recovered, was also the most susceptible. Although 34.1% of the Salmonella isolates was susceptible to all compounds, 60.1% was resistant to more than 11 compounds. However, multiple resistances were predominately observed among Typhimurium isolates. Antimicrobial drugs for
Figure 1. Percentage of Salmonella Typhimurium, Heidelberg, and Kentucky isolated from shell eggs and wash water collected at commercial shell egg processing plants (n = 41) resistant to 1, 2, 11, 12, or 13 antimicrobial agents.
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Table 2. Distribution of minimum inhibitory concentrations (%) for Salmonella isolated from shell eggs and water samples collected at commercial shell egg-processing plants (n = 41) Distribution of minimum inhibitory concentrations1 (%)
Drug (g/mL)
No. (%) resistant
≤0.02
0.06
0.12
0.25
0.50
1
2
4
8
16
32
64
128
256
512
Amikacin Amoxicillin/clavulanic acid Ampicillin Cefoxitin Ceftiofur Ceftriaxone Cephalothin Chloramphenicol Ciprofloxacin Gentamicin Kanamycin Nalidixic acid Streptomycin Sulfamethoxazole Tetracycline Trimethoprim/sulfa
0 (0.0) 25 (61.0) 25 (61.0) 25 (61.0) 25 (61.0) 1 (2.4) 25 (61.0) 25 (61.0) 0 (0.0) 25 (61.0) 25 (61.0) 26 (63.4) 26 (63.4) 23 (56.1) 26 (63.4) 0 (0.0)
— — — — — — — — 36.6 — — — — — — —
— — — — — — — — 2.4 — — — — — — —
— — — — 0.0 — — — 0.0 — — — — — — 51.2
— — — — 0.0 36.6 — — 58.5 24.4 — — — — — 48.8
9.8 — — 0.0 36.6 0.0 — — 2.4 14.6 — 0.0 — — — 0.0
78.0 39.0 34.1 9.8 0.0 2.4 — — 0.0 0.0 — 0.0 — — — 0.0
9.8 0.0 4.9 26.8 2.4 0.0 26.8 0.0 0.0* 0.0 — 2.4 — — — 0.0*
2.4 0.0 0.0 2.4 0.0* 0.0 12.2 29.3 0.0 0.0 — 34.1 — — 34.1 0.0
— 0.0 0.0 0.0 0.0 2.4 0.0 9.8 — 0.0* 39.0 0.0 — — 2.4* —
— 0.0* 0.0* 0.0* 61.0 36.6 0.0* 0.0* — 26.8 0.0 0.0* — 39.0 2.4 —
—* 2.4 0.0 61.0 — 19.5* 2.4 2.4 — 34.1 0.0* 0.0 36.6* 0.0 0.0 —
— 58.5 61.0 — — 2.4 58.5 58.5 — — 4.9 63.4 2.4 0.0 61.0 —
— — — — — — — — — — 56.1 — 61.0 2.4 — —
— — — — — — — — — — — — — 2.4* — —
— — — — — — — — — — — — — 26.8 — —
Asterisk indicates that the breakpoint for resistance occurs between this and the next highest concentration.
resistant to one or more antimicrobial agents, and multidrug resistances were observed in 35% of the isolates (Icgen et al., 2002). Resistance was serotype-dependent in the current study. Salmonella Typhimurium isolates demonstrated the greatest degree of multiple resistances, followed by Heidelberg isolates. In the 2003 NARMS summary, only 2.6, 0.8, and 0.1% of 5,353 isolates were resistant to 11, 12, and 13 antimicrobial drugs, respectively, over all serotypes (NARMS, 2005). However, in the current study, most of the S. Typhimurium isolates were recovered from a single shell egg-processing plant and on the same sample collection day, increasing the likelihood that the isolates would be related. However, further characterization by pulsedfield gel electrophoresis will be required to confirm this suspicion. Escherichia coli and Salmonella isolates analyzed in the current study displayed resistance to antimicrobial drugs. However, as expected, Salmonella resistance was serotypedependent. Salmonella isolates were more likely to be resistant to antimicrobial drugs and displayed a larger number of multiple resistances. This observation was influenced by the fact that many of the isolates were S. Typhimurium, a serotype that has been previously reported as exhibiting multiple resistances. These data indicate that shell eggs and shell egg processing water can harbor resistant foodborne and commensal bacteria.
ACKNOWLEDGMENTS We gratefully acknowledge the excellent technical assistance of Patsy Mason, Jordan Shaw, Susan Akins, Kathy Orr, Jovita Haro, Takiyah Ball, Sherry Turner, Stanford Jones, Jerrie Bennett, and Manju Amin. This work was assisted in part by a grant from the US Poultry & Egg Association (project no. 501), and their support is greatly appreciated.
REFERENCES Anderson, A. D., J. M. Nelson, S. Rossiter, and F. J. Angulo. 2003. Public health consequences of use of antimicrobial agents in food animals in the United States. Microb. Drug Resist. 9:373–379. Antunes, P., C. Reu, J. C. Sousa, L. Peixe, and N. Pestana. 2003. Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents. Int. J. Food Microbiol. 82:97–103. Bager, F., and R. Helmuth. 2001. Epidemiology of resistance of quinolones in Salmonella. Vet. Res. 32:285–290. Bajaj, B. K., V. Sharma, and R. L. Thakur. 2003. Prevalence and antibiotic resistance profiles of Salmonella spp. in poultry eggs. J. Food Sci. Technol. 40:682–684. Busani, L., C. Graziani, A. Battisti, A. Franco, A. Ricci, D. Vio, E. Digiannatale, F. Paterlini, M. D’Incau, S. Owczarek, A. Caprioli, and I. Luzzi. 2004. Antibiotic resistance in Salmonella enterica serotypes Typhimurium, Enteritidis and Infantis from human infections, food stuffs and farm animals in Italy. Epidemiol. Infect. 132:245–251. Chung, Y. H., Y. I. Kwon, S. Y. Kim, S. H. Kim, B. K. Lee, and Y. H. Chang. 2004. Antimicrobial susceptibilities and epidemiological analysis of Salmonella enteritidis isolates in Korea by phage typing and pulsed-field gel electrophoresis. J. Food Prot. 67:264–270. Dargatz, D. A., P. J. Fedorka-Cray, S. R. Ladely, C. A. Kopral, K. E. Ferris, and M. L. Headrick. 2003. Prevalence and antimicrobial susceptibility of Salmonella spp. isolates from US cattle in feedlots in 1999 and 2000. J. Appl. Microbiol. 95:743–761. Del Cerro, A., S. M. Soto, and M. C. Mendoza. 2003. Virulence and antimicrobial-resistance gene profiles determined by PCR-based procedures for Salmonella isolated from samples of animal origin. Food Microbiol. 20:431–438. Dias de Oliveira, S., F. Siqueira Flores, L. Ruschel dos Santos, and A. Brandelli. 2005. Antimicrobial resistance in Salmonella enteritidis strains isolated from broiler carcasses, food, human and poultry-related samples. Int. J. Food Microbiol. 97:297–305. Icgen, B., G. C. Gurakan, and G. Ozcengiz. 2002. Characterization of Salmonella enteritidis isolates of chicken, egg and human origin from Turkey. Food Microbiol. 19:375–382. Kaye, K. S., J. J. Engemann, H. S. Fraimow, and E. Abrutyn. 2004. Pathogens resistant to antimicrobial agents: Epidemiology,
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 30, 2015
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ed. NCCLS M100-S14. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA. Northcutt, J. K., M. T. Musgrove, and D. R. Jones. 2005. Chemical analyses of commercial shell egg wash water. J. Appl. Poult. Res. 14:289–295. Salyers, A. A., and D. D. Whitt. 2005. Revenge of the Microbes: How Bacterial Resistance is Undermining the Antibiotic Miracle. ASM Press, Washington, DC. Schroeder, C. M., D. G. White, and J. Meng. 2004. Retail meat and poultry as a reservoir of antimicrobial-resistant Escherichia coli. Food Microbiol. 21:249–255. Suresh, T., A. A. M. Hatha, D. Sreenivasan, N. Sangeetha, and P. Lashmanaperumalsamy. 2005. Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retail markets of Coimbatore, South India. Food Microbiol. 23:294–299. Walsh, C. 2003. Antibiotics: Actions, Origins, Resistance. ASM Press, Washington, DC. Yang, S. J., K. Y. Park, S. H. Kim, K. M. No, T. E. Besser, H. S. Yoo, S. H. Kim, B. K. Lee, and Y. H. Park. 2002. Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: Comparison of phenotypic and genotypic resistance characterization. Vet. Microbiol. 86:295–301. Zhao, S., A. R. Datta, S. Ayers, S. Friedman, R. D. Walker, and D. G. White. 2003. Antimicrobial-resistance Salmonella serovars isolated from imported foods. Int. J. Food Microbiol. 84:87–92.
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molecular mechanisms, and clinical management. Infect. Dis. Clin. North Am. 18:467–511. Lanz, R., P. Kuhnert, and P. Boerlin. 2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet. Microbiol. 91:37–84. Musgrove, M. T. 2004. Effects of processing on the microbiology of commercial shell eggs. PhD Dissertation. Univ. Georgia, Athens. Musgrove, M. T., D. R. Jones, J. K. Northcutt, M. A. Harrison, and N. A. Cox. 2005b. Impact of commercial processing on the microbiology of shell eggs. J. Food Prot. 68:2367–2375. National Antimicrobial Resistance Monitoring System. 2005. Subject: NARMS data. http://www.ars.usda.gov/Main/ docs.htm?docid=6750 Accessed Dec. 2005. National Committee for Clinical Laboratory Standards. 2003. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 2nd ed. NCCLS M31-A2. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA. National Committee for Clinical Laboratory Standards. 2004a. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 2nd ed. NCCLS M31-S1. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA. National Committee for Clinical Laboratory Standards. 2004b. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; 2nd
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Key words: Salmonella, Escherichia coli, egg, antimicrobial resistance 2006 Poultry Science 85:1665–1669
INTRODUCTION Antimicrobial resistance is an increasingly global problem, and emerging antimicrobial resistance has become a public health issue worldwide (Kaye et al., 2004). A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in human or veterinary medicine and in food-animal production (Bager and Helmuth, 2001; Anderson et al., 2003; Schroeder et al., 2004). Though many bacteria recovered from poultry or poultry-related samples have been monitored, few published studies have reported on antimicrobial resistance in bacteria, particularly Salmonella and Escherichia coli recovered from shell eggs (Yang et al., 2002; Antunes et al., 2003; Bajaj et al., 2003; Dargatz et
©2006 Poultry Science Association Inc. Received January 5, 2006. Accepted April 6, 2006. 1 Corresponding author: [email protected]
al., 2003; Zhao et al., 2003; Busani et al., 2004; Chung et al., 2004; Del Cerro et al., 2003; Dias de Oliveira et al., 2005). Studies were conducted in 2003 to monitor microbial populations, including Salmonella and other Enterobacteriaceae, along the shell egg-processing chain (Musgrove, 2004; Musgrove et al., 2005a,b). This paper reports on the antimicrobial susceptibility profiles of E. coli and Salmonella recovered in that study.
MATERIALS AND METHODS Description of Shell Egg Processing Plants A survey of in-line egg-processing facilities in the southeastern United States was conducted. Three plants were selected for sampling on 3 separate processing days. These plants were designated as X, Y, and Z to protect the anonymity of the participating companies. A more detailed description of the plants has been previously published (Musgrove et al., 2005b).
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Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 30, 2015
ABSTRACT The development of antimicrobial resistance in bacteria has become a global problem. Isolates of Salmonella and Escherichia coli recovered from shell egg samples, collected at 3 commercial plants, were analyzed for resistance to 16 antimicrobial agents (n = 990). Eggs were sampled by rinsing in a saline solution. Pooled samples were preenriched in buffered peptone water and then selectively isolated using standard broths and agars. Salmonella-positive isolates were serogrouped immunologically before being serotyped. Enterobacteriaceae were enumerated from individual samples using violet red bile glucose agar plates. Escherichia coli were identified biochemically from presumptive Enterobacteriaceae isolates. Salmonella and generic E. coli antimicrobial-susceptibility testing was conducted using a semiautomated broth microdilution system. More resistance was observed in the
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Shell Egg-Sample Collection Eggs were collected from commercial plants at the following points of processing: at the accumulator, at prewash wetting, after the first washer, after the second washer, at the sanitizing rinse, at drying, at oiling, at check detection and weighing, at packaging (at 2 different packer head belts), at the entrance of the rewash belt, and at the exit of the rewash belt. Eggs were collected after the line had been operating for at least 2 h but during the midmorning break so as not to interfere with processing. This also allowed samples to be taken simultaneously from all sampling sites. Twelve eggs from each collection site were aseptically placed into clean foam cartons, packed into half-cases, and transported back to the laboratory.
As described in Musgrove et al. (2005b), 10 of the 12 eggs collected at each site were sampled using a shell rinse technique. Each egg was placed into a sterile WhirlPak bag (Nasco Modesto, Modesto, CA) with 10 mL of sterile PBS and rinsed by shaking for 1 min. Rinsate from the rinse and the crush method for every egg was then subjected to microbiological analyses as previously described (Musgrove et al., 2005b).
Water-Sampling Methodology Water samples were collected from the washer tanks in each of the 3 shell egg-processing plants and analyzed for pH, temperature, chlorine, protein, solids, and minerals, including iron, using procedures described previously (Northcutt et al., 2005). Samples were enriched for Salmonella. Collection procedures and other details are described in a previously published article (Northcutt et al., 2005).
Direct Plating Microbiology Microbial populations from the individual samples described above were enumerated for Enterobacteriaceae on violet red bile glucose agar (Becton, Dickinson and Co., Franklin Lakes, NJ) plates with overlay (purple-red colonies) as previously described. Presumptive colonies were counted and reported as log cfu/mL of egg rinsate. Up to 5 isolates for each positive sample were randomly selected for further analysis. An isolate from the third streak plate was saved on brain heart infusion agar slants at 37°C and Protect beads (Technical Service Consultants Ltd., Heywood, Lancashire, UK) at −20°C until further analyses for identification could be performed. Identification of Isolates A more detailed description of sampling methodology has been published previously (Musgrove, 2004). Each stored isolate was streaked onto plate count agar and incubated overnight at 37°C. A cultural suspension using 5 mL of physiological saline was prepared from each isolate. BioMe´rieux API 20 E strips (bioMe´r-
Salmonella Enrichment For each of the 12 collection sites, 2 pooled samples were formed by combining shell egg rinses or crushed shells and membranes from 5 eggs. Samples were preenriched in buffered peptone water at 35°C for 18 to 24 h, followed by enrichment in TT broth (Becton, Dickinson and Co.) and Rappaport-Vassiliadis broth (Becton, Dickinson and Co.) overnight at 42°C. Enriched samples were plated onto BG Sulfa (Becton, Dickinson and Co.) and XLT-4 (Becton, Dickinson and Co.) agar plates and incubated at 37°C for 24 h. Presumptive positive colonies were inoculated into lysine iron agar (Becton, Dickinson and Co.) and triple sugar iron slants (Becton, Dickinson and Co.) and incubated at 35°C for 18 to 24 h. Those samples giving presumptive results on each of these media were confirmed using serogrouping antisera (Becton, Dickinson and Co.). Confirmed isolates were then streaked for purity and stocked onto agar slants and ceramic beads in cryogenic protective media. A copy of each isolate was provided to the National Veterinary Services Laboratories (Ames, IA) for serotyping. A sample was recorded as positive if it was confirmed and serotyped from either the shell rinse or crushed shell and membrane composite samples.
Antibiogram Methodology Salmonella and generic E. coli were tested for antimicrobial susceptibility using a semiautomated broth microdilution system (Sensititre, TREK Diagnostic Systems Inc., Cleveland, OH). Custom-made panels of 16 antimicrobial drugs were configured in 96-well plates (National Antimicrobial Resistance Monitoring System, 2005). The minimum inhibitory concentration for each isolate was determined according to Clinical and Laboratory Standards Institute (National Committee for Clinical Laboratory Standards, 2003, 2004a,b). Breakpoint interpretations used in the National Antimicrobial Resistance Monitoring System (NARMS) were used when Clinical and Laboratory Standards Institute guidelines were not available (NARMS, 2005).
RESULTS AND DISCUSSION Since their discovery in the 1940s, antibiotics have been widely used in both human and veterinary medical practice (Salyers and Whitt, 2005). However, many important human and animal pathogens have developed resistance to these compounds (Walsh, 2003). Resistant bacteria are routinely isolated from a variety of foods, including poultry meat and eggs (NARMS, 2005). Since 1997, pathogenic and commensal organisms, such as Salmonella and E. coli,
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 30, 2015
Shell Egg-Sampling Methodologies
ieux, Marcy l’Etoile, France) were inoculated, incubated, handled, and analyzed according to the manufacturer’s instructions. Reactions were recorded, and identifications were determined using Apilab Plus software (bioMerieux).
ANTIMICROBIAL RESISTANCE IN SALMONELLA AND ESCHERICHIA COLI
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Table 1. Antimicrobial resistance patterns for Escherichia coli isolated from shell eggs at commercial shell egg processing plants (n = 194)
Antimicrobial agent None Cephalothin Cephalothin-tetracycline Gentamicin-streptomycin-sulfamethoxazole-tetracycline Gentamicin-tetracycline Streptomycin Streptomycin-tetracycline Tetracycline
No. of resistant isolates (%) 142 (73.2) 2 (1.0) 1 (0.5) 2 (1.0) 4 (2.1) 5 (2.6) 5 (2.6) 33 (17.0)
which many Salmonella isolates exhibited resistance were tetracycline (63.4%), nalidixic acid (63.4%), and streptomycin (61.0%). Few studies have reported on antimicrobial resistance of Salmonella isolates collected from eggs or the egg-processing environment. In a study conducted in India, all of the 66 Salmonella isolates were susceptible to at least one of the compounds tested (Bajaj et al., 2003). Highest resistance was observed for penicillin (96.9%), vancomycin (83.3%), and erythromycin (81.8%). Resistance was also noted for trimethoprim (42.4%), streptomycin (24.2%), tetracycline (9.0%), and gentamycin (3.0%). Bajaj et al. (2003) did not report on the serotype(s) of Salmonella isolated in their study. In the NARMS summary report for 2003, when considering all Salmonella serotypes, 27.1% were resistant to tetracycline, 0.4% were resistant to nalidixic acid, and 19.5% were resistant to streptomycin (NARMS, 2005). Figure 1 depicts the distribution of resistance in Salmonella by serotype. In a study conducted in India of 38 salmonellae isolated from eggs and egg packing materials, 95% were Salmonella Enteritidis (Suresh et al., 2005). All of the isolates were resistant to tetracycline, and 40% were resistant to nalidixic acid. There were 5,353 Salmonella isolates in the 2003 NARMS report (2005). The most frequently isolated serotypes were Kentucky (n = 555, 10.4%), Typhimurium-Copenhagen (n = 533, 10.0%), Newport (n = 483, 9.0%), Heidelberg (n = 439, 8.2%), and Typhimurium (n = 411, 7.7%). Serotypes Typhimurium, Heidelberg, and Kentucky are 3 of the most commonly isolated serotypes collected from poultry clinical and slaughter samples (NARMS, 2005), and they were the most commonly identified isolates in our study. No S. Enteritidis were recovered in the current study, whereas only 2.5% of S. Enteritidis (n = 139) was reported to NARMS in 2003 (NARMS, 2005). An Italian survey recovered antimicrobial-resistant Salmonella Typhimurium, Enteritidis, and Infantis from humans, food, and animal samples (Busani et al., 2004). In the Italian study, the highest rates of multiple resistances were detected for S. Typhimurium, as was the case with the current study. In a Turkish study involving S. Enteritidis isolated from chicken, eggs, and humans, 58 of 82 isolates (71%) were
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are monitored by the NARMS. However, little attention has been given to antimicrobial resistance of bacteria isolated from commercial shell eggs or the egg-processing environment. Antibiogram patterns for E. coli isolates are summarized in Table 1. Most of the 194 isolates in this study (73.2%) were pan susceptible. Antimicrobial agents for which many E. coli isolates exhibited resistance were tetracycline (29.9%), streptomycin (6.2%), and gentamicin (3.1%). Only 1% (n = 2) of the E. coli isolates were resistant to 4 compounds. Many E. coli isolated from meat and poultry have demonstrated resistance to at least one antimicrobial drug (Schroeder et al., 2004; NARMS, 2005). Lanz et al. (2003) analyzed E. coli isolates from veterinary clinical sources in Switzerland, including from laying hens. Of the 16 antimicrobial drugs tested, the isolates in the Swiss study were most resistant to sulfonamides, tetracycline, and streptomycin. Sulfonamides, tetracycline, and streptomycin are the oldest drugs used in infectious disease, and it is not surprising that some level of resistance would have emerged over time (Salyers and Whitt, 2005). In a NARMS summary of antimicrobial resistance in E. coli collected from a variety of species from 1998 to 2003, gentamicin resistance ranged from 14.7 to 26.5%, streptomycin resistance ranged from 35.4 to 62.2%, and tetracycline resistance ranged from 36 to 79.9% (NARMS, 2005). Levels of resistance for these compounds were considerably lower in the E. coli isolates analyzed in the present study compared with the published reports. In our study, prevalence of multiple resistances was 20.6% for 1 compound, 5.2% for 2 compounds, and 1.0% for 4 compounds. Salmonella antibiogram results are summarized in Table 2. From the perspective of total numbers, more resistance was observed in the Salmonella isolates than in the E. coli isolates. However, resistance in Salmonella is largely serotype-dependent (NARMS, 2005). Salmonella Typhimurium was the most prevalent (69.0%) serotype and demonstrated the greatest multiple resistance. Salmonella Kentucky, the least prevalent (5.0%) serotype recovered, was also the most susceptible. Although 34.1% of the Salmonella isolates was susceptible to all compounds, 60.1% was resistant to more than 11 compounds. However, multiple resistances were predominately observed among Typhimurium isolates. Antimicrobial drugs for
Figure 1. Percentage of Salmonella Typhimurium, Heidelberg, and Kentucky isolated from shell eggs and wash water collected at commercial shell egg processing plants (n = 41) resistant to 1, 2, 11, 12, or 13 antimicrobial agents.
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Table 2. Distribution of minimum inhibitory concentrations (%) for Salmonella isolated from shell eggs and water samples collected at commercial shell egg-processing plants (n = 41) Distribution of minimum inhibitory concentrations1 (%)
Drug (g/mL)
No. (%) resistant
≤0.02
0.06
0.12
0.25
0.50
1
2
4
8
16
32
64
128
256
512
Amikacin Amoxicillin/clavulanic acid Ampicillin Cefoxitin Ceftiofur Ceftriaxone Cephalothin Chloramphenicol Ciprofloxacin Gentamicin Kanamycin Nalidixic acid Streptomycin Sulfamethoxazole Tetracycline Trimethoprim/sulfa
0 (0.0) 25 (61.0) 25 (61.0) 25 (61.0) 25 (61.0) 1 (2.4) 25 (61.0) 25 (61.0) 0 (0.0) 25 (61.0) 25 (61.0) 26 (63.4) 26 (63.4) 23 (56.1) 26 (63.4) 0 (0.0)
— — — — — — — — 36.6 — — — — — — —
— — — — — — — — 2.4 — — — — — — —
— — — — 0.0 — — — 0.0 — — — — — — 51.2
— — — — 0.0 36.6 — — 58.5 24.4 — — — — — 48.8
9.8 — — 0.0 36.6 0.0 — — 2.4 14.6 — 0.0 — — — 0.0
78.0 39.0 34.1 9.8 0.0 2.4 — — 0.0 0.0 — 0.0 — — — 0.0
9.8 0.0 4.9 26.8 2.4 0.0 26.8 0.0 0.0* 0.0 — 2.4 — — — 0.0*
2.4 0.0 0.0 2.4 0.0* 0.0 12.2 29.3 0.0 0.0 — 34.1 — — 34.1 0.0
— 0.0 0.0 0.0 0.0 2.4 0.0 9.8 — 0.0* 39.0 0.0 — — 2.4* —
— 0.0* 0.0* 0.0* 61.0 36.6 0.0* 0.0* — 26.8 0.0 0.0* — 39.0 2.4 —
—* 2.4 0.0 61.0 — 19.5* 2.4 2.4 — 34.1 0.0* 0.0 36.6* 0.0 0.0 —
— 58.5 61.0 — — 2.4 58.5 58.5 — — 4.9 63.4 2.4 0.0 61.0 —
— — — — — — — — — — 56.1 — 61.0 2.4 — —
— — — — — — — — — — — — — 2.4* — —
— — — — — — — — — — — — — 26.8 — —
Asterisk indicates that the breakpoint for resistance occurs between this and the next highest concentration.
resistant to one or more antimicrobial agents, and multidrug resistances were observed in 35% of the isolates (Icgen et al., 2002). Resistance was serotype-dependent in the current study. Salmonella Typhimurium isolates demonstrated the greatest degree of multiple resistances, followed by Heidelberg isolates. In the 2003 NARMS summary, only 2.6, 0.8, and 0.1% of 5,353 isolates were resistant to 11, 12, and 13 antimicrobial drugs, respectively, over all serotypes (NARMS, 2005). However, in the current study, most of the S. Typhimurium isolates were recovered from a single shell egg-processing plant and on the same sample collection day, increasing the likelihood that the isolates would be related. However, further characterization by pulsedfield gel electrophoresis will be required to confirm this suspicion. Escherichia coli and Salmonella isolates analyzed in the current study displayed resistance to antimicrobial drugs. However, as expected, Salmonella resistance was serotypedependent. Salmonella isolates were more likely to be resistant to antimicrobial drugs and displayed a larger number of multiple resistances. This observation was influenced by the fact that many of the isolates were S. Typhimurium, a serotype that has been previously reported as exhibiting multiple resistances. These data indicate that shell eggs and shell egg processing water can harbor resistant foodborne and commensal bacteria.
ACKNOWLEDGMENTS We gratefully acknowledge the excellent technical assistance of Patsy Mason, Jordan Shaw, Susan Akins, Kathy Orr, Jovita Haro, Takiyah Ball, Sherry Turner, Stanford Jones, Jerrie Bennett, and Manju Amin. This work was assisted in part by a grant from the US Poultry & Egg Association (project no. 501), and their support is greatly appreciated.
REFERENCES Anderson, A. D., J. M. Nelson, S. Rossiter, and F. J. Angulo. 2003. Public health consequences of use of antimicrobial agents in food animals in the United States. Microb. Drug Resist. 9:373–379. Antunes, P., C. Reu, J. C. Sousa, L. Peixe, and N. Pestana. 2003. Incidence of Salmonella from poultry products and their susceptibility to antimicrobial agents. Int. J. Food Microbiol. 82:97–103. Bager, F., and R. Helmuth. 2001. Epidemiology of resistance of quinolones in Salmonella. Vet. Res. 32:285–290. Bajaj, B. K., V. Sharma, and R. L. Thakur. 2003. Prevalence and antibiotic resistance profiles of Salmonella spp. in poultry eggs. J. Food Sci. Technol. 40:682–684. Busani, L., C. Graziani, A. Battisti, A. Franco, A. Ricci, D. Vio, E. Digiannatale, F. Paterlini, M. D’Incau, S. Owczarek, A. Caprioli, and I. Luzzi. 2004. Antibiotic resistance in Salmonella enterica serotypes Typhimurium, Enteritidis and Infantis from human infections, food stuffs and farm animals in Italy. Epidemiol. Infect. 132:245–251. Chung, Y. H., Y. I. Kwon, S. Y. Kim, S. H. Kim, B. K. Lee, and Y. H. Chang. 2004. Antimicrobial susceptibilities and epidemiological analysis of Salmonella enteritidis isolates in Korea by phage typing and pulsed-field gel electrophoresis. J. Food Prot. 67:264–270. Dargatz, D. A., P. J. Fedorka-Cray, S. R. Ladely, C. A. Kopral, K. E. Ferris, and M. L. Headrick. 2003. Prevalence and antimicrobial susceptibility of Salmonella spp. isolates from US cattle in feedlots in 1999 and 2000. J. Appl. Microbiol. 95:743–761. Del Cerro, A., S. M. Soto, and M. C. Mendoza. 2003. Virulence and antimicrobial-resistance gene profiles determined by PCR-based procedures for Salmonella isolated from samples of animal origin. Food Microbiol. 20:431–438. Dias de Oliveira, S., F. Siqueira Flores, L. Ruschel dos Santos, and A. Brandelli. 2005. Antimicrobial resistance in Salmonella enteritidis strains isolated from broiler carcasses, food, human and poultry-related samples. Int. J. Food Microbiol. 97:297–305. Icgen, B., G. C. Gurakan, and G. Ozcengiz. 2002. Characterization of Salmonella enteritidis isolates of chicken, egg and human origin from Turkey. Food Microbiol. 19:375–382. Kaye, K. S., J. J. Engemann, H. S. Fraimow, and E. Abrutyn. 2004. Pathogens resistant to antimicrobial agents: Epidemiology,
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 30, 2015
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ed. NCCLS M100-S14. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA. Northcutt, J. K., M. T. Musgrove, and D. R. Jones. 2005. Chemical analyses of commercial shell egg wash water. J. Appl. Poult. Res. 14:289–295. Salyers, A. A., and D. D. Whitt. 2005. Revenge of the Microbes: How Bacterial Resistance is Undermining the Antibiotic Miracle. ASM Press, Washington, DC. Schroeder, C. M., D. G. White, and J. Meng. 2004. Retail meat and poultry as a reservoir of antimicrobial-resistant Escherichia coli. Food Microbiol. 21:249–255. Suresh, T., A. A. M. Hatha, D. Sreenivasan, N. Sangeetha, and P. Lashmanaperumalsamy. 2005. Prevalence and antimicrobial resistance of Salmonella enteritidis and other salmonellas in the eggs and egg-storing trays from retail markets of Coimbatore, South India. Food Microbiol. 23:294–299. Walsh, C. 2003. Antibiotics: Actions, Origins, Resistance. ASM Press, Washington, DC. Yang, S. J., K. Y. Park, S. H. Kim, K. M. No, T. E. Besser, H. S. Yoo, S. H. Kim, B. K. Lee, and Y. H. Park. 2002. Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: Comparison of phenotypic and genotypic resistance characterization. Vet. Microbiol. 86:295–301. Zhao, S., A. R. Datta, S. Ayers, S. Friedman, R. D. Walker, and D. G. White. 2003. Antimicrobial-resistance Salmonella serovars isolated from imported foods. Int. J. Food Microbiol. 84:87–92.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 30, 2015
molecular mechanisms, and clinical management. Infect. Dis. Clin. North Am. 18:467–511. Lanz, R., P. Kuhnert, and P. Boerlin. 2003. Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland. Vet. Microbiol. 91:37–84. Musgrove, M. T. 2004. Effects of processing on the microbiology of commercial shell eggs. PhD Dissertation. Univ. Georgia, Athens. Musgrove, M. T., D. R. Jones, J. K. Northcutt, M. A. Harrison, and N. A. Cox. 2005b. Impact of commercial processing on the microbiology of shell eggs. J. Food Prot. 68:2367–2375. National Antimicrobial Resistance Monitoring System. 2005. Subject: NARMS data. http://www.ars.usda.gov/Main/ docs.htm?docid=6750 Accessed Dec. 2005. National Committee for Clinical Laboratory Standards. 2003. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 2nd ed. NCCLS M31-A2. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA. National Committee for Clinical Laboratory Standards. 2004a. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals. 2nd ed. NCCLS M31-S1. Clinical Laboratory Standards Institute (formerly NCCLS), Wayne, PA. National Committee for Clinical Laboratory Standards. 2004b. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; 2nd
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