Surveillance of Campylobacter spp., Escherichia coli O157, Listeria monocytogenes, Salmonella spp., Shigella spp., and Methicillin-Resistant Staphylococcus aureus In Poultry

ABSTRACT

Poultry meat has been implicated as a significant source of food-borne pathogens that can cause fatal illness among consumers. This study was undertaken to determine the level of contamination of Campylobacter spp., Escherichia coli O157, Listeria monocytogenes, Salmonella spp., Shigella spp., and Methicillin-Resistant Staphylococcus aureus in poultry across 10 states in the United States. A total of 100 samples (10 from each state) were shipped to the laboratory in cool packs and were immediately screened for the following pathogens: Campylobacter spp., E. coli O157, L. monocytogenes, Salmonella spp., Shigella spp., and MRSA. Screening methods user were multiplex PCR, either of individual pathogens (Campylobacter spp., MRSA, and L. monocytogenes) or multiple pathogens in a single PCR analysis (Salmonella spp., Shigella spp., and E. coli O157). Initial reactive samples were further confirmed using a secondary PCR and cultural confirmation methods using USDA-FSIS or FDA-BAM procedures. Background micro-flora was also determined by using an IEH MPN method that simultaneously enumerates total aerobic bacteria, total coliforms, and E. coli. The background flora for chicken parts and ground chicken were as follows (MPN/g): 67 to 10^6 aerobic bacteria, 3 to 10^5 total coliforms and < 2.2 to 500 E. coli O157, Salmonella spp., L. monocytogenes, and Campylobacter spp. were detected in 37, 26, and 72 samples, respectively. None of the samples tested positive for E. coli O157, Shingella spp., or MRSA. Multiple pathogens were detected in 32% of the positive samples. Salmonella spp. incidence was higher in ground chicken that in chicken parts, while Campylobacter spp. incidence was higher in chicken parts than in ground meat. All the positive results were confirmed using the USDA-FSIS methods specific for each pathogen. Results from this study reaffirm earlier reports on the high incidence of Campylobacter and Salmonella in poultry products, despite quality control testing performed by the meat processors. A significant number of samples contained two or more pathogens, which can potentially cause serious human illness if not eliminated through proper handling and adequate cooking.

OBJECTIVE

This study was undertaken to determine the level of contamination of Campylobacter spp., Escherichia coli O157, Listeria monocytogenes, Salmonella spp., Shigella spp., and Methicillin-Resistant Staphylocuccus aureus in poultry across 10 states in the United States.

INTRODUCTION

Bacterial food borne pathogens, such as Salmonella and Campylobacter, have been associated with the highest percentage of reported outbreaks involving diarrheal illness in developed countries such as the United States. Annually, 3.2% and 1.8% of all food borne illnesses in the U.S. are attributed to Salmonella and Campylobacter, respectively. Poultry meat consumption in the United States has increased dramatically. There is thus a need to investigate the level of contamination of Campylobacter spp., Escherichia coli O157, Listeria monocytogenes, Salmonella spp., Shigella spp., and Methicillin-Resistant Staphylococcus aureus in poultry products across 10 states in the United Sates in order to understand the potential public health risks.

MATERIALS AND METHODS

Sample Collection

Fresh poultry products were obtained from retail locations in ten different states and were transported to the laboratory for analysis.

Microbiological analysis

Microbial Counts. Samples were analyzed for Aerobic Plat Count (APC), Total Coliform Count (TCC), and biotype I E. coli Count (ECC) using IEH MPN method (Molecular Epidemiology, Inc., Seattle, WA). Plates were incubated at 37ºC for 24h and subsequently enumerated.

E. coli O157 Analysis. Samples were enriched in IEH Medium (Molecular Epidemiology, Inc. Seattle, WA) and incubated at 42ºC for 24h. Following incubation, enrichments were screened using Muliplex PCR for E. coli O157, Salmonella, and Shigella (Molecular Epidemiology, Inc., Seattle, WA).

Salmonella Analysis. Samples were enriched in IEH Medium (Molecular Epidemiology, Inc., Seattle, WA) and incubated at 42ºC for 24h. Following incubation, enrichments were screened using Multiplex PCR for E. coli O157, Salmonella, and Shigella (Molecular Epidemiology, Inc., Seattle, WA). Enrichments were transferred to secondary enrichment mediums, Rappaport-Vassiliadis R10 Broth (RV) and Tetra-Thionate Broth (TT), and incubated for 24h at 42ºC and 37ºC, respectively. Following incubation, the secondary enrichments were screened again using Multiplex PCR for Salmonella (Molecular Epidemiology, Inc., Seattle, WA). Presumptive positive enrichments were streaked onto Xylose Lysine Tergitol 4 (XLT4) agar. Suspect colonies were picked from XLT4 to Tryptic Soy Agar (TSA) with 5% Sheep Blood and incubated at 37ºC for 24h. Isolated cultures were subsequently tested by the Multiplex PCR for Salmonella. Those samples displaying positive PCR results following the secondary PCR test on pure cultures were analyzed using Vitek.

Shigella Analysis. Samples were enriched in IEH Medium (Molecular Epidemiology, Inc., Seattle, WA) and incubated at 42ºC for 24h. Following incubation, enrichments were screened using Multiplex PCR for E. coli O157, Salmonella, and Shigella (Molecular Epidemiology, Inc., Seattle, WA).

Listeria monocytogenes Analysis. Samples were enriched in M7 Medium (Molecular Epidemiology, Inc., Seattle, WA) and incubated at 35ºC for 24h. Following incubation, enrichments were screened using Multiplex PCR for L. monocytogenes (Molecular Epidemiology, Inc., Seattle, WA). Enrichments were transferred to secondary enrichment medium, Fraser Broth, and incubated at 35ºC for 24h. Following incubation, the secondary enrichments were screened using Multiplex PCR for L. monocytogenes. Initial reactives were streaked to modified Oxford Listeria Agar base(MOX), and incubated at 35ºC for 24h. Following incubation, suspect colonies were re-tested using the multiplex PCR for confirmation of initial reactives. Those samples displaying positive PCR results following the secondary PCR test on pure cultures, were considered positive for L. monocytogenes.

Methicillin-Resistant Staphylococcus aureus Analysis. Samples were enriched in Brain Heart Infusion Broth (BHI) with 10% NaCl and incubated at 35ºC for 24h. Following incubation, enrichments were screened using Multiplex PCR for MRSA (Molecular Epidemiology, Inc., Seattle, WA). Initial reactives from this screening were streaked onto modified Mannitol Salt Agar (mMSA) with Cefoxitin for isolation and incubated at 35ºC for 18h. Following incubation, suspect colonies were re-tested using the multiplex PCR for confirmation of initial reactives. Those samples displaying positive PCR results following the secondary PCR test on pure cultures, were analyzed using Vitek for antibiotic susceptibility. Samples exhibiting antibiotic resistant, and identified as S. aureus were considered positive Methicillin-Resistant Staphylococcus aureus.

Campylobacter Analysis. Samples were enriched in Bolton Broth with antibiotic supplement, incubated at 35ºC for 48 h under micro-aerophilic conditions. Following enrichment, all samples were screened using a multiplex PCR reaction designed to detect Campylobacter spp. (Molecular Epidemiology, Inc.). Initial reactives from this screening were streaked onto modified Charcoal Cefaperazone Desoxycholate Agar (mCCDA) plates for isolation and incubated at 35ºC for 48h under microa-erophilic conditions. Following incubation, suspect colonies were re-tested using the multiplex PCR for confirmation of initial reactives. Those samples displaying positive PCR results following the secondary PCR test on pure cultures, were considered positive for Campylobacter.

Pulsed Field Gel Electrophoresis (PFGE). Samonella isolates and Listeria monocytogenes isolates were analyzed by PFGE performed according to CDC Pulse Net protocol for molecular subtyping of Salmonella and Listeria monocytogenes with slight modifications (Specifications for PFGE Molecular Epidemiology, Inc., Seattle, WA). PFGE gel images were analyzed with BioNumerics software (Applied Maths, Inc., Texas, USA). For cluster analysis we used the Dice similarity coefficient to calculate pattern similarity (with optimization 0.5%, and position tolerance 1%), and UPGMA (Unweighted Pair Group Method with Arithmetic mean) algorithm to construct a dendrogram.

Figure 1. Microbial populations of chicken parts and ground chicken

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Figure 2. Incidence of Salmonella, Listeria monocytogenes, and Campylobacter on/in chicken parts and ground chicken

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Figure 3.Incidence of Salmonella, Listeria monocytogenes, and Campylobacter on store-bought chicken by State of purchase

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Table 1. Antibiotic resistance of analysis of Salmonella isolates from chicken parts and ground chicken

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Figure 4. Relatedness of Listeria isolates analyzed by pulsed-field gel electrophoresis using Apal restriction enzyme

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Figure 5. Relatedness of Salmonella isolates analyzed by pulsed-field gel electrophoresis using Xbal restriction enzyme

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CONCLUSIONS

  • E. coli loads on ground chicken was 1.2 log CFU/g, which is below the National Baseline average of 2.46 log CFU/g
  • None of the samples tested positive for E. coli O157, Shingella spp., or MRSA
  • Incidence rates of Salmonella spp,. L. monocytogenes, and Campylobacter spp. on chicken meats were 37%, 26%, and 72% respectively.
  • Multiple pathogens were detected in 32% of the positive samples Salmonella spp. incidence was higher in ground chicken (54%) than in chicken parts (20%)
  • Campylobacter spp. incidence was higher in chicken parts (86%) than in ground chicken (58%)
  • Sixteen DNA restriction patterns were observed by PFGE analysis of Apal digest of 26 L. monocytogenes isolates. Except for having the same pattern K (see Fig. 4), the ground isolates showed patterns distinguishable from those of the chicken parts. There were three ground chicken isolates in MA, GA, and FL sharing the same pattern E (see Fig.4)
  • Twenty DNA restriction patterns were observed by PFGE analysis of Xbal digest of 34 Salmonella isolates. Six Salmonella isolates from ground chicken and two Salmonella isolates from chicken parts in IL, MA, CA, Fl, and NY had the same pattern E (see Fig. 5)
  • Antibiotic susceptibility testing by Vitek on Salmonella isolates showed 4 multiple-drug resistant strains isolated from ground chicken and 1 multiple drug resistant strain isolated from chicken parts. These strains had distinguishable PFGE patterns.