Food Protection Trends - September/October 2016 - 358

(4, 18). Higher mortality rates are typically associated with immunocompromised persons, the elderly, pregnant women, and neonates (9). Of the recognized Listeria species, only L. monocytgenes is associated with human listeriosis outbreaks, with serotype 4b being the most commonly implicated serotype (2). Although serotypes 1/2a and 1/2b are more frequently isolated from food products, serotype 4b is more frequently isolated from clinical specimens (8). Hence, serotyping and subtyping of L. monocytogenes isolates are epidemiologically important steps in identification and classification of this pathogen during human listeriosis outbreaks as well as in routine regulatory surveillance. L. monocytogenes in food products is believed to be derived from the food processing environment despite a lack of direct evidence linking a specific foodborne L. monocytogenes strain to the food processing plant environment (7, 19). Listeria species that persist in the food processing environment may develop resistance to chemicals used in cleaning and sanitation (3, 11, 14). Therefore, persistent monitoring of this pathogen in foodrelated environments is important to prevent or minimize contamination of the final food product. In addition, microbiological environmental monitoring provides useful information for food safety control programs such as Hazard Analysis Critical Control Point (HACCP) and good manufacturing practices (GMPs) (5). The National Antimicrobial Resistance Monitoring System (NARMS) has monitored the antimicrobial resistance of all of the major foodborne pathogens, except L. monocytogenes, since 1996. L. monocytogenes is associated with high hospitalization and mortality rates, and antimicrobial resistance appears to be increasing (4, 15, 18). The contamination of food products by L. monocytogenes is a major concern to regulatory agencies and the food industry, both of which seek to minimize consumer exposure to this organism. Therefore, the purpose of this study was to genotypically and phenotypically characterize food-related environmental L. monocytogenes isolates and determine their antimicrobial resistance. MATERIALS AND METHODS Isolation and identification of Listeria spp. strains The samples were collected from food processing facilities in the U.S. during 2007 to 2011 by the Pacific Regional Laboratory-Southwest of the FDA (PRL, Irvine, CA), using FDA guidance. The guidance is available at http://www.fda.gov/Food/GuidanceRegulation/ GuidanceDocumentsRegulatoryInformation/ FoodProcessingHACCP/ucm073110.htm#app4. Nineteen L. monocytogenes strains were recovered, identified, and serotyped as described in the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) and in previous studies (1, 10). 358 Food Protection Trends September/October Bacterial cultivation and pulsed-field gel electrophoresis (PFGE) L. monocytogenes isolates were cultured in BHI broth (Difco Laboratories, Detroit, MI). Turbidity measurements and PFGE analysis for subtyping were based on the CDC standard protocol (http://www.cdc.gov/pulsenet/protocols/ pulsenet_listeria_protocol%20.pdf) as modified for a previous study (1). The restriction enzymes AscI and ApaI were used to digest the DNA plugs. Antimicrobial susceptibility assays and MIC determination Antimicrobial susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (http://www.microbiolab-bg.com/CLSI. pdf). Broth microdilution assays were used to determine the minimum inhibitory concentration (MIC) of each antimicrobial: ampicillin, cephalothin, cefoxitin, ceftriaxone, cefepime, gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, vancomycin, rifampicin, ciprofloxacin, sulfamethoxazole-trimethoprim, and chloramphenicol. Antibiotic diffusion disks (BD, Franklin Lakes, NJ) were used to confirm antimicrobial resistance of all strains. Staphylococcus aureus (ATCC 25923) and L. monocytogenes EGD-e were used as reference strains (1). Detection of genes involved in antimicrobial resistance The polymerase chain reaction (PCR) was used to detect genes conferring aminoglycoside, ß-lactam, or tetracycline resistance. Primers used in this study are shown in Table 1. Genomic DNA was extracted from resistant strains by use of the Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, CA), or plasmid DNA was extracted by a previously described method (12). Amplification reactions were done with the Taq PCR Master Mix Kit (Qiagen) and 400 nM primers (Table 1) by use of an Applied Biosystem Veriti™ 96 well thermal cycler (Life Technologies: Grand Island, NY, USA). PCR for tetM was performed under the following conditions: initially incubated at 95°C for 10 min, then subjected to 35 cycles of 95°C for 15 sec, 56°C for 15 sec, and 72°C for 30 sec, with a final extension at 72°C for 5 min. PCR conditions for other antimicrobial-resistance genes were published previously (15). RESULTS AND DISCUSSION Phenotypic and genotypic diversity of L. monocytogenes isolates Nineteen L. monocytogenes strains were recovered from food-processing environments in the U.S.: nine, six, and four strains of serotypes 1/2a, 4b, and 1/2b, respectively. L. monocytogenes isolates were grouped into 10 pulse-types by dendrogram analysis of the PFGE banding patterns from AscI restriction enzyme digestion of total DNA, using a threshold of > 90% genetic similarity among the strains within each pulse- http://www.cdc.gov/pulsenet/protocols/pulsenet_listeria_protocol%20.pdf http://www.cdc.gov/pulsenet/protocols/pulsenet_listeria_protocol%20.pdf http://www.microbiolab-bg.com/CLSI.pdf http://www.microbiolab-bg.com/CLSI.pdf http://www.fda.gov/Food/GuidanceRegulation/guidancedocumentsregulatoryinformation/foodprocessingHACCP/ucm073110.htm#app4 http://www.fda.gov/Food/GuidanceRegulation/guidancedocumentsregulatoryinformation/foodprocessingHACCP/ucm073110.htm#app4 http://www.fda.gov/Food/GuidanceRegulation/guidancedocumentsregulatoryinformation/foodprocessingHACCP/ucm073110.htm#app4

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