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Little information has been published on the microbiological aspects of U.S. commercial duck processing. The objective of this study was to measure prevalence and/or levels of bacteria in duck samples representing the live bird and partially or fully processed oven-ready duck meat. At 12 monthly sampling times, samples were collected at six sites along the processing line in a commercial duck slaughter plant. Crop and cecum samples were collected at the point of evisceration. Whole carcass rinse samples were collected before and after carcass immersion chilling plus application of an antimicrobial spray. Leg quarters were collected from the cut-up line before and after application of an antimicrobial dip treatment. All samples (five from each site per monthly replication) were directly plated and/or enriched for Salmonella and Campylobacter. For the last 10 replications, carcass and leg quarter rinse samples were also evaluated for enumeration of total aerobic bacteria, Escherichia coli, and coliforms. Most cecum, crop, and prechill carcass rinse samples were positive for Campylobacter (80, 72, and 67%, respectively). Carcass chilling and chlorinated spray significantly lowered Campylobacter prevalence (P < 0.01), and even fewer leg quarters were positive for Campylobacter (P < 0.01). Passage through a chlorinated dip did not further reduce Campylobacter prevalence on leg quarters. Salmonella was infrequently found in any of the samples examined (≤10%). Total aerobic bacteria, coliforms, and E. coli levels were reduced (P < 0.01) on whole carcasses by chilling but were not different after cut-up or leg quarter dip treatment. Overall, current commercial duck processing techniques as applied in the tested plant were effective for reducing the prevalence and levels of Campylobacter on duck meat products. HIGHLIGHTS

Foodborne campylobacteriosis has been linked to undercooked chicken liver. We have detected Campylobacter in chicken livers available at retail. The objective of the current project was to determine the prevalence and subtype of Campylobacter associated with livers and ceca of the same broiler carcasses at commercial slaughter. Within 2 min of commercial evisceration, we collected liver and ceca of one broiler carcass from each of 70 discreet flocks over a 12-mo period. Liver surface, liver internal tissue, and cecal contents were cultured for Campylobacter using standard methods. One example of the predominant colony type was selected from each positive sample for whole genome sequencing and multilocus sequence typing. We detected Campylobacter in at least one sample from 58 of 70 (83%) carcasses/flocks; 41 ceca, 57 liver surface samples, and 19 liver internal tissue samples were positive. For 11 of 18 carcasses from which all samples were positive, the predominant colony types were indistinguishable. However, some carcasses did have multiple subtypes of Campylobacter. Of carcasses with Campylobacter on the surface of the liver and within the ceca, it was more likely that the subtypes be the same than different (P 0.05). We detected different subtypes of Campylobacter from internal liver tissue and liver surface of seven broiler carcasses/flocks. Livers from a large percentage of broiler carcasses/flocks can have one or more subtypes of Campylobacter.

We performed whole-genome multi-locus sequence typing for 2554 genes in a large and heterogenous panel of 180Listeria monocytogenesstrains having diverse geographical and temporal origins. The subtyping data was used for characterizing genetic variation and evaluating patterns of linkage disequilibrium in the pan-genome ofL. monocytogenes. Our analysis revealed the presence of strong linkage disequilibrium inL. monocytogenes, with ∼99% of genes showing significant non-random associations with a large majority of other genes in the genome. Twenty-seven loci having lower levels of association with other genes were considered to be potential “hot spots” for horizontal gene transfer (i.e., recombination via conjugation, transduction, and/or transformation). The patterns of linkage disequilibrium inL. monocytogenessuggest limited exchange of foreign genetic material in the genome and can be used as a tool for identifying new recombinant strains. This can help understand processes contributing to the diversification and evolution of this pathogenic bacteria, thereby facilitating development of effective control measures.

We performed whole-genome multi-locus sequence typing for 2554 genes in a large and heterogenous panel of 180Listeria monocytogenesstrains having diverse geographical and temporal origins. The subtyping data was used for characterizing genetic variation and evaluating patterns of linkage disequilibrium in the pan-genome ofL.monocytogenes. Our analysis revealed the presence of strong linkage disequilibrium inL.monocytogenes, with ~99% of genes showing significant non-random associations with a large majority of other genes in the genome. Twenty-seven loci having lower levels of association with other genes were considered to be potential “hot spots” for horizontal gene transfer (i.e., recombination via conjugation, transduction, and/or transformation). The patterns of linkage disequilibrium inL.monocytogenessuggest limited exchange of foreign genetic material in the genome and can be used as a tool for identifying new recombinant strains. This can help understand processes contributing to the diversification and evolution of this pathogenic bacteria, thereby facilitating development of effective control measures.

Chicken has recently overtaken beef as the most-consumed meat in the United States. The growing popularity of chicken is accompanied by frequent occurrences of foodborne pathogens and increasing concerns over antibiotic usage. Our study represents a proof-of-concept investigation into the possibility and practicality of leveraging microbiome-informed food safety and quality. Through a longitudinal and cross-sectional survey, we established the chicken microbiome as a robust and multifaceted food microbiology attribute that could provide a variety of safety and quality information and retain systematic signals characteristic of overall processing environments., Microorganisms and their communities on foods are important determinants and indicators of food safety and quality. Despite growing interests in studying food and food-related microbiomes, how effective and practical it is to glean various food safety and quality information from food commodity microbiomes remains underinvestigated. Microbiomes of retail chicken breast from 4 processing establishments in 3 major U.S. broiler production states displayed longitudinal consistency over 7 months and cross-sectional distinctiveness associated with individual processing environments. Packaging type and processing environment but not antibiotic usage and seasonality affected composition and diversity of the microbiomes. Low abundances of antimicrobial resistance genes were found on chicken breasts, and no significant resistome difference was observed between antibiotic-free and conventional products. Benchmarked by culture enrichment, shotgun metagenomics sequencing delivered sensitive and specific detection of Salmonella enterica from chicken breasts. IMPORTANCE Chicken has recently overtaken beef as the most-consumed meat in the United States. The growing popularity of chicken is accompanied by frequent occurrences of foodborne pathogens and increasing concerns over antibiotic usage. Our study represents a proof-of-concept investigation into the possibility and practicality of leveraging microbiome-informed food safety and quality. Through a longitudinal and cross-sectional survey, we established the chicken microbiome as a robust and multifaceted food microbiology attribute that could provide a variety of safety and quality information and retain systematic signals characteristic of overall processing environments.

A reliable and standardized classification of Listeria monocytogenes is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS)-based approaches for strain characterization are either difficult to standardize, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool, Haplo-ST, to improve standardization and provide maximum discriminatory potential to WGS data tied to a multilocus sequence typing (MLST) framework. Haplo-ST performs whole-genome MLST (wgMLST) for L. monocytogenes while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user-specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, and (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely used BIGSdb-Lm database. Tests of Haplo-ST’s performance with simulated reads from L. monocytogenes reference strains demonstrated high sensitivity (97.5%), and coverage depths of ≥20× were found to be sufficient for wgMLST profiling. We then used Haplo-ST to characterize and differentiate between two groups of L. monocytogenes isolates derived from the natural environment and poultry processing plants. Phylogenetic reconstruction identified lineages within each group, and no lineage specificity was observed with isolate phenotypes (transient versus persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of L. monocytogenes within poultry processing plants. IMPORTANCE We have developed an open-source tool (https://github.com/swarnalilouha/Haplo-ST) that provides allele-based subtyping of L. monocytogenes isolates at the whole-genome level. Along with allelic profiles, this tool also generates allele sequences and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low-resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of L. monocytogenes isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlight the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates in studies of bacterial population genetics.

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first derive a stable source of inoculum. Subsequently, we compared microbiome development between chicks receiving this passaged cecal material, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa were identified across treatment types that may inform future studies aimed at identifying strains associated with beneficial phenotypes.

A reliable and standardized classification of Listeria monocytogenes (Lm) is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS) based approaches for strain characterization either lack standardization, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool Haplo-ST to improve standardization and provide maximum discriminatory potential to WGS data tied to an MLST (multi locus sequence typing) framework. Haplo-ST performs whole-genome MLST (wgMLST) for Lm while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely-used BIGSdb-Lm database. Tests on Haplo-ST’s performance with simulated reads from Lm reference strains yielded a high sensitivity of 97.5%, and coverage depths of ≥ 20× was found to be sufficient for wgMLST profiling. We used Haplo-ST to characterize and differentiate between two groups of Lm isolates, derived from the natural environment and poultry processing plants. Phylogenetic reconstruction showed sharp delineation of lineages within each group and no lineage-specificity was observed with isolate phenotypes (transient vs. persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of Lm within poultry processing plants.IMPORTANCEWe have developed an open-source tool that provides allele-based subtyping of Lm isolates at the whole genome level. Along with allelic profiles, this tool also generates allele sequences, and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of Lm isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlights the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates for routine surveillance, outbreak investigations and source tracking.

Bactivory by protozoa is a major factor that limits the number of bacteria in nature and may control the presence of Listeria monocytogenes. The effectiveness of Tetrahymena pyriformis destruction of L. monocytogenes was measured. Within 1 hr, 35–40 T. pyriformis cells ingested an average of 1,219 CFU of L. monocytogenes. Gentamicin was then added to kill un‐ingested Listeria. In 24 hr, the recoverable bacteria were reduced at an exponential rate to undetectable levels (

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

SUMMARY Campylobacter is a human food-borne pathogen frequently associated with poultry and poultry products and can be found in high numbers within the gut contents of colonized broilers. The number of Campylobacter on broiler carcasses increases markedly during automated defeathering as rubber picker fingers press on the abdomen and can force gut contents out of the cloaca. One method to prevent this increase is to plug the cloaca and prevent escape of gut contents. For a plug to be effective, it must resist expulsion during picking. In the current work, we test shredded sponge or paper as a means to plug broiler carcass cloacae during automated feather removal. Broiler carcass cloacae were plugged before scalding and carcasses were sampled to determine the bacterial numbers present on breast skin after scalding (before feather picking) and again after feather picking. Neither 50 cc of shredded paper nor 25 cc of shredded sponge was effective as a cloacal plug. However, a plug made of 50 cc shredded sponge material was effective to occlude broiler carcass vents during automated defeathering. Significantly fewer Campylobacter cells were detected on the breast skin of post pick carcasses plugged with 50 mL of shredded sponge than on post pick unplugged control carcasses.

Foodborne campylobacteriosis has been traced to undercooked chicken liver. The objectives of this study were to determine the prevalence of Campylobacter associated with chicken livers at retail and to determine which subtypes are detected on the surface and in the internal tissues of the livers. Fifteen packages of fresh chicken livers, each representing a unique combination of processing plant and sell-by date, were collected at each of three retail grocery stores in Georgia. Three intact, undamaged livers per container ( n = 45) were selected and sampled using each three methods: outside swab, inside swab accessed by pressing through a heat-sterilized outer surface, and whole liver blended in enrichment broth. Each liver sample with 0.1 mL of exudate from packages was cultured for Campylobacter by plating on Campy-Cefex agar. The most prevalent Campylobacter colony type from each positive sample was subjected to whole genome sequencing and multilocus sequence typing. Campylobacter was detected in at least one sample from every package. Surface swabs were positive for 29 of 45 livers, but significantly fewer swabs of internal tissue were positive, 14 of 45 ( P < 0.01). Campylobacter was detected in 30 of 45 blended whole liver samples. Multiple subtypes were detected from eight livers. In four livers, a different subtype was dominant on the surface than was dominant internally. In one liver, three subtypes were detected. Various subtypes of Campylobacter can be readily isolated from fresh retail chicken livers; therefore, undercooked chicken livers pose a food safety risk.

Campylobacter can be difficult to recover from complex samples due to overgrowth by background bacteria. A 0.45- or 0.65-μm-pore-size filter overlaid on agar plates can be used as a means to separate Campylobacter from confounding non-Campylobacter cells, facilitating detection on solid plating media. It is unclear what percentage of cells in a Campylobacter suspension passes through a filter and results in visible colonies. The objective of this study was to compare the number of Campylobacter cells detected by the filter method with those detected by direct plating and determine if the filter method can be used to estimate cellular density of an unknown Campylobacter in suspension. Overnight liquid cultures of six subtypes of Campylobacter jejuni and six of Campylobacter coli, all originally detected in chicken samples, were used for this study. Motility of isolates was tested by using a stab into soft agar, incubating plates, and measuring colony size. Each subtype was applied to Campy-Cefex agar directly and through a 0.45- or 0.65-μm-pore-size filter. Filters were removed, plates were incubated, and colonies were counted; three replications were conducted. Mean recovery by direct plating was 8.3 log CFU/mL. Regardless of pore size, the overall mean number of Campylobacter detected by using the filter method was significantly less than that using direct plating (P < 0.05). The mean difference between direct plating and plating though a 0.65-μm-pore-size filter for motile Campylobacter was log 2.4 CFU/mL, with a 95% confidence interval of ±0.2 log CFU/mL.

Campylobacter is associated with live broilers and chicken meat products. There is some discussion in the literature about the possibility that Campylobacter prevalence in broilers could be affected by season or weather conditions. The objective of this study was to measure the flock prevalence of Campylobacter by sampling cecal contents from multiple flocks in one commercial slaughter plant over the course of 3 years. Two-hundred-and eleven discrete cecal samples, each from a different flock, were cultured for Campylobacter. Weather data, collected daily at a nearby University of Georgia experiment station, was used for testing for potential relationships between environmental conditions and Campylobacter detection. Fifty-five percent of flocks were found to be Campylobacter-positive. No clear trend was uncovered for Campylobacter prevalence related to mo of yr or daily maximum temperature. Furthermore, no significant relationship was noted between prevalence of Campylobacter and rainfall on the d of slaughter (P = 0.52) or the total rainfall during the grow-out period (P = 0.37).

Sixteen sites in the watershed of the South Fork of the Broad River (SFBR) in Northeastern Georgia, USA, were sampled in two seasons to detect Campylobacter. Sites were classified as mostly influenced by forest, pasture, wastewater pollution control plants (WPC) or mixed use. Sampling was repeated in the late spring and late fall for 2 years for a total of 126 samples. Free-catch water and sediment grab samples were taken at each site; Moore's swabs were placed for up to 3 days at most sites. A total of 56 isolates of thermophilic Campylobacter were recovered. Thirteen samplings were positive by two or three methods, and 26 samplings were positive by only one method; once by Moore's swab only and 25 times by free-catch water only. Campylobacter was detected at 58% of cattle pasture sites, 30% of forested sites and 81% of WPC sites. Twenty-one of the isolates carried antimicrobial resistance genes, mostly blaOXA-61. Free-catch water samples were more efficient than Moore's swabs or sediment samples for recovery of Campylobacter, which was more likely to be detected in streams near cattle pastures and human communities than in forested land. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of environmental water in transmitting Campylobacter was investigated, and methods for recovery of the organism were compared. The sequence types of recovered Campylobacter correlated with adjacent land use without regard to the method used to isolate the organisms. Sequence types and antimicrobial resistance genes associated with cattle were most prevalent near pastures. Even though types were recurrent at a given site, types appeared to be lost or replaced as the water flowed downstream.

Bed sediments of streams and rivers may store high concentrations of fecal indicator bacteria (FIB) and pathogens. Due to resuspension events, these contaminants can be mobilized into the water column and affect overall water quality. Other bacterial indicators such as microbial source tracking (MST) markers, developed to determine potential sources of fecal contamination, can also be resuspended from bed sediments. The primary objective of this study was to predict occurrence of waterborne pathogens in water and streambed sediments using a simple statistical model that includes traditionally measured FIB, environmental parameters and source allocation, using MST markers as predictor variables. Synoptic sampling events were conducted during baseflow conditions downstream from agricultural (AG), forested (FORS), and wastewater pollution control plant (WPCP) land uses. Concentrations of FIB and MST markers were measured in water and sediments, along with occurrences of the enteric pathogens Campylobacter, Listeria and Salmonella, and the virulence gene that carries Shiga toxin, stx2. Pathogens were detected in water more often than in underlying sediments. Shiga toxin was significantly related to land use, with concentrations of the ruminant marker selected as an independent variable that could correctly classify 76% and 64% of observed Shiga toxin occurrences in water and sediment, respectively. FIB concentrations and water quality parameters were also selected as independent variables that correctly classified Shiga toxin occurrences in water and sediment (54%–87%), and Salmonella occurrences in water (96%). Relationships between pathogens and indicator variables were generally inconsistent and no single indicator adequately described occurrence of all pathogens. Because of inconsistent relationships between individual pathogens and FIB/MST markers, incorporating a combination of FIB, water quality measurements, and MST markers may be the best way to assess microbial water quality in mixed land use systems.

Salmonella Kentucky has become the predominant serovar recovered from broilers slaughtered in the United States, and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serovar. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 Salmonella Kentucky isolates recovered from chicken carcasses from 2004 to 2013. Pulsed-field gel electrophoresis cluster analysis revealed 112 unique types sharing 79% similarity. Over half of the isolates studies were assigned to two large clusters (unique restriction patterns) consisting of 190 (A) and 151 (B) isolates. The remaining (n = 259) more diverse isolates (110 unique patterns) shall be designated cluster C for discussion. Clusters A had significantly more (p

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first determine if serial transfers exploiting the ceca in vivo, rather than the external environment or artificial incubations, can produce a stable microbial community. Subsequently, we compared microbiome development between chicks receiving this passaged, i.e. host-selected, cecal material orally, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa identified across treatment types may inform future studies aimed at identifying strains associated with beneficial phenotypes.

Broilers can carry Campylobacter in their intestine during live haul transport to the slaughter plant; feces from positive birds will contaminate the floor surface of transport containers. Such contamination can be transferred to previously negative broilers when transported in unwashed cages. We tested the use of a water rinse and 15 s steam heat to lessen the numbers of coliforms, Escherichia coli and Campylobacter within broiler intestinal contents spread on 5 × 5 cm squares of broiler transport cage flooring. Floor squares were covered with 1 g of fresh broiler intestinal contents and subjected to either no treatment, 15 s water rinse, 15 s steam heat or a combination of water rinse followed by steam treatment. All squares were sampled by a sponge wipe method; sponges were used to prepare serial dilutions, which were plated for numbers of coliforms, E. coli and Campylobacter. Four replications were conducted with four samples of each treatment (n = 16). Spray washing alone was ineffective to lessen the numbers of bacteria detected on floor squares. Steam treatment resulted in a significant (P

Flea-borne diseases (FBDs) impact both human and animal health worldwide. Because adult fleas are obligately hematophagous and can harbor potential pathogens, fleas act as ectoparasites of vertebrates, as well as zoonotic disease vectors. Cat fleas (Ctenocephalides felis) are important vectors of two zoonotic bacterial genera listed as priority pathogens by the National Institute of Allergy and Infectious Diseases (NIAID-USA): Bartonella spp. and Rickettsia spp., causative agents of bartonelloses and rickettsioses, respectively. In this study, we introduce the first microbiome analysis of C. felis samples from California, determining the presence and abundance of relevant pathogenic genera by characterizing the cat flea microbiome through 16S rRNA next-generation sequencing (16S-NGS). Samples from both northern (NoCal) and southern (SoCal) California were assessed to expand current knowledge regarding FBDs in the state. We identified Rickettsia and Bartonella, as well as the endosymbiont Wolbachia, as the most abundant genera, followed by less abundant taxa. In comparison to our previous study screening Californian cat fleas for rickettsiae using PCR/digestion/sequencing of the ompB gene, the 16S-NGS approach applied herein showed a 95% level of agreement in detecting Rickettsia spp. There was no overall difference in microbiome diversity between NoCal and SoCal samples. Bacterial taxa identified by 16S-NGS in this study may help to improve epidemiological investigations, pathogen surveillance efforts, and clinical diagnostics of FBDs in California and elsewhere.

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Campylobacter is a natural member of the gut microflora in many commercial broilers and as such can become a contaminant on edible surfaces during processing. Culturing gut contents or feces can be a means to determine flock status prior to live-haul. The wide variety of non-Campylobacter background bacteria in these complex samples contaminates growth media and can make it very difficult to isolateCampylobacter. Over the course of 17 months, we cultured cecal contents from 100 different broiler flocks. For the last 50 flocks, we tested 3 selective plating media with and without the additional selection of a 0.45-μm filter for detection ofCampylobacter from cecal contents. Furthermore, from the last 50 flocks we also collected and cultured carcass rinse samples. Growth media tested included: Campy–Cefex Agar, Campy–Line Agar, and RF-Campylobacter jejuni/coli agar. About half (52%) of the 100 tested flocks were positive forCampylobacter; positive flocks were detected during each month of the year. Overall, theCampylobacter status of cecal contents from one carcass was predictive of the status of a carcass rinse from the same flock. Placing a complex sample such as cecal contents onto a 0.45-μm filter laid on top of the plating medium improved the detection ofCampylobacter by eliminating non-Campylobacter background colonies. All media allowed for detection ofCampylobacter from less complex carcass rinse samples without filtering. However, Campy–Cefex agar had higher numbers of competing bacterial colonies than did Campy–Line agar or RF-Campylobacter jejuni/coli agar.

Next-generation DNA sequencing is rapidly becoming a powerful tool for food animal management. One valuable use of this technology is to re-examine long-standing observations of performance differences associated with animal husbandry practices to better understand how these differences may be modulated by the gastrointestinal (GI) microbiome. The influences of environmental parameters such as air temperature and relative humidity on broiler chicken performance have commonly been observed, but how the GI microbiome may respond to seasonal environmental changes remains largely unknown. The purposes of this study were therefore to: (1) characterize the cecal microflora of commercial broilers (N = 87) collected at harvest across all 4 seasons, and (2) identify any significant changes of the GI microbiome and specific taxa according to season and Campylobacter status. Finding taxa with significant positive or negative correlations with Campylobacter could be useful by identifying indicator or antagonistic taxa and could also inform inferences regarding the ecological niche of Campylobacter. Whole GI tracts were removed from commercial broilers representing 87 independent flocks between April 2013 and May 2014 in the U.S. state of Georgia. Intact ceca were separated, cultured for Campylobacter and cecal contents were frozen. The cecal microbiome was characterized using barcoded sequencing of 16S rRNA genes on the Illumina MiSeq platform. The composition of the microbiome measured at processing was generally not affected by Campylobacter status but was most significantly affected by season of grow-out. Significantly fewer bacterial genera were found in winter than spring or summer. Bacterial genera with prior evidence for both positive or negative influences on gut health outcomes were significantly less abundant in the fall. Identifying specific members of the GI microbiota that vary according to season may help develop novel interventions to improve husbandry practices and growth performance.

Campylobacter jejuni clone SA is the major cause of sheep abortion and contributes significantly to foodborne illnesses in the United States. Clone SA is hypervirulent because of its distinct ability to produce systemic infection and its predominant role in clinical sheep abortion. Despite the importance of clone SA, little is known about its distribution and epidemiological features in cattle. Here we describe a prospective study on C. jejuni clone SA prevalence in 35 feedlots in 5 different states in the United States and a retrospective analysis of clone SA in C. jejuni isolates collected by National Animal Health Monitoring System (NAHMS) dairy studies in 2002, 2007, and 2014. In feedlot cattle feces, the overall prevalence of Campylobacter organisms was 72.2%, 82.1% of which were C. jejuni . Clone SA accounted for 5.8% of the total C. jejuni isolates, but its prevalence varied by feedlot and state. Interestingly, starlings on the feedlots harbored C. jejuni in feces, including clone SA, suggesting that these birds may play a role in the transmission of Campylobacter . In dairy cattle, the overall prevalence of clone SA was 7.2%, but a significant decrease in the prevalence was observed from 2002 to 2014. Whole-genome sequence analysis of the dairy clone SA isolates revealed that it was genetically stable over the years and most of the isolates carried the tetracycline resistance gene tet (O) in the chromosome. These findings indicate that clone SA is widely distributed in both beef and dairy cattle and provide new insights into the molecular epidemiology of clone SA in ruminants. IMPORTANCE C. jejuni clone SA is a major cause of small-ruminant abortion and an emerging threat to food safety because of its association with foodborne outbreaks. Cattle appear to serve as a major reservoir for this pathogenic organism, but there is a major gap in our knowledge about the epidemiology of clone SA in beef and dairy cattle. By taking advantage of surveillance studies conducted on a national scale, we found a wide but variable distribution of clone SA in feedlot cattle and dairy cows in the United States. Additionally, the work revealed important genomic features of clone SA isolates from cattle. These findings provide critically needed information for the development of preharvest interventions to control the transmission of this zoonotic pathogen. Control of C. jejuni clone SA will benefit both animal health and public health, as it is a zoonotic pathogen causing disease in both ruminants and humans.

The objective of this study was to compare subtypes of Campylobacter jejuni and Campylobacter coli detected on three selective Campylobacter plating media to determine whether each medium selected for different subtypes. Fifty ceca and 50 carcasses (representing 50 flocks) were collected from the evisceration line in a commercial broiler processing plant. Campylobacter was cultured and isolated from cecal contents and carcass rinses on Campy-Cefex, Campy Line, and RF Campylobacter jejuni/coli agars. When a positive result was obtained with all three media, one colony of the most prevalent morphology on each medium was selected for further analysis by full genome sequencing and multilocus sequence typing. Sequence types were assigned according to PubMLST. A total of 49 samples were positive for Campylobacter on all three media. Forty samples contained only C. jejuni , three had only C. coli , and both species were detected in six samples. From 71% of samples, Campylobacter isolates of the same sequence type were recovered on all three media. From 81.6% of samples, isolates were all from the same clonal complex. From significantly fewer samples (26%, P0.01), one medium recovered an isolate with a sequence type different from the type recovered on the other two media. When multiple sequence types were detected, six times the medium with the odd sequence type was Campy-Cefex, four times it was Campy-Line, and six times it was RF Campylobacter jejuni/coli . From one sample, three sequence types were detected. In most cases, all three plating media allowed detection of the same type of Campylobacter from complex naturally contaminated chicken samples.

A survey of 2,003 cecal content samples from chickens, turkeys, cattle, and swine at slaughter facilities in the United States was conducted to estimate the prevalence of the mcr-1 gene conferring resistance to colistin in Enterobacteriaceae . Two cecal samples from swine had Escherichia coli with IncI2 plasmids bearing the mcr-1 gene.

IncI2 type plasmids are medium-sized (~55–80 kb) conjugative plasmids that have been found carrying important antimicrobial resistance genes but have also been frequently found as cryptic plasmids. The DNA sequences for 147 fully sequenced IncI2 plasmids were studied by a whole-plasmid multi-locus sequence typing (wpMLST) scheme. A total of 171 loci were identified of which 52 were considered core (carried by greater than 95% of the plasmids). Most of the plasmids carrying the antimicrobial gene mcr-1 were in a distinct clade while most of the antimicrobial gene free plasmids were more distantly related. However, the host strains of bacteria were disparate for both groups of plasmids, showing that conjugal transfer of IncI2 plasmid is frequent. The mcr-1 gene was likely to have been introduced into IncI2 plasmids multiple times. It was also observed that the genes for conjugation showed significant linkage disequilibrium despite substantial diversity for most of those genes. Genes associated with biofilm formation were also among the core genes. The core genes can be considered the cohesive unit that defines the IncI2 plasmid group. Given the role conjugation can play in biofilm formation, it was concluded that conjugation is an active survival strategy for IncI2 plasmids. The IncI2 plasmid will have selective advantage when the plasmid-bearing bacteria are introduced to a new animal host that carries potential conjugal mates.

IncA/C plasmids are a class of plasmids from the Enterobacteriaceae that are relatively large (49 to >180 kbp), that are readily transferred by conjugation, and that carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remodeling by recombination-mediated horizontal gene transfer (HGT). We hypothesized that evaluation of nucleotide polymorphisms relative to the rate of HGT would help to develop a clock to show whether anthropic practices have had significant influences on the lineages of the plasmid. A system was developed to rapidly sequence up to 191 known open reading frames from each of 39 recently isolated IncA/C plasmids from a diverse panel of Salmonella enterica and Escherichia coli strains. With these data plus sequences from GenBank, we were able to distinguish six distinct lineages that had extremely low numbers of polymorphisms within each lineage, especially among the largest group designated as group 1. Two regions, each about half the plasmid in size, could be distinguished with a separate lineal pattern. The distribution of group 1 showed that it has migrated extremely rapidly with fewer polymorphisms than can be expected in 2,000 years. Remodeling by frequent HGT was evident, with a pattern that appeared to have the highest rate just upstream of the putative conjugation origin of transfer (oriT). It seems likely that when an IncA/C plasmid is transferred by conjugation there is an opportunity for plasmid remodeling adjacent to the oriT, which was also adjacent to a multiple antimicrobial resistance gene cassette.

SUMMARY Poultry processing plants can become colonized with Listeria monocytogenes, resulting in long-term residence in floor drains. Earlier work showed that water spray into a contaminated floor drain causes airborne dissemination of a nonvirulent surrogate, Listeria innocua. The objective of the current study was to determine the extent to which a hose spray can result in the transfer of L. innocua from an established floor drain biofilm to raw poultry meat. Model floor drains were inoculated and subjected to a 2-s water spray during which broiler breast fillets were left uncovered on a table 2.4 m away. Other fillets were indirectly exposed by placement on the table 10 min after the drain spray. The number of Listeria on each fillet was determined on the day of exposure and after 4 d of refrigerated storage. An average of approximately 18 Listeria cells was detected per air-exposed filet; about half that number was detected on indirectly exposed fillets. The number of Listeria did not change substantially during 4 d of cold storage. An inadvertent 2-s spray into a contaminated floor drain could result in transfer of low numbers of Listeria to raw meat on a work surface.

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Transmissible colistin resistance conferred by the mcr-1 gene-bearing IncI2 plasmid has been recently reported in Escherichia coli in the United States. We report here the completed genome sequence of a second E. coli strain isolated from swine in the United States that carried the mcr-1 gene on an IncI2-type plasmid.

Transmissible colistin resistance in the form of an mcr-1 -gene-bearing plasmid has been recently reported in Enterobacteriaceae in several parts of the world. We report here the completed genome sequence of an Escherichia coli strain isolated from swine in the United States that carried the mcr-1 gene on an IncI2-type plasmid.

Waterways should be considered in the migration routes of Campylobacter, and the genus has been isolated from several water sources. Inferences on migration routes can be made from tracking genetic types in populations found in specific habitats and testing how they are linked to other types. Water samples were taken over a 4-year period from waterways in the Upper Oconee River Watershed, Georgia, to recover isolates of thermophilic Campylobacter. The isolates were typed by multilocus sequence typing (MLST) and analyzed to determine the overall diversity of Campylobacter in that environment. Forty-seven independent isolates were recovered from 560 samples (8.4 %). Two (~4 %) isolates were Campylobacter coli, three (~6 %) isolates were putatively identified as Campylobacter lari, and the remaining 42 (~90 %) were Campylobacter jejuni. The C. jejuni and C. coli isolates were typed by the Oxford MLST scheme. Thirty sequence types (STs) were identified including 13 STs that were not found before in the MLST database, including 24 novel alleles. Of the 17 previously described STs, 10 have been isolated from humans, 6 from environmental water, and 6 from wild birds (five types from multiple sources). Seven sites had multiple positive samples, and on two occasions, the same ST was isolated at the same site. The most common type was STST61 with four isolates, and the most common clonal complex was CC179 with nine isolates. CC179 has been commonly associated with environmental water. Although some Campylobacter STs that were found in the Oconee River engage in widespread migration, most are tightly associated with or unique to environmental water sources.

Multi-drug-resistant (MDR) bacteria in food animals are a potential problem in both animal and human health. In this study, MDR commensal Escherichia coli isolates from poultry were examined. Thirty-two E. coli isolates from broiler carcass rinses were selected based on their resistance to aminoglycosides, β-lactams, chloramphenicols, tetracyclines, and sulfonamide antimicrobials. Microarray analysis for the presence of antimicrobial resistance and plasmid genes identified aminoglycoside [aac(6), aac(3), aadA, aph, strA, and strB], β-lactam (bla(AmpC), bla(TEM), bla(CMY), and bla(PSE-1)), chloramphenicol (cat, flo, and cmlA), sulfamethoxazole (sulI and sulII), tetracycline [tet(A), tet(C), tet(D), and tetR], and trimethoprim (dfrA) resistance genes. IncA/C plasmid core genes were detected in 27 isolates, while IncHI1 plasmid genes were detected in one isolate, indicating the likely presence of these plasmids. PCR assays for 18 plasmid replicon types often associated with MDR in Enterobacteriaceae also detected one or more replicon types in all 32 isolates. Class I integrons were investigated by PCR amplification of the integrase I gene, intI1, and the cassette region flanked by conserved sequences. Twenty-five isolates were positive for the intI1 gene, and class I integrons ranging in size from ~1,000 to 3,300 bp were identified in 19 of them. The presence of class I integrons, IncA/C plasmid genes, and MDR-associated plasmid replicons in the isolates indicates the importance of these genetic elements in the accumulation and potential spread of antimicrobial resistance genes in the microbial community associated with poultry.

SUMMARY Because of fecal shedding from Campylobacter-positive birds, broiler transport cages can be contaminated with Campylobacter, leading to cross-contamination of previously Campylobacter-negative broilers during live haul. Earlier work has shown that drying soiled or washed cages for 24 to 48 h can lower or even eliminate detectable Campylobacter on cage flooring. Because extended drying times are impractical, we tested the use of hot flowing air to speed the process. To determine if the effect was due to heat alone or flowing air alone, hot flowing air was compared with unheated flowing air and static hot air as well as with a benchtop control. The numbers of Campylobacter, Escherichia coli, and coliforms on small squares of washed or unwashed fecally soiled transport cage flooring were measured after drying treatments. When applied after a water spray wash treatment, flowing air at approximately 50°C for 15 min lowered the numbers of Campylobacter to an undetectable level. This represents a tremendous savings in drying time and suggests a potential commercial application. Static heat at similar temperatures was not nearly as effective, and unheated flowing air was moderately effective, but less so than hot air. Processors may be able to use a forced hot air treatment to dry cages between flocks, lessening the number of Campylobacter on cage flooring, thereby decreasing the potential for cross-contamination during live haul.

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.

SUMMARY Campylobacter numbers increase on broiler carcasses during defeathering because of leakage of gut contents through the vent. We tested several processing modifications designed to interfere with the transfer of Campylobacter from gut contents to the carcass surface. The numbers of Campylobacter detected on the breast skin of carcasses treated with each modification was compared with the numbers on control broilers processed using a standard method. Filling the vent and colon with commercially available canned spray foam did not consistently form an effective plug, and Campylobacter numbers increased during picking. Likewise, hanging carcasses with the vent pointed downward during defeathering was not effective in preventing the increase in Campylobacter numbers. Eviscerating carcasses by hand immediately before defeathering eliminated the increase in Campylobacter during automated feather picking. However, inadvertent contamination during hand evisceration led to higher numbers before feather removal. Therefore, we tested hand evisceration before scalding, allowing the scald water to kill and wash away Campylobacter spilled on the carcass during evisceration. Prescald evisceration was effective in significantly moderating the increase in Campylobacter on broiler carcasses during automated defeathering. Changing the order of standard broiler processing may help to control contamination with Campylobacter.

A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ², p

SUMMARY Because of the escape of contaminated gut contents, the number of Campylobacter spp. recovered from broiler carcasses increases during defeathering. Chlorine dioxide is approved for use as an antimicrobial treatment during poultry processing. A study was designed to test if application of 50 ppm of ClO2 during defeathering could prevent the expected increase in Campylobacter numbers on carcasses. Three replications were conducted, each using carcasses from different Campylobacter-positive flocks. Carcasses were collected from the shackle line immediately before and after defeathering with and without ClO2 spray; all carcasses were subjected to a whole-carcass rinse. Rinsate was cultured for Campylobacter, Escherichia coli, and Salmonella. Carcasses sprayed with ClO2 during defeathering had significantly lower numbers of Campylobacter and E. coli than carcasses treated with the water spray control defeathering. The ClO2 defeathering treatment also resulted in a lower prevalence of Salmonella than did control defeathering. Antimicrobial resistance profiles of Campylobacter and Salmonella isolates collected from ClO2-treated carcasses were not different from those collected from control carcasses. Application of ClO2 during feather removal may have potential as a means to mitigate the increase in bacterial contamination associated with broiler defeathering.

Aims: The intergenic sequence regions (ISR) between the 16S and 23S genes of Campylobacter jejuni and Campylobacter coli are markedly different for each species. However, in the genomic sequence for Camp. coli RM2228, two rRNA operons have an ISR that is characteristic of Camp. coli, and the third operon is characteristic of Camp. jejuni. The aim of this study was to determine the prevalence of ISR heterogeneity in these organisms. Methods and Results: PCR primers were designed to yield a 327-base pair (bp) product for Camp. coli and 166-bp product for Camp. jejuni. A strain like Camp. coli RM2228 should yield products of both sizes. DNA from a panel of Camp. coli (n = 133) and Camp. jejuni (n = 134) isolates were tested. All of the isolates yielded products of the predicted size for the species. To verify the data for Camp. coli RM2228, each ribosomal operon from the isolate was individually amplified by PCR and tested with the ISR primer pair. Products of both sizes were produced as predicted. Conclusions: The cross-species heterogeneity of the ISR seen in Camp. coli RM2228 is uncommon. Significance and Impact of the Study: The heterogeneity must have been caused by horizontal gene transfer at a frequency lower than predicted from housekeeping gene data. Thus, it can be expected that species identification based on the ISR can be confused in rare isolates.

Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replicon type Inc A/C and Inc H1. It is important to understand the transmission of these MDR plasmids because the genes they carry can affect the outcome of antimicrobial therapy. The aim of this study was to design a microarray with oligonucleotide probes for every gene in the six Inc A/C and one Inc H1 plasmids of interest while representing all redundant sequences only once. The microarray is printed in triplicate with 493 unique oligonucleotide probes 70 nucleotides in length. Salmonella enterica and Escherichia coli control strains and test plasmids (in the parent strain and transformed into a known E. coli background strain) were hybridized to the plasmid microarray. This hybridization arrays presents a rapid and cost effective method for high-density screening of isolates to evaluate the gene content of Inc A/C and H1 plasmids and will show how plasmids can change content with transmission.

To understand the mechanisms and epidemiology of antimicrobial resistance (AR), the genetic elements responsible must be identified. Due to the myriad of possible genes, a high-density genotyping technique is needed for initial screening. To achieve this, AR genes in the National Center for Biotechnology Information GenBank database were identified by their annotations and compiled into a nonredundant list of 775 genes. A DNA microarray was constructed of 70mer oligonucelotide probes designed to detect these genes encoding resistances to aminoglycosides, beta-lactams, chloramphenicols, glycopeptides, heavy metals, lincosamides, macrolides, metronidazoles, polyketides, quaternary ammonium compounds, streptogramins, sulfonamides, tetracyclines, and trimethoprims as well as resistance transfer genes. The microarray was validated with two fully sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and Typhi CT18 (multidrug resistance [MDR]). All resistance genes encoded on the MDR plasmid, pHCM1, harbored by CT18 were detected in that strain, whereas no resistance genes were detected in LT2. The microarray was also tested with a variety of bacteria, including MDR Salmonella enterica serovars, Escherichia coli, Campylobacter spp., Enterococcus spp., methicillin-resistant Staphylococcus aureus, Listeria spp., and Clostridium difficile. The results presented here demonstrate that a microarray can be designed to detect virtually all AR genes found in the National Center for Biotechnology Information database, thus reducing the subsequent assays necessary to identify specific resistance gene alleles.

This study was undertaken to determine potential sources of Listeria monocytogenes in a newly constructed chicken further processing plant and document the eventual colonization of the facility by this pathogen. To ascertain the colonization status of the plant, floor drains were sampled after a production shift and again after a cleanup shift on roughly a monthly basis for 21 months. Potential sources of L. monocytogenes to the plant included incoming raw meat, incoming fresh air, and personnel. Nearby environment and community samples were also examined. All L. monocytogenes detected were subjected to DNA sequence-based subtyping. L. monocytogenes was not detected in the plant before the commencement of processing operations. Within 4 months, several subtypes of L. monocytogenes were detected in floor drains, both before and after cleaning and sanitizing operations. No L. monocytogenes was detected on filters for incoming air, samples associated with plant employees, or a nearby discount shopping center. One subtype of L. monocytogenes was detected in a natural stream near the plant; however, this subtype was never detected inside the plant. Eight subtypes of L. monocytogenes were detected in raw meat staged for further processing; one of the raw meat subtypes was indistinguishable from a persistent drain subtype recovered after cleaning on eight occasions in four different drains. Poultry further processing plants are likely to become colonized with L. monocytogenes; raw product is an important source of the organism to the plant.

The objective of this study was to measure the effect of broiler processing on the prevalence, serotype, and antimicrobial resistance profiles of salmonellae. Twenty U.S. commercial processing plants representing eight integrators in 13 states were included in the survey. In each of four replications, 10 carcasses from one flock were collected at rehang and 10 more carcasses were collected at postchill; each carcass was sampled by whole-carcass rinse. Salmonella organisms were isolated from carcass rinses by standard cultural techniques, serotypes were determined, and the resistance to 15 antimicrobials was measured. Overall, Salmonella was detected on 72% of carcasses at rehang (ranging from 35 to 97%) and on 20% of carcasses postchill (ranging from 2.5 to 60%). In every instance, a significant (P < 0.05) decrease in Salmonella prevalence was noted between rehang and postchill. The four most common serotypes, accounting for 64% of all Salmonella isolates, were Kentucky, Heidelberg, Typhimurium, and Typhimurium var. 5-; most isolates of Kentucky (52%), Heidelberg (79%), and Typhimurium (54%) serotypes were susceptible to all antimicrobial drugs tested. However, only 15% of the Typhimurium var. 5- isolates were pansusceptible; more than one-half of the isolates of this serotype were resistant to three or more drugs. No isolate of any serotype exhibited resistance to amikacin, ceftriaxone, ciprofloxacin, or trimethoprim-sulfamethoxazole. These data demonstrate that although processing lessens carcass contamination with Salmonella, antimicrobial-resistant isolates may still be present.

Although enterococci are considered opportunistic nosocomial pathogens, their contribution to foodborne illnesses via dissemination through retail food remains undefined. In this study, prevalence and association of antimicrobial resistance and virulence factors of 80 Enterococcus faecalis isolates from retail food items were investigated. The highest rates of resistance were observed for lincomycin (73 of 80 isolates, 91%) and bacitracin (57 of 80 isolates, 71%), and lower rates of resistance (or =40%) were found for chloramphenicol, ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, nitrofurantoin, penicillin, and tylosin. Overall resistance to antimicrobials was low for most isolates tested. Of the virulence factors tested, the majority of isolates were positive for ccf (78 of 80 isolates, 98%), efaAfs (77 of 80, 96%), and cpd (74 of 80, 93%). Isolates also commonly contained cob (72 of 80 isolates, 90%) and gelE (68 of 80, 85%). Very few isolates contained cylMBA (12 of 80 isolates [15%] for cylM and 9 of 80 isolates [11%] for both cylB and cylA) and efaAfm (2 of 80 isolates, 3%). Positive statistical associations (significance level of 0.05) were found between agg and tetracycline resistance, cylM and erythromycin resistance, and gelE and efaAfs and lincomycin resistance. The presence of the cylB and cylA alleles also was positively correlated with bacitracin and tetracycline resistance. Negative correlations were observed between many of the virulence attributes and resistance to ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, and tylosin. These data suggest that both positive and negative associations exist between antimicrobial resistance genes and virulence factors in E. faecalis isolates from foods commonly purchased from grocery stores.

Rivers may serve as reservoirs for enteric organisms. Very little is known about the boundaries of microbial communities in moving bodies of water so this study was undertaken to find the limits of distribution of some bacteria, focusing on enteric organisms. The presence of Salmonella, Campylobacter, and Enterococcus spp. and the antimicrobial resistance phenotypes carried by these organisms was evaluated for the Upper Oconee River basin, a small river in the lower Piedmont of northeastern Georgia, USA. Samples were obtained from 83 sites during a 3-h period on a spring day (April 2005) in an approximately 30 x 20 km region. Campylobacter spp. was isolated at 12 sites. The Campylobacter isolates from three sites were resistant to tetracycline. Of the five short-variable region (SVR) subtypes of Campylobacter that were found, three were found at more than one site, two types were found twice, and one subtype was found three times. Enterococcus was isolated at 71 sites. E. casseliflavus was the most common species. Based on species identification and antimicrobial resistance patterns, 24 types of Enterococcus were found. Salmonella was isolated from 62 sites. Of the 19 Salmonella serovars that were isolated, serovar Muenchen accounted for about 20% of the isolates. The next three most common serovars isolated, Rubislaw, Hartford, and Give, accounted for about 44% of the river isolates. Antimicrobial resistance profiling offered limited differentiation of Salmonella isolates because only seven isolates were resistant to any antimicrobial. The sites at which Salmonella, Campylobacter, or Enterococcus were isolated did not correlate with each other or with the total coliform number or Escherichia coli count for the site. However, isolates of some of the same species and type occurred in clusters that were restricted to areas within 5 to 6 km.

The use of antimicrobials in food animal production, particularly those commonly used to treat infections in humans, has become a source of debate in recent years. However, limited data are available regarding the development of resistance following the subtherapeutic or therapeutic administration of antimicrobials in animal production. The objective of this study was to evaluate the effect of the administration of therapeutic and subtherapeutic concentrations of tylosin on the erythromycin susceptibility of Campylobacter jejuni and Campylobacter coli isolated from the ceca of treated broilers. In three replicated studies, day-of-hatch chicks were exposed to macrolide-susceptible C. jejuni or C. coli. At 2 weeks of age, tylosin was administered at subtherapeutic (22 ppm, continuously in the diet) or therapeutic concentrations (529 ppm, in the drinking water for 5 days). Broilers were sacrificed weekly. Total and erythromycin-resistant Campylobacter spp. were enumerated from individual ceca plus cecal contents. Overall erythromycin resistance was observed at a higher frequency (P < 0.01) among C. coli isolates (70.8%) than among C. jejuni isolates (36.8%) following tylosin administration. Across Campylobacter species, erythromycin resistance was observed at a higher frequency (P < 0.001) when tylosin was administered at subtherapeutic (62.7%) than at therapeutic (11.4%) concentrations. Subtherapeutic administration resulted in the recovery of 83.3 and 56.1% erythromycin-resistant isolates compared with only 33.3 and 7.9% of the isolates expressing erythromycin resistance following the administration of therapeutic concentrations for C. coli and C. jejuni, respectively. Further studies are needed to determine the factors involved in the apparent difference in the acquisition of macrolide resistance in C. coli compared with C. jejuni.

Campylobacter lari is frequently isolated from shore birds and can cause illness in humans. Here, we report the draft whole-genome sequence of a urease-positive strain of C. lari that was isolated in estuarial water on the coast of Delaware, USA.

Most foodborne outbreaks of listeriosis have been found to involve a small number of closely related strains of Listeria monocytogenes serotype 4b. The ecology of these organisms and their reservoirs in nature or in the processing plant environment, however, remain poorly understood. Surveys of environmental samples from two turkey processing plants in the United States indicated presence of L. monocytogenes of the serotype 4b complex (serotype 4b and the closely related serotypes 4d and 4e). In addition, environmental and raw product samples from one plant repeatedly yielded isolates with genetic markers typical of two major serotype 4b epidemic clonal groups, ECI and ECII. The pulsed field gel electrophoresis (PFGE) profiles of these isolates, however, were clearly distinct from those of confirmed epidemic-associated strains. Furthermore, we observed minor but consistent differences in PFGE profiles of isolates that harbored ECI- or ECII-specific genetic markers, and that were obtained at different sampling times from the same plant. The findings suggest processing plant persistence (or repeated introductions) and genomic diversification of L. monocytogenes serotype 4b isolates that harbor ECI- or ECII-specific genetic markers. Such diversification would need to be taken into consideration in further efforts to elucidate the evolution and epidemiology of these organisms.

In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes , the sequences of all of the annotated genes from the fully sequenced strain L. monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L. monocytogenes ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes ( hisJ , cbiE , truB , ribC , comEA , purM , aroE , hisC , and addB ) in the 80 to 90% identity group and two genes ( gyrB and rnhB ) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L. monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of L. monocytogenes that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the L. monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of L. monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.