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We have found an avian pathogenic Escherichia coli (APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli (UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. coli strain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coli and their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.

A metal oxide sensor-based electronic nose system consisting of nine sensors was designed and used to analyze the volatile compounds emanating from beef strip loins (M. Longisimmus lumborum ) stored at 4 °C and 10 °C. Two statistical techniques, i.e., linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA) were used to develop classification models from the collected sensor signals. The performances of the developed models were validated by two different methods, i.e., leave-1-out cross validation and bootstrapping. Also, each classification model was developed using 6 and 10 features. The classification models classified meat samples based on the microbial population into “unspoiled” (microbial counts 10 cfu/g) and “spoiled” (microbial counts ≥ 6 log 10 cfu/g). For the meat samples stored at 10 °C, the highest classification accuracies obtained by LDA method with leave-1-out and bootstrapping validations were 83.8% and 89.1%, respectively. On the other hand, classification by QDA and subsequent validation by leave-1-out and bootstrapping provided highest accuracies of 81.5% and 93.2%, respectively. For samples stored at 4 °C, the LDA method provided highest classification accuracies of 80% and 86.6% using leave-1-out and bootstrapping validation, respectively. The highest classification accuracies obtained for the samples stored at 4 °C were 85% and 96% by using QDA method with leave-1-out and bootstrapping validations, respectively.

A commercially available Cyranose-320™ conducting polymer-based electronic nose system was used to analyse the headspace from fresh beef strip loins (M. Longissimus lumborum ) stored at 4° and 10 °C. The raw signals obtained from the electronic nose system were pre-processed by various signal-processing techniques to extract area-based features. Principal component analysis was subsequently performed on the processed signals to further reduce the dimensionalities. Classification models using radial basis function neural networks were developed using the extracted features. The performance of the developed models was validated using leave-1-out cross-validation method. The developed models classified meat samples stored at two storage temperatures into two groups, i.e., “unspoiled” (microbial counts 10 cfu/g) and “spoiled” (microbial counts ⩾6.0 log 10 cfu/g). Maximum total classification accuracies of 100% were obtained for both the samples stored at 10 and 4 °C. Classification models based on “Area scaled” feature showed higher accuracies than that obtained using “Area unscaled feature.”

This study investigated the growth and survival of Escherichia coli O157:H7 on minced and whole pieces of bison meat. Growth curves of native microflora, including Pseudomonas spp. and Enterobacteriaceae were also generated. A marked E. coli O157:H7 strain was inoculated onto minced and whole pieces of bison meat at an initial level of 1.5 log10 cfu g−1. The inoculated meat was stored at either 5 °C for 28 days or 10 °C for 21 days. Survival, but no growth, of E. coli O157:H7 was observed on both forms of bison meat stored at 5 °C, while significant growth of the organism was observed at 10 °C. E. coli O157:H7 counts on whole pieces were generally higher than counts observed on minced bison meat, and reached their highest population by 14 days, with a total increase of 3.36 log10 cfu g−1 on whole pieces and 2.12 log10 cfu g−1on minced bison meat stored at 10 °C. Under the same storage temperature, Pseudomonas spp. and total counts displayed similar growth patterns on both pieces and minced bison meat, while the Enterobacteriaceae showed a slower growth rate. This study showed that the growth of E. coli O157:H7 on bison meat is similar to that observed in studies of beef.

A commercially available Cyranose-320TM (Cyrano Sciences, Pasadena, CA) conducting polymer–based electronic nose system was used to analyze the volatile organic compounds emanating from vacuum-packaged beef strip loins (Longissimus Lamborum) that were repackaged simulating retail store conditions and stored at 4 and 10°C after inoculating them with Salmonella Typhimurium. Two statistical techniques, i.e., linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA), were used to develop classification models from the collected sensor signals. The models were developed for the captured signals, which were scaled and not scaled. The performances of the developed models were validated by two different methods, leave-one-out cross validation and bootstrapping. The developed models classified the meat samples based on the Salmonella population into “No Salmonella” (microbial counts

This study was carried out to determine the prevalence of some virulence characteristics associated with Salmonella isolates recovered from processed turkey carcasses in the Midwestern region of the United States. A total of 94 Salmonella isolates recovered from turkey carcasses from two processing plants (A and B) were examined to determine the prevalence of invA, pagC, and spvC genes. Bioassays also were used to evaluate aerobactin and colicin production. All isolates (100%) were positive for the presence of invA and pagC but were negative for spvC. Overall, 19.1% of all isolates tested were positive for aerobactin production, and 25.5% of all isolates were positive for colicin. Aerobactin and colicin production differed among isolates recovered from the two plants; more isolates from plant B produced these compounds. The Salmonella isolates examined in this study possess significant potential for causing human illness.

The efficacy of rinse, excision, and swab methods for the microbiological analysis of prechill turkey carcasses was investigated. Aerobic plate counts from a 50-cm2 area of the breast sampled by excision and by swabbing were compared. Escherichia coli and Salmonella recoveries were determined from turkeys sampled by a carcass rinse (CR), a modified rinse with the carcass supported in a swing (MCR), a two-site swab of 50 cm2 at the back and thigh (2S), a one-site swab of 50 cm2 beneath the wing (1S), a whole-carcass swab of the inner and outer carcass surface (WS), and excision of 25 g of neck skin tissue (NE). The effect of diluent volume (25, 50, and 100 ml) on E. coli counts from swab samples was also assessed. The aerobic plate count from breast tissue sampled by excision was greater than that by swabbing (P0.05). E. coli recoveries by the MCR method were similar to those by CR. E. coli counts from IS and WS samples were higher when swabs were stomached in 50 rather than 25 ml of diluent (P0.05). For swabs stomached in 50 ml of diluent, E. coli recoveries by the MCR, 2S, 1S, and WS methods were similar. For swabs stomached in 50 ml of diluent, Salmonella recoveries by the WS and MCR methods were higher than those by the 2S and 1S methods. Excision was more effective than swabbing for obtaining total bacterial counts from reduced turkey carcass areas. Whole-carcass sampling by rinsing or swabbing is necessary for optimum Salmonella recovery. Sampling a reduced area of the carcass is sufficient for E. coli analysis.

The current study was carried out to determine the antimicrobial resistance profiles and evaluate some molecular characteristics of a set of Salmonella and Campylobacter isolates recovered from production line turkeys in the Midwest region of the United States. A total of 94 birds identified as being positive for both Salmonella and Campylobacter spp. were selected for study. All Salmonella isolates were examined for antimicrobial resistance using the methods employed in the National Antimicrobial Resistance Monitoring System (NARMS). Campylobacter isolates were subjected to similar analysis using the Etest ® . In addition, polymerase chain reaction (PCR) was carried out to determine the presence of the antimicrobial resistance associated genes, integrase ( int1 ), class 1 integrons ( Salmonella and Campylobacter ) and a multidrug efflux pump ( Campylobacter spp.). Results from the study showed that the Salmonella and Campylobacter isolates examined displayed resistance to a number of antimicrobials, with Salmonella and Campylobacter isolates being resistant to at least three antimicrobials while some isolates showed resistances to 6 or 8 different antimicrobials. In addition, 68.1% of the Salmonella isolates tested were found to be positive for the class I integrase gene ( int1 ), 28.7% possessed a 1000 bp gene cassette and 17% possessed an 800 bp gene cassette. All Campylobacter isolates were negative for int1 , but 36.2% tested positive for the Campylobacter multidrug efflux pump (CmeB). A considerable number of Salmonella and Campylobacter isolates tested displayed varying degrees of antimicrobial resistance as well as the presence of some factors associated with the carriage and persistence of antimicrobial resistance. Similarities in the types of antimicrobial resistance observed in Campylobacter and Salmonella strains was evident. The results of this study suggest that prescribing practice at the farm level may be a factor in promoting antimicrobial resistance in more than one species of organism. Such practices may, therefore, contribute to the potential health risk for consumers should micro-organisms carrying multiple antimicrobial resistances enter the food chain. This study may be one of the first to report on the incidence of the multidrug efflux pump (CmeB) in Campylobacters recovered from processed turkeys. The antimicrobial resistance and molecular characteristics of Salmonella and Campylobacter is discussed.

Bison meat is a relatively new, emerging meat species gaining increased popularity in the US and European meat markets, but little is known of its microflora or pathogens that may be present. This study was carried out to determine the incidence of the foodborne pathogens Listeria, Salmonella, Escherichia coli/E. coli O157:H7 on slaughtered bison and to evaluate the bison slaughter process. Bison carcass sampling was carried out at monthly intervals over a period of 1 year at a Bison processing facility in the Midwestern United States. A total of 355 Bison carcasses were sampled by surface swabbing the carcasses at five points on the production line: pre-dehiding, post-evisceration, post-USDA inspection, post-washing and 24 h chilled carcass. Overall, the prevalence of Listeria spp., Salmonella spp., E. coli and E. coli O157:H7 was 18.3%, 3.94%, 38.3% and 1.13%, respectively. The prevalence of Listeria spp. at each sampling point tested was 42.24%, 18.1%, 6.03%, 1.72% and 3.77% while the prevalence of E. coli at each sampling point was: 88.79%, 73.28%, 52.59%, 56.89% and 11.3%, respectively. The data obtained suggests that current antimicrobial intervention strategies used at the plant are relatively effective in reducing Listeria and E. coli contamination on bison carcasses to some extent, however further study is required to determine the influence of current slaughter practices on carcass contamination. The data reported in this study to the authors’ knowledge is some of the first information reporting on the bacteriological status of Bison, and provides some useful baseline information for future research.

A commercially available Cyranose-320. conducting polymer-based electronic nose system was used to analyze the volatile organic compounds emanating from fresh beef strip loins (M. Longisimmus lumborum) stored at 4°C and 10°C. Two statistical techniques, i.e., linear discriminant analysis (LDA) and quadratic discriminant analysis (QDA), were used to develop classification models from the collected sensor signals. The performances of the developed models were validated by two different methods: leave-1-out cross-validation, and bootstrapping. The developed models classified meat samples based on the microbial population into “unspoiled” (microbial counts 6.0 log10 cfu/g). Overall, quadratic discriminant-based classification models performed better than linear discriminant analysis based models. For the meat samples stored at 10°C, the highest classification accuracies obtained by the LDA method with leave-1-out and bootstrapping validations were 87.10% and 85.87%, respectively. On the other hand, classification by QDA and subsequent validation by leave-1-out and bootstrapping provided highest accuracies of 87.5% and 97.38%, respectively. For samples stored at 4°C, the LDA method provided highest classification accuracies of 79.17% and 85.64% using leave-1-out and bootstrapping validation, respectively. When the QDA method was used, the highest classification accuracies obtained for the samples stored at 4°C were 87.50% and 98.48%, respectively, with leave-1-out and bootstrapping validations.

Aim: The primary aim of this study was to determine the incidence of Campylobacter spp. on turkey, presented for processing at participating production plants located in the midwest region of the United States. Methods and Results: The two participating plants were visited on a monthly basis for a period of 1 year. Sampling of carcasses was carried out using a surface swab technique. Swabs were obtained from carcasses at two points on the production line – prechill and postchill. In addition, samples of chill water were also obtained for examination. Isolation and detection of Campylobacter was carried out using enrichment in Preston broth with recovery of the organism on blood free Campylobacter selective agar (CCDA). Isolates recovered were screened and identified using the API Campy® identification system. The study found that 34·9% of all samples tested were positive for Campylobacter spp. The overall, contamination rates observed for both plants were relatively similar (39·2% for plant A and 30·6% for plant B). Differences were observed in the incidence of Campylobacter spp. on prechill vs postchill carcasses (i.e. 40·8% prechill vs 37·6% postchill for plant A and 41·8% prechill vs 19·8% postchill for plant B). Campylobacter species most often isolated included Camp. jejuni and Camp. coli. Other species recovered were Camp. fetus fetus, Camp. upsaliensis and Camp. lari. Conclusions: The incidence of Campylobacter spp. on processed poultry was relatively common. Factors such as the processing plant examined, season and the farms presenting birds for processing influenced the incidence of the pathogen. Significance and Impact of the Study: Differences were observed in the prevalence of Campylobacter spp. isolated from the two plants examined. The study suggests a seasonal prevalence of Campylobacter in the cooler months with processing conditions also influencing the overall occurrence of the organism. The incidence, isolation and detection of Campylobacter spp. from processed poultry are discussed.

Not since the emergence of enterohemorrhagic E. coli (EHEC) in the 1980s have bacteria gained such attention in the public eye, among food producers, healthcare workers and researchers worldwide. Currently, antimicrobial resistant microorganisms are now recognized as one of the most serious global health threats to both humans and animals worldwide by many national and international agencies alike including the World Health Organization (WHO), the Food and Agricultural Organization of the United Nations (FAO), World Organization for Animal Health (OIE), Centers for Disease Control (CDC), United States Department of Agriculture (USDA), and the Food and Drugs Administration (FDA) etc. These agencies all recognize the importance of antimicrobial resistance control and education in how to stem the flow of antimicrobial resistance in animal production and human health.

This study examined the individual and combined effects of the selective agents normally present in Yersinia-selective agar (i.e. cefsulodin, irgasan and novobiocin) on the growth kinetics of plasmid-bearing (P+) and plasmid-cured (P-) Yersinia enterocolitica serotype O:3 at 25 and 37 degrees C. Growth studies were carried out in pure culture, and the data obtained were subjected to linear regression analysis to determine lag phase duration(s) and growth rates of the examined strains. In general, the presence of selective agents increased the duration of the lag phase at 37 degrees C, with longer lag phases noted in all cases in which two or more selective agents were present. Growth rates in CIN broth base (CIN NA) and CIN NA plus commercial supplement (SR 109) (CIN) were faster at 37 than 25 degrees C, but in some cultures of incomplete CIN NA broth with less than three supplements added, growth tended to be faster at 25 than 37 degrees C. Generally, plasmid-bearing strains grew slower than plasmid-cured strains in most media at 37 degrees C due to virulence plasmid expression retarding growth. In some instances at 37 degrees C, it was observed that the growth rates of both plasmid-bearing and plasmid-cured strains were comparable, indicating the influence of added selective agent/s negating any effects associated with virulence plasmid expression. The effects of selective agents, incubation temperature and virulence plasmid carriage on the growth kinetics of Y. enterocolitica are discussed.

A novel PCR primer pair was used to detect the presence of cmeC in 131 Campylobacter jejuni and C. coli strains isolated from various hosts (cattle, turkeys, humans, and pigs). DNA sequence analysis revealed a high degree of genetic variation between the two species, while extremely limited genetic variation among isolates of the same species was detected.

Made available in DSpace on 2015-03-18T15:56:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-12Bitstream added on 2015-03-18T16:29:28Z : No. of bitstreams: 1 WOS000341230400114.pdf: 1415775 bytes, checksum: b12917aec8bc7f33d0431c69c4f02631 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Dean's Office College of Veterinary Medicine, Iowa State University (ISU) VPR Office, ISU Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitD(chrom), fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpV(Sakai) and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some "zoonotic-related'' STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential. State Univ Campinas UNICAMP, Inst Biol, Dept Genet Evolut & Bioagents, Bacterial Mol Biol Lab, Campinas, SP, Brazil Iowa State Univ, Coll Vet Med, Dept Vet Microbiol & Prevent Med, Ames, IA USA Adolfo Lutz Inst, Sao Paulo, Brazil CAPTAA, Unidade Pesquisa & Desenvolvimento Bastos, Inst Biol, Bastos, SP, Brazil Univ Estadual Paulista, Fac Med, Dept Clin Med, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Microbiol & Imunol, Botucatu, SP, Brazil Univ Estadual Campinas, Dept Internal Med, Campinas, SP, Brazil Univ Estadual Paulista, Fac Med, Dept Clin Med, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Microbiol & Imunol, Botucatu, SP, Brazil FAPESP: 12/05073-3 FAPESP: 13/09167-5 CAPES: PNPD/CAPES 23038.042588/2008-11 FAPESP: 12/04931-6

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a significant disease, causing extensive animal and financial losses globally. Because of the significance of this disease, more knowledge is needed regarding APEC's mechanisms of virulence. Here, we present the fully closed genome sequence of a typical avian pathogenic E. coli strain belonging to the serogroup O78.

Two-component signaling systems (TCSs) are major mechanisms by which bacteria adapt to environmental conditions. It follows then that TCSs would play important roles in the adaptation of pathogenic bacteria to host environments. However, no pathogen-associated TCS has been identified in uropathogenic Escherichia coli (UPEC). Here, we identified a novel TCS, which we termed KguS/KguR (KguS: α-ketoglutarate utilization sensor; KguR: α-ketoglutarate utilization regulator) in UPEC CFT073, a strain isolated from human pyelonephritis. kguS/kguR was strongly associated with UPEC but was found only rarely among other E. coli including commensal and intestinal pathogenic strains. An in vivo competition assay in a mouse UTI model showed that deletion of kguS/kguR in UPEC CFT073 resulted in a significant reduction in its colonization of the bladders and kidneys of mice, suggesting that KguS/KguR contributed to UPEC fitness in vivo. Comparative proteomics identified the target gene products of KguS/KguR, and sequence analysis showed that TCS KguS/KguR and its targeted-genes, c5032 to c5039, are encoded on a genomic island, which is not present in intestinal pathogenic E. coli. Expression of the target genes was induced by α-ketoglutarate (α-KG). These genes were further shown to be involved in utilization of α-KG as a sole carbon source under anaerobic conditions. KguS/KguR contributed to the regulation of the target genes with the direct regulation by KguR verified using an electrophoretic mobility shift assay. In addition, oxygen deficiency positively modulated expression of kguS/kguR and its target genes. Taken altogether, this study describes the first UPEC-associated TCS that functions in controlling the utilization of α-ketoglutarate in vivo thereby facilitating UPEC adaptation to life inside the urinary tract., Author Summary Successful colonization requires bacterial pathogens to adapt their metabolism to the conditions encountered in particular infection sites. Two-component signaling systems (TCSs) enable bacterial pathogens to sense and respond to environmental cues, thus mediating their adaptation to environmental change. Though many TCSs that have been characterized in commensal E. coli have been associated with UPEC pathogenesis, no characterized TCS has been significantly associated with UPEC strains. Here, we characterized a UPEC-associated TCS that was localized to a genomic island. This novel TCS and its target genes were involved in anaerobic utilization of α-ketoglutarate, an abundant metabolite in UPEC infection site-renal proximal tubules, thus contributed to UPEC fitness in vivo. Our results also suggest that this TCS controls a variant tricarboxylic acid (TCA) cycle, the first described in E. coli, which links the oxidative and reducing branches under anaerobic conditions. Similar TCA branches have been identified in other bacterial pathogens that have adapted to oxygen-limited, obligate anaerobic conditions, and/or intracellular carbon sources. Therefore, this study provides new insight into the adaptation of bacterial pathogens to nutrient availability in vivo and makes possible the discovery of targets for antimicrobial treatment.

SUMMARY. Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 mg of GST-Iss, and 1/12 chickens immunized with 50 mg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

Background Extraintestinal pathogenic Escherichia coli are important pathogens of human and animal hosts. Some human and avian extraintestinal pathogenic E. coli are indistinguishable on the basis of diseases caused, multilocus sequence and phylogenetic typing, carriage of large virulence plasmids and traits known to be associated with extraintestinal pathogenic E. coli virulence. Results The gene tkt1 identified by a previous signature-tagged transposon mutagenesis study, was found on a 16-kb genomic island of avian pathogenic Escherichia coli (APEC) O1, the first pathogenic Escherichia coli strain whose genome has been completely sequenced. tkt1 was present in 39.6% (38/96) of pathogenic Escherichia coli strains, while only 6.25% (3/48) of E. coli from the feces of apparently healthy chickens was positive. Further, tkt1 was predominantly present in extraintestinal pathogenic E. coli belonging to the B2 phylogenetic group, as compared to extraintestinal pathogenic E. coli of other phylogenetic groups. The tkt1-containing genomic island is inserted between the metE and ysgA genes of the E. coli K12 genome. Among different extraintestinal pathogenic E. coli of the B2 phylogenetic group, 61.7% of pathogenic Escherichia coli, 80.6% of human uropathogenic E.coli and 94.1% of human neonatal meningitis-causing E. coli, respectively, harbor a complete copy of this island; whereas, only a few avian fecal E. coli strains contained the complete island. Functional analysis showed that Tkt1 confers very little transketolase activity but is involved in peptide nitrogen metabolism. Conclusion These results suggest tkt1 and its corresponding genomic island are frequently associated with avian and human ExPEC and are involved in bipeptide metabolism.

A subset of extraintestinal pathogenic Escherichia coli is zoonotic and has developed strategies to adapt to different host-specific environments. However, the underlying mechanisms of these adaptive strategies have yet to be discerned. Here, the proteomic response of an avian pathogenic E. coli strain, which appears indistinguishable from neonatal meningitis E. coli, was compared following growth in human and avian sera to determine whether it uses the same mechanisms to overcome the antibacterial effects of sera from different host species. Proteins involved in biosynthesis of iron receptors were up-regulated under both sera, suggesting that serum, regardless of the host of origin, is an iron-limited environment. However, several proteins involved in synthesis of nucleic acids, sulfur-containing amino acids and fatty acids, were differentially expressed in response to the sera from different hosts. Mutational analysis showed that this APEC strain required nucleotide biosynthesis during incubation in human, but not avian serum, and deletion of genes involved in the biosynthesis of sulfur-containing amino acids increased its resistance to human serum. Continued investigation of the proteome of ‘zoonotic’ ExPEC strains, grown under other ‘dual’ host conditions, will contribute to our understanding of ExPEC pathogenesis and host specificity and development of effective therapies and control strategies.

The emergence of plasmid-mediated multidrug resistance (MDR) among enteric bacteria presents a serious challenge to the treatment of bacterial infections in humans and animals. Recent studies suggest that avian Escherichia coli commonly possess the ability to resist multiple antimicrobial agents, and might serve as reservoirs of MDR for human extraintestinal pathogenic Escherichia coli (ExPEC) and commensal E. coli populations. We determined antimicrobial susceptibility profiles for 2202 human and avian E. coli isolates, then sought for associations among resistance profile, plasmid content, virulence factor profile, and phylogenetic group. Avian-source isolates harbored greater proportions of MDR than their human counterparts, and avian ExPEC had higher proportions of MDR than did avian commensal E. coli. MDR was significantly associated with possession of the IncA/C, IncP1-alpha, IncF, and IncI1 plasmid types. Overall, inferred virulence potential did not correlate with drug susceptibility phenotype. However, certain virulence genes were positively associated with MDR, including ireA, ibeA, fyuA, cvaC, iss, iutA, iha, and afa. According to the total dataset, isolates segregated significantly according to host species and clinical status, thus suggesting that avian and human ExPEC and commensal E. coli represent four distinct populations with limited overlap. These findings suggest that in extraintestinal E. coli, MDR is most commonly associated with plasmids, and that these plasmids are frequently found among avian-source E. coli from poultry production systems

To understand the continuous problems that Escherichia coli O157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC of E. coli K-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of an E. coli O157:H7 parent strain to that of its isogenic flhC mutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in the flhC mutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity of E. coli O157:H7 on meat by interfering with the signal transduction pathways.

Background Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS) panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST) analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA). PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. Results The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14) with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. Conclusion The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar.

Vacuum packaged beef strip-loins (fresh and aged) were repackaged on polystyrene trays and over-wrapped with food grade cling film for the storage study. Several volatile compounds such as 3-methyl-1-butanol, 2,3-butanedione, 2-butanone, 3-hydroxy-2-butanone, acetic acid and a few hydrocarbons were detected in the headspace of these tray packaged fresh and aged beef strip loins both in the control and Salmonella typhimurium inoculated samples, in varying concentrations. These compounds were identified using manual headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography/mass spectrometry (GC-MS) over a storage period of 4 days and samples were incubated at 20°C. No naturally occurring Salmonella was present in the control samples. Hexanal (r = 0.99), carbon dioxide (r = 0.98), 3-hydroxy-2-butanone (r = 0.93) and 2-methyl propane (r = 0.95) showed positive correlations with Salmonella population for fresh beef samples. In aged beef samples, 3-methyl-1-butanol (r = 0.99), 3-hydroxy-2-butanone (r = 0.98), carbon dioxide (r = 0.98) and acetic acid (r = 0.86) showed similar trends. In fresh beef samples, F values were significant at p < 0.05 for 3-hydroxy-2-butanone and for carbon dioxide with storage time for fresh beef samples; they were significant for 3-hydroxy-2-butanone, acetic acid and carbon dioxide for aged beef samples.

SUMMARY. The prevalence of Escherichia coli, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis isolated from the feces of wild European starlings (Sturnus vulgaris) humanely trapped at a feedlot in central Kansas was assessed. All E. coli and Salmonella isolates recovered were tested for antimicrobial susceptibility using National Antimicrobial Resistance Monitoring System panels and the E. coli isolates were classified as to their content of genes associated with pathogenic E. coli of birds and cattle, including cvaC, iroN2, ompTp, hlyF2, eitC, iss, iutA, ireA, papC, stxI, stxII, sta, K99, F41, and eae. Escherichia coli O157:H7 and Mycobacterium avium subsp. paratuberculosis were not detected and Salmonella was isolated from only three samples, two of which displayed antimicrobial resistance. Approximately half of the E. coli isolates were resistant to antimicrobial agents with 96% showing resistance to tetracycline. Only one isolate was positive for a single gene associated with bovine pathogenic E. coli .A n interesting finding of this study was that 5% of the E. coli isolates tested met the criteria established for identification as avian pathogenic E. coli (APEC). Thus these findings suggest that starlings are not a significant source of Salmonella spp., Mycobacterium avium subsp. paratuberculosis, E. coli O157, or other shiga toxin–producing E. coli in this feedlot. However, they may have the potential to spread APEC, an important pathogen of poultry and a potential pathogen to human beings. RESUMEN. Presencia de Escherichia coli, Salmonella y Mycobacterium avium subesp. paratuberculosis en estorninos pintos silvestres en un lote de engorde de ganado en Kansas.

Salmonella enterica serovar Heidelberg strains are frequently associated with food-borne illness, with recent isolates showing higher rates of resistance to multiple antimicrobial agents. One hundred eighty S. enterica serovar Heidelberg isolates, collected from turkey-associated production and processing sources, were tested for antimicrobial susceptibility and compared by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis. The potential for the transfer of resistance between strains was studied by conjugation experiments. PFGE analysis using XbaI digestion identified eight clusters (based on 90% similarity), with the largest containing 71% of the isolates. Forty-two percent of the isolates were resistant to at least 1 of the 15 antimicrobial agents tested, and 4% of the isolates were resistant to 8 or more antimicrobial agents. Resistances to streptomycin (32%), tetracycline (30%), and kanamycin (24%) were most commonly detected. Interestingly, the XbaI PFGE profiles of selective multidrug-resistant strains ( n = 22) of S. enterica serovar Heidelberg from turkey-associated sources were indistinguishable from the predominant profile (JF6X01.0022) detected in isolates associated with human infections. These isolates were further differentiated into seven distinct profiles following digestion with the BlnI enzyme, with the largest cluster comprising 15 isolates from veterinary diagnostic and turkey processing environments. Conjugation experiments indicated that resistance to multiple antimicrobial agents was transferable among strains with diverse PFGE profiles.

This study was aimed at comparing the ability of conventional culture, the iQ-Check real-time PCR kit, and invA PCR to detect Salmonella in naturally contaminated premarket and retail turkey parts. Premarket (n = 120) turkey parts collected from a commercial turkey processing plant, and retail turkey parts (n = 138) were examined. Both PCR methods detected a significantly greater (P < 0.05) number of positive samples when compared with the conventional culture method for the premarket turkey parts. The indices of total agreement between the conventional culture method and the iQ-Check kit for the premarket and retail parts were 79.2% (95% CI: 70.8, 86) and 90.6% (95% CI: 84.4, 94.9), respectively. When the conventional culture method was compared with invA PCR for Salmonella detection in the premarket and retail parts, the indices of total agreement were 75.8% (95% CI: 67.2, 83.2) and 84.1% (95% CI: 76.9, 89.7), respectively. The rates of false positives (premarket: 31.9%, retail: 9.7%) and false negatives (premarket: 5.9%, retail: 9.7%) were determined between the culture method and the iQ-Check kit. When invA PCR was compared with the culture method, the rates of false positives (premarket: 37.7%, retail: 11.1%) and false negatives (premarket: 5.9%, retail: 18.3%) were obtained. The higher total agreement and the lower rates of both false positives and false negatives for the iQ-Check kit compared with invA PCR for both premarket and retail turkey parts corroborates the use of the iQ-Check kit as a screening tool for Salmonella in poultry meat.

A novel metal oxide based electronic nose system was designed and developed to detect Salmonella contamination in packaged meat (beef). This system contained an array of metal oxide detectors and custom made electronics for acquiring olfactory signature of the volatile organic compounds in the headspace of the meat packages produced by detector arrays. The acquired signals were processed using three wavelet packet transforms (WPT) for simultaneous noise reduction and compression. The coefficients of wavelet packets were used as features for developing statistical classification models using bootstrapped linear and quadratic discriminant analysis techniques. The average total classification accuracies for classifying Salmonella contaminated meat samples were higher than 82% with highest average accuracy of about 88%.

Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

SUMMARY. Colibacillosis, caused by avian pathogenic Escherichia coli (APEC) is a major problem for the poultry industry resulting in significant losses annually. Previous work in our lab and by others has shown that the increased serum survival gene (iss) is a common trait associated with the virulence of APEC. This gene was first described for its contributions to E. coli serum resistance. However, recently published research has called the contribution of iss to this trait into question. In the present study, the level of serum resistance conferred on an E. coli isolate by iss is examined. Additionally, the contribution of l bor gene to E. coli serum resistance is studied, as iss is thought to be derived from bor and bor occurs commonly among E. coli. To better understand the iss and bor contributions to serum resistance, a series of iss and bor mutants was generated. An iss deletion (iss 2 ) mutant showed a significant drop in its resistance to serum. Similarly, a bor mutant showed a drop in serum resistance but not as drastic as that observed with the iss mutant, suggesting that iss contributes more to serum resistance than bor in this E. coli strain. Also, when iss was reintroduced into the iss 2 mutant the wild-type level of serum resistance was restored, confirming that the deletion of iss was responsible for the change in resistance seen in the mutant.

The Yersinia high pathogenicity island (HPI) encodes a yersiniabactin-mediated iron acquisition system present in highly pathogenic strains of Yersinia and several members of the Enterobacteriaceae. In this study, 420 salmonellae representing multiple serovars recovered from diverse hosts were investigated for the presence of the HPI. The isolates were initially screened via PCR with primers specific for irp2, a conserved gene involved in yersiniabactin biosynthesis. Seventeen isolates produced an amplicon of the expected size. These isolates were further investigated using PCR primers spanning the HPI core region and all isolates produced identical results. HPI-positive isolates were recovered only from turkeys (feces) or production samples (feed and water) from a single flock and identified as Salmonella enterica serovar Senftenberg, a Subspecies I. Southern hybridization and genome sequencing of isolate 3-70-31 revealed that the island is plasmid-borne and is 92-97% identical to the core region in Yersinia species. To our knowledge, this is the first report of the HPI in Salmonella Subspecies I. This study illustrates the presence of the HPI in a new species, and the continued importance of poultry as a reservoir and vehicle for the dissemination of zoonotic pathogens.

Aims: To determine the antimicrobial susceptibility profiles, distribution of class 1 integrons, virulence genes and genes encoding resistance to tetracycline (tetA, tetC, tetD and tetE) and streptomycin (strA, strB and aadA1) in Salmonella recovered from turkeys. Methods and Results: The antimicrobial susceptibility of 80 isolates was determined using National Antimicrobial Resistance Monitoring System. The distribution of resistance genes, class 1 integrons and virulence genes was determined using PCR. Resistances to tetracycline (76·3%) and streptomycin (40%) were common. Sixty-two (77·5%) isolates displayed resistance against one or more antimicrobials and 33 were multi-drug resistant. tetA was detected in 72·5% of the isolates, while tetC, tetD and tetE were not detected. The strA and strB genes were detected in 73·8% of the isolates. Two isolates possessed class 1 integrons of 1 kb in size, containing the aadA1 gene conferring resistance to streptomycin and spectinomycin. Fourteen of the virulence genes were detected in over 80% of the isolates. Conclusions: This study shows that continuous use of tetracycline and streptomycin in poultry production selects for resistant strains. The Salmonella isolates recovered possess significant ability to cause human illness. Significance and Impact of the Study: Information from this study can be employed in guiding future strategies for the use of antimicrobials in poultry production.

A novel PCR primer pair was used to detect the presence of cmeC in 131 Campylobacter jejuni and C. coli strains isolated from various hosts (cattle, turkeys, humans, and pigs). DNA sequence analysis revealed a high degree of genetic variation between the two species, while extremely limited genetic variation among isolates of the same species was detected.

Vol. 73, no. 6, p. 1976-1983, 2007. Page 1982, column 2: Acknowledgments should read as follows. This project was funded, in part, by The Alliance for the Prudent Use of Antibiotics (APUA; supported by NIH grant no. U24 AI 50139) and the Iowa State University Biotechnology Council, Provost's Office

Aims: To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. Methods and Results: Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Δbor mutants, and Bor was detected on Δiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. Conclusions: These results demonstrate that Bor and Iss are exposed on E. coli’s outer membrane where they may be recognized by the host's immune system. Significance and Impact of the Study: To our knowledge, this is the first report confirming Iss’ location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.

Contaminated poultry meat has been identified as one of the principal foodborne sources of Salmonella. Molecular characterization of Salmonella is important in addressing methods to control this pathogen. Seventy-four retail turkey meat samples were collected from various stores in Fargo, North Dakota in the fall of 2003. Salmonella was recovered from 30 samples using the standard conventional culture method (FSIS, USDA). Isolated Salmonella were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) analysis, plasmid analysis, and antibiotic resistance profiling. Five serotypes were identified among the isolates: Newport (n = 12), Hadar (n = 8), Heidelberg (n = 7), 4,12:nonmotile (n = 2), and Reading (n = 1). XbaI PFGE analysis revealed 13 PFGE types and succeeded in grouping the isolates according to their serotypes. Plasmid profiling identified 5 plasmid types (with 1 or 2 plasmids) among eleven isolates that harbored plasmids. Seventeen isolates were resistant to antibiotics. The Heidelberg serotype showed resistance to multiple antibiotics: 1 isolate had resistance to gentamicin, sulfamethoxazole, and streptomycin, and 6 isolates had resistance to tetracycline, gentamycin, sulfamethoxazole, kanamycin, and streptomycin. The Hadar serotype isolates were resistant to 2 or 3 antibiotics: tetracycline and streptomycin (1 isolate); tetracycline and kanamycin (1 isolate); and tetracycline, kanamycin, and streptomycin (6 isolates). The 4,12:nonmotile serotype isolates showed resistance to tetracycline only. The Newport and the Reading serotypes were susceptible to all 16 of the antimicrobials tested.

The volatile organic compounds (VOCs) generated by microorganisms provide important information about the presence of bacteria in meat and their proliferation over time. Our previous meat spoilage and contamination experiments have used headspace of meat packages at every 24 h interval. Distribution of VOCs at different time scale (< 24 h) of packaged meat (beef) under aerobic conditions have been studied and reported here. The headspace was acquired using an automated headspace sampling system at 0, 4, 8 and 12 h (4 h intervals) from the meat samples that was spoiled at room temperature. The acquired headspace samples were analyzed by Gas Chromatography/Mass Spectroscopy (GC/MS). The temporal analysis revealed that the compounds such as carbon dioxide, acetone, acetic acid, 2-butanone-3-OH, toluene, N,N-dimethyl-formamide and hexanal were present thought-out the meat spoilage period. The microbial analysis revealed that microflora such as Pseudomonas, lactic acid bacteria and enterobacteroasese were found to be growing throughout the spoilage period.

In case of meat contamination studies, the volatile organic compounds (VOCs) generated by microorganisms provide important information about the presence of pathogenic bacteria in meat. Our current meat contaminations experiments have used headspace output of meat packages at 24 h intervals. However, it is also important to observe the VOC pattern change at every 3-4 h interval. This paper describes a specially designed automated headspace sampler to extract headspace from control and Salmonella spiked meat at user defined time intervals (4h, 8h and 12h) under anaerobic conditions. The collected headspace samples were analyzed by Gas Chromatography/Mass Spectroscopy (GC/MS) and TF-module electronic nose sensor.

In this study, whole carcasses were sampled at eight stages on a turkey-processing line and Salmonella prevalence was determined using enrichment techniques. Recovered Salmonella was further characterized using serotyping and the molecular profiles were determined using pulsed-field gel electrophoresis (PFGE). Prevalence data showed that contamination rates varied along the line and were greatest after defeathering and after chilling. Analysis of contamination in relation to serotypes and PFGE profiles found that on some visits the same serotype was present all along the processing line while on other days, additional serotypes were recovered that were not detected earlier on the line, suggesting that the birds harbored more than one serotype of Salmonella or there was cross-contamination occurring during processing. Overall, this study found fluctuations in Salmonella prevalence along a turkey-processing line. Following washing, Salmonella prevalence was significantly reduced, suggesting that washing is critical for Salmonella control in turkey processing and has significant application for controlling Salmonella at the postdefeathering and prechill stages where prevalence increased.

Aims: The aim of this study was to compare the real-time iQ-CheckTMSalmonella kit (Bio-Rad) with the immunocapture assay RapidCheck®Salmonella method, and a conventional culture method (FSIS, USDA) in detecting Salmonella in naturally contaminated turkey meat products. This study was also designed to determine if a selective enrichment step might improve the real-time detection of Salmonella. Methods and Results: Using the culture method, Salmonella was recovered from 49 out of 99 retail turkey meat samples collected. RapidCheck® failed to detect 11 Salmonella samples that were positive by the culture method. The iQ-CheckTM real-time PCR also failed to detect three samples that were positive by the culture method. However, when carried out after a selective enrichment step, the iQ-CheckTM real-time PCR detected all 49 Salmonella samples recovered by the culture method. The iQ-CheckTM real-time PCR detected the presence of Salmonella in some samples that were not recovered by the culture method. Conclusions: Adding a selective enrichment step to the iQ-CheckTM real-time PCR improves the detection of Salmonella in naturally contaminated turkey meat samples. Significance and Impact of the Study: The iQ-CheckTMSalmonella real-time PCR can be used as a rapid method to monitor Salmonella in turkey meat, together with conventional culture methodology.

The purpose of this study was to develop a multiplex polymerase chain reaction (PCR) protocol useful in the virulence genotyping of Salmonella spp. with the idea that genotyping could augment current Salmonella characterization and typing methods. Seventeen genes associated with Salmonella invasion, fimbrial production, toxin production, iron transport, and intramacrophage survival were targeted by three PCR reactions. Most of these genes are required for full Salmonella virulence in a murine model, and many are also located on Salmonella pathogenicity islands (PAIs) and are associated with type III secretion systems (TTSSs). Once the success of procedures that used positive and negative control strains was verified, the genotypes of 78 Salmonella isolates incriminated in avian salmonellosis (primarily from sick, commercially reared chickens and turkeys) and 80 Salmonella isolates from apparently healthy chickens or turkeys were compared. Eleven of the 17 genes tested (invA, orgA, prgH, tolC, spaN [invJ], sipB, sitC, pagC, msgA, spiA, and iroN) were found in all of the isolates. Another (sopB) was present in all isolates from sick birds and all but one isolate from healthy birds. The remaining five genes (lpfC, cdtB, sifA, pefA, and spvB) were found in 10%-90% of the isolates from sick birds and 3.75%-90% of the healthy birds. No significant differences in the occurrence of these genes between the two groups of isolates were detected. These results suggest that these virulence genes, and presumably the PAls and TTSSs with which they are associated, are widely distributed among Salmonella isolates of birds, regardless of whether their hosts of origin have been identified as having salmonellosis.

Seventeen Salmonella enterica serovar Hadar isolates recovered from bison were found to possess a range of virulence genes and resistance to tetracycline, gentamicin, sulfamethoxazole, and streptomycin simultaneously. A 1-kb class 1 integron containing the aadA1 gene was identified in all isolates. Pulsed-field gel electrophoresis found that all isolates were closely related, indicating the possibility of cross-contamination during processing.

Nontyphoidal salmonellae are among the leading causes of food-borne disease in the United States. Because of the importance of Salmonella enterica in food-borne disease, numerous typing methodologies have been developed. Among the several molecular typing methods, pulsed-field gel electrophoresis (PFGE) is currently considered the “gold standard” technique in typing Salmonella . The aim of this study was to compare the discriminatory power of PFGE to multilocus sequence typing (MLST) in typing Salmonella enterica serovar Typhimurium clinical isolates. A total of 85 Salmonella Typhimurium clinical isolates from cattle were used in this study. PFGE using XbaI was performed on the 85 isolates by the Centers for Disease Control and Prevention method, and data were analyzed using the BioNumerics software package. Fifty PFGE profiles were observed among the isolates, and these grouped into three major clusters. For the MLST analysis, the manB , pduF , glnA , and spaM genes were amplified by PCR from the same 85 isolates. DNA sequencing of these four genes, manB , pduF , glnA , and spaM , showed no genetic diversity among the isolates tested, with a 100% identity in nucleotide sequence. Moreover, the DNA sequences of the aforementioned genes showed 100% identity to the sequence reported in GenBank for the S. enterica serovar Typhimurium LT2 strain. Therefore, MLST, using these genes, lacks the discriminatory power of PFGE for typing Salmonella enterica serovar Typhimurium.

The prevalence of the Campylobacter multi-drug efflux pump (CmeABC) was evaluated in Campylobacter isolates recovered from freshly processed turkeys at two Midwestern processing plants. A total of 94 Campylobacter isolates recovered from processed turkeys were examined using polymerase chain reaction (PCR) to determine the presence of the multi-drug efflux pump genes cmeA, cmeB, and cmeC. Results from this study found that 51% of all isolates tested were positive for CmeABC. 46.6% of these positive isolates were from plant A and 55.1% from plant B. Differences were observed in the prevalence of individual genes found among Campylobacter isolates from each plant. Additional analysis found that among the isolates positive for CmeABC, 85.5% were identified as C. jejuni and 14.5% identified as C. coli. There was a relatively high occurrence of the Campylobacter multi-drug efflux pump genes in Campylobacter spp. recovered from processed turkeys, however, the presence of the genes could not be significantly linked to antimicrobial resistance observed in the test strains and suggests that the CmeABC genes are only one factor associated with antimicrobial resistance in Campylobacter spp.

Aim: This study was carried out to determine the survival of Escherichia coli O157:H7 and subsequent shelf life of beef subjected to subatmospheric steam at differing temperatures. Methods and Results: A specifically built, laboratory scale decontamination apparatus was used in decontamination trials to examine the effect of condensing steam at differing subatmospheric pressures on the survival of E. coli O157:H7 on meat. Beef slices were inoculated with a nontoxigenic E. coli O157:H7 strain and subjected to condensing steam at temperatures of 55, 65 and 75°C. Following treatment, the decontaminated meat was packaged and stored in air or under vacuum at temperatures of 10 or 0°C for up to 42 days. Microbiological analysis of the decontaminated and a control product (not subjected to any heat treatment) was carried out at regular intervals over the storage time of the product. Overall, significant reductions (ca 1·5 log10 CFU cm−2) in pathogen numbers were observed at a steam treatment temperature of 75°C, however, postprocess storage conditions were important in ensuring no re-growth of the pathogen and this was best achieved by storage under vacuum at 0°C. Conclusions: Steam had a significant impact in reducing E. coli O157:H7 populations, but storage conditions post-treatment were important for ensuring inhibition of the pathogen. Significance and Impact of the Study: This study indicated that subatmospheric steam could have significant application in the decontamination of meat primals postfabrication, immediately prior to packaging thus ensuring a safer product for consumers.

A method using gas chromatography-mass spectrometry (GC-MS) with headspace solid-phase microextraction (SPME) was developed for the study of raw beef storage under two different temperatures (10oC and 4oC) in refrigerators. The headspace volatiles were extracted by 75-¦Im Carboxen-Polydimethylsiloxane (CAR/PDMS) fibers in a home-made extraction chamber, and separated by an XTI-5 capillary column. By studying the relationships between the amounts of certain volatiles formed on the beef samples and the storage days, as well as the bacterial counts, a strong correction (r iÝ 0.88) existed between the peak area response of 3-hydroxy-2-butanone and the storage days at both temperatures, showing that 3-hydroxy-2-butanone can be used for a quantitative indicator compound for monitoring the raw beef spoilage.