Beilei Ge

Prevalence of Campylobacter spp., Escherichia coli, and Salmonella Serovars in Retail Chicken, Turkey, Pork, and Beef from the Greater Washington, D.C., Area

ABSTRACT A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence of Escherichia coli and Salmonella serovars, and 719 of these samples were also tested for Campylobacter spp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples (n = 184) were contaminated withCampylobacter, and a large percentage of the stores visited (91%) had Campylobacter-contaminated chickens. Approximately 14% of the 172 turkey samples yieldedCampylobacter, whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722Campylobacter isolates were obtained from 159 meat samples; 53.6% of these isolates were Campylobacter jejuni, 41.3% were Campylobacter coli, and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yielded E. coli, while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive for E. coli. However, only 25 (3.0%) of the retail meat samples tested were positive for Salmonella. Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.

Identification and Characterization of Integron-Mediated Antibiotic Resistance among Shiga Toxin-Producing Escherichia coli Isolates

ABSTRACT A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons. Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes. Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed. Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM. The majority of the amplified integron fragments were 1 kb in size with the exception of one E. coli O111:H8 isolate which possessed a 2-kb amplicon. DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin. Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similaraadA gene. However, DNA sequencing revealed only 86 and 88% homology, respectively. The 2-kb integron of the E. coli O111:H8 isolate contained three genes, dfrXII,aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found inCitrobacter freundii and Klebsiella pneumoniae. Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E. coli O157:H7 and to several strains of Hafnia alvei. To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E. coliO157:H7.

Antimicrobial-Resistant Campylobacter Species from Retail Raw Meats

ABSTRACT The antimicrobial susceptibilities of 378 Campylobacter isolates were determined. Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%). Campylobacter coli displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than Campylobacter jejuni, and Campylobacter isolates from turkey meat showed a greater resistance than those from chicken meat.

Role of Efflux Pumps and Topoisomerase Mutations in Fluoroquinolone Resistance in Campylobacter jejuni and Campylobacter coli

ABSTRACT Point mutations in the topoisomerase (DNA gyrase A) gene are known to be associated with fluoroquinolone resistance in Campylobacter. Recent studies have shown that an efflux pump encoded by cmeABC is also involved in decreased susceptibilities to fluoroquinolones, as well as other antimicrobials. Genome analysis suggests that Campylobacter jejuni contains at least nine other putative efflux pumps. Using insertional inactivation and site-directed mutagenesis, we investigated the potential contributions of these pumps to susceptibilities to chloramphenicol, ciprofloxacin, erythromycin, and tetracycline in C. jejuni and Campylobacter coli. Insertional inactivation of cmeB resulted in 4- to 256-fold decreases in the MICs of chloramphenicol, ciprofloxacin, erythromycin, and tetracycline, with erythromycin being the most significantly affected. In contrast, inactivation of all other putative efflux pumps had no effect on susceptibility to any of the four antimicrobials tested. Mutation of gyrA at codon 86 (Thr-Ile) caused 128- and 64-fold increases in the MICs of ciprofloxacin and nalidixic acid, respectively. The replacement of the mutated gyrA with a wild-type gyrA allele resulted in a 32-fold decrease in the ciprofloxacin MIC and no change in the nalidixic acid MIC. Our findings indicate that CmeABC is the only efflux pump among those tested that influences antimicrobial resistance in Campylobacter and that a point mutation (Thr-86-Ile) in gyrA directly causes fluoroquinolone resistance in Campylobacter. These two mechanisms work synergistically in acquiring and maintaining fluoroquinolone resistance in Campylobacter species.

Current Trends in Detecting Non-O157 Shiga Toxin–Producing Escherichia coli in Food

Non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC) strains are increasingly recognized as important foodborne pathogens worldwide. Together with E. coli O157:H7, six additional STEC serogroups (O26, O45, O103, O111, O121, and O145) are now regulated as adulterants in certain raw beef products in the United States. However, effective detection and isolation of non-O157 STEC strains from food matrices remain challenging. In the past decade, great attention has been paid to developing rapid and reliable detection methods for STEC in general (targeting common virulence factors) and specific STEC serogroups in particular (targeting serogroup-specific traits). This review summarizes current trends in detecting non-O157 STEC in food, including culture, immunological, and molecular methods, as well as several novel technologies.

Retrospective Analysis of Salmonella, Campylobacter, Escherichia coli, and Enterococcus in Animal Feed Ingredients

The presence and antimicrobial susceptibility of foodborne pathogens and indicator organisms in animal feed are not well understood. In this study, a total of 201 feed ingredient samples (animal byproducts, n=122; plant byproducts, n=79) were collected in 2002 and 2003 from representative rendering plants and the oilseed (or cereal grain) industry across the United States. The occurrence and antimicrobial susceptibility of four bacterial genera (Salmonella, Campylobacter, Escherichia coli, and Enterococcus) were determined. Salmonella isolates were further characterized by serotyping and pulsed-field gel electrophoresis (PFGE). None of the samples yielded Campylobacter or E. coli O157:H7, whereas Salmonella, generic E. coli, and Enterococcus were present in 22.9%, 39.3%, and 86.6% of samples, respectively. A large percentage (47.8%) of Salmonella-positive samples harbored two serovars, and the vast majority (88.4%) of Enterococcus isolates were E. faecium. Animal byproducts had a significantly higher Salmonella contamination rate (34.4%) than plant byproducts (5.1%) (p<0.05). Among 74 Salmonella isolates recovered, 27 serovars and 55 PFGE patterns were identified; all were pan-susceptible to 17 antimicrobials tested. E. coli isolates (n=131) demonstrated similar susceptibility to these antimicrobials except for tetracycline (15.3% resistance), sulfamethoxazole (7.6%), streptomycin (4.6%), ampicillin (3.8%), and nalidixic acid (1.5%). Enterococcus isolates (n=362) were also resistant to five of 17 antimicrobials tested, ranging from 1.1% to penicillin to 14.6% to tetracycline. Resistance rates were generally higher among isolates recovered from animal byproducts. Taken together, our findings suggest that diverse populations of Salmonella, E. coli, and Enterococcus are commonly present in animal feed ingredients, but antimicrobial resistance is not common. Future large-scale studies to monitor these pathogenic and indicator organisms in feed commodities is warranted.

Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce.

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 105 to 106 CFU per 25 g (i.e., 103 to 104 CFU per g) in produce, except for strains harboring the stx 2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx 2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.

Multilaboratory validation of a luminex microbead-based suspension array for the identification of the 11 most clinically relevant Shiga toxin-producing Escherichia coli O serogroups.

Rapid and high-throughput identification and serotyping of Shiga toxin-producing Escherichia coli (STEC) O serogroups is important for detecting, investigating, and controlling STEC infection outbreaks and removing hazardous products from commerce. A Luminex microbead-based suspension array has been developed to identify the 11 most clinically relevant STEC serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. Here we present results of a blinded multilaboratory collaborative study involving 10 participants from nine laboratories using 55 unknown strains. From the total 495 analyses, two false-positive and three false-negative results were obtained, indicating the assay to be a rapid, high-throughput, and robust method for identifying clinically relevant STEC serogroups.

Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce

Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.

Validation of a Salmonella loop-mediated isothermal amplification assay in animal food

Loop-mediated isothermal amplification (LAMP) has emerged as a promising alternative to PCR for pathogen detection in food testing and clinical diagnostics. This study aimed to validate a Salmonella LAMP method run on both turbidimetry (LAMP I) and fluorescence (LAMP II) platforms in representative animal food commodities. The U.S. Food and Drug Administration (FDA)s culture-based Bacteriological Analytical Manual (BAM) method was used as the reference method and a real-time quantitative PCR (qPCR) assay was also performed. The method comparison study followed the FDAs microbiological methods validation guidelines, which align well with those from the AOAC International and ISO. Both LAMP assays were 100% specific among 300 strains (247 Salmonella of 185 serovars and 53 non-Salmonella) tested. The detection limits ranged from 1.3 to 28 cells for six Salmonella strains of various serovars. Six commodities consisting of four animal feed items (cattle feed, chicken feed, horse feed, and swine feed) and two pet food items (dry cat food and dry dog food) all yielded satisfactory results. Compared to the BAM method, the relative levels of detection (RLODs) for LAMP I ranged from 0.317 to 1 with a combined value of 0.610, while those for LAMP II ranged from 0.394 to 1.152 with a combined value of 0.783, which all fell within the acceptability limit (2.5) for an unpaired study. This also suggests that LAMP was more sensitive than the BAM method at detecting low-level Salmonella contamination in animal food and results were available 3days sooner. The performance of LAMP on both platforms was comparable to that of qPCR but notably faster, particularly LAMP II. Given the importance of Salmonella in animal food safety, the LAMP assays validated in this study holds great promise as a rapid, reliable, and robust method for routine screening of Salmonella in these commodities.