Search

Antimicrobial resistance (AMR) developed by Salmonella within animals used for food products is a major global issue. Monitoring AMR in animals destined for slaughter is, therefore, critical. Abatt...

Most Escherichia coli (E. coli) strains do not cause disease, naturally living in the lower intestine and is expelled into the environment within faecal matter. Escherichia coli can utilize citrate under anaerobic conditions but not aerobic conditions. However, the underlying regulatory mechanisms are poorly understood. In this study, we explored regulatory mechanisms of citrate fermentation genes by global regulators ArcA and Fnr under anaerobic conditions. A gel mobility shift assay showed that the regulator proteins ArcA and Fnr binded to the promoter region localized between the citAB and citCDEFXGT operons. Subsequent assays confirmed that ArcA indirectly controled the expression of citrate fermentation genes via regulating CitA-CitB system, while Fnr directly regulated but also indirectly modulated citrate fermentation genes via controling CitA-CitB system. Deletions of arcA and fnr significantly reduced the growth of Escherichia coli in M9 medium with a citrate carbon source. We conclude that both ArcA and Fnr can indirectly control the citrate utilization via CitA-CitB system, while Fnr can also directly regulate the expression of citrate fermentation genes in E. coli under anaerobic conditions.

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for the severe disease listeriosis and notorious for its ability to persist in food processing plants, leading to contamination of processed, ready-to-eat foods. L. monocytogenes persistence in various food processing environments (FPEs) has been extensively investigated by various subtyping tools, with increasing use of whole genome sequencing (WGS). However, major knowledge gaps remain. There is a need for facility-specific molecular signatures not only for adequate attribution of L. monocytogenes to a specific FPE but also for improved understanding of the ecology and evolution of L. monocytogenes in the food processing ecosystem. Furthermore, multiple strains can be recovered from a single FPE sample, but their diversity can be underestimated with common molecular subtyping tools. In this study we investigated a panel of 54 L. monocytogenes strains from four turkey processing plants in the United States. A combination of WGS and phenotypic assays was employed to assess strain persistence as well as identify facility-specific molecular signatures. Comparative analysis of allelic variation across the whole genome revealed that allelic profiles have the potential to be specific to individual processing plants. Certain allelic profiles remained associated with individual plants even when closely-related strains from other sources were included in the analysis. Furthermore, for certain sequence types (STs) based on the seven-locus multilocus sequence typing scheme, presence and location of premature stop codons in inlA, inlB length, prophage sequences, and the sequence content of a genomic hotspot could serve as plant-specific signatures. Interestingly, the analysis of different isolates from the same environmental sample revealed major differences not only in serotype and ST, but even in the sequence content of strains of the same ST. This study highlights the potential for WGS data to be deployed for identification of facility-specific signatures, thus facilitating the tracking of strain movement through the food chain. Furthermore, deployment of WGS for intra-sample strain analysis allows for a more complete environmental surveillance of L. monocytogenes in food processing facilities, reducing the risk of failing to detect strains that may be clinically relevant and potentially novel.

Neonatal meningitis E. coli (NMEC) is the second leading cause of sepsis and meningitis in neonates worldwide. Here, we report the genome sequence of NMEC15, belonging to serotype O18:K1, isolated from the cerebrospinal fluid (CSF) of an infant with neonatal bacterial meningitis (NBM) in the Netherlands.

This chapter includes a collection of miscellaneous organisms that have caused disease in poultry or are a public health concern. The less common pathogens implicated in poultry loss included are Staphylococcosis, Streptococcus and Enterococcus, Erysipelas, avian intestinal spirochetosis, tuberculosis. Disease syndromes included in the chapter include beak necrosis, venereal disease of geese, and liver granulomas, but are not identified to a specific organism responsible because of the multifactorial nature of the disease. Staphylococcosis is diagnosed by culturing suspected clinical material including exudate from joints, yolk material, and stab swabs of internal organs. Staphylococcosis can resemble infection with Escherichia coli, Pasteurella multocida, Salmonella gallinarum, Mycoplasma synoviae, reoviruses, or any other infection of bones or joints that is hatchery‐related, associated with mechanical trauma, or causes septicemia. Prevention and control of Enterococcus infections require reducing stress and preventing immunosuppressive diseases and conditions. Proper cleaning and disinfection can reduce environmental enterococcal resident flora to minimize external exposure.

Staphylococcus aureus is an important pathogen involved in subclinical bovine mastitis, causing high economic losses for the dairy industry. The successful persistence of this pathogen in the host occurs due to a series of factors associated with colonization ability and the acquisition of virulence factors. This bacterial species carries genetic heterogeneity, and genetically diverse strains characterize the population. Analysis of the genetic variation is an important tool for epidemiological studies. For this study, S. aureus strains were randomly selected by molecular profiling. All strains were originated from the milk of cows of subclinical mastitis on farms in the State of Rio de Janeiro. Strains of S. aureus were profiled using virulence gene analysis profiles, agr and spa typing, Pulsed Field Gel Electrophoresis (PFGE), and Multilocus Sequence Typing (MLST). 47% (8/17) of the tested strains were positive strains for icaA gene; 82.3% (14/17) for icaD gene; 41% (7/17) for fbnA gene; 47% (8/17) positive for fbnB gene; 94% (16/17) for the hlA gene and 70.5% (12/17) for hlB gene. These virulence results generated 11 different profiles. Most strains (58.8% - 10/17) were classified as type-II by agr system. spa typing identified seven different spa types. PFGE analysis found extensive genetic heterogeneity and no clones were observed. MLST analysis generated five different types of ST/CC. Considering the results observed at the present study, a high genetic variety of S. aureus strains associated of the presence of different virulence factors justified the absence of clonal strains at the properties evaluated. Besides, that, the definition of S. aureus clonal strain as well dynamic population in not fully understood since there are a limited number of studies in S. aureus associated with bovine mastitis., Staphylococcus aureus é um importante patógeno envolvido na mastite subclínica bovina, causando altas perdas econômicas para a indústria de laticínios. A persistência bem-sucedida desse patógeno no hospedeiro ocorre devido a uma série de fatores associados à capacidade de colonização e aquisição de fatores de virulência. Esta espécie bacteriana possui heterogeneidade genética e a população é caracterizada por cepas geneticamente diversas. A análise da variação genética é uma ferramenta importante para estudos epidemiológicos. Para este estudo, cepas de S. aureus foram selecionadas aleatoriamente por perfil molecular. Todas as cepas foram originadas do leite de vacas de mastite subclínica em fazendas do Estado do Rio de Janeiro. As cepas de S. aureus foram caracterizadas usando perfis de análise de genes de virulência, tipagem agr e spa, eletroforese em gel de campo pulsado (PFGE) e tipagem de sequência de foco múltiplo (MLST). 47% (8/17) das cepas testadas foram positivas para o gene icaA; 82.3% (14/17) para o gene icaD; 41% (7/17) para o gene fbnA; 47% (8/17) positivo para o gene fbnB; 94% (16/17) para o gene hlA e 70.5% (12/17) para o gene hlB. Esses resultados de virulência geraram 11 perfis diferentes. A maioria das cepas (58.8% - 10/17) foi classificada como tipo II pelo sistema agr. A tipificação por spa identificou sete tipos diferentes. A análise por PFGE encontrou extensa heterogeneidade genética e nenhum clone foi observado. A análise do MLST gerou cinco tipos diferentes de ST / CC. Considerando os resultados observados no presente estudo, uma elevada variedade genética de cepas de S. aureus associada à presença de diferentes fatores de virulência justificou a ausência de cepas clonais nas propriedades avaliadas. Além disso, a definição de cepas clonais assim como a dinâmica populacional em S. aureus não é totalmente compreendida, pois há um número limitado de estudos em S. aureus associados à mastite bovina.

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world’s protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1–9. The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB, and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA, was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel (iroN, ompT, hlyF, iss, iutA). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective.

Passive immunization with specific egg yolk antibodies (immunoglobulin Y, IgY) is emerging as a promising alternative to antibiotics to control bacterial infections. Recently, we developed a novel conjugate vaccine that could trigger a strong immune response in rabbits directed against enterobactin (Ent), a highly conserved siderophore molecule utilized by different Gram-negative pathogens. However, induction of Ent-specific antibodies appeared to be affected by the choice of animal host and vaccination regimen. It is still unknown if the Ent conjugate vaccine can trigger a specific immune response in layers for the purpose of production of anti-Ent egg yolk IgY. In this study, three chicken vaccination trials with different regimens were performed to determine conditions for efficient production of anti-Ent egg yolk IgY. Purified Ent was conjugated to three carrier proteins, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and CmeC (a subunit vaccine candidate), respectively. Intramuscular immunization of Barred Rock layers with KLH-Ent conjugate four times induced strong immune response against whole conjugate vaccine but the titer of Ent-specific IgY did not change in yolk with only a 4 fold increase detected in serum. In the second trial, three different Ent conjugate vaccines were evaluated in Rhode Island Red pullets with four subcutaneous injections. The KLH-Ent or CmeC-Ent conjugate consistently induced high level of Ent-specific IgY in both serum (up to 2,048 fold) and yolk (up to 1,024 fold) in each individual chicken. However, the Ent-specific immune response was only temporarily and moderately induced using a BSA-Ent vaccination. In the third trial, ten White Leghorn layers were subcutaneously immunized three times with KLH-Ent, leading to consistent and strong immune response against both whole conjugate and the Ent molecule in each chicken; the mean titer of Ent-specific IgY increased approximately 32 and 256 fold in serum and yolk, respectively. Consistent with its potent binding to various Ent derivatives, the Ent-specific egg yolk IgY also inhibited in vitro growth of a representative Escherichia coli strain. Together, this study demonstrated that the novel Ent conjugate vaccine could induce strong, specific, and robust immune response in chickens. The Ent-specific hyperimmune egg yolk IgY has potential for passive immune intervention against Gram-negative infections.

Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed-field gel electrophoresis (PFGE) and whole-genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended-spectrum beta-lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1-PFGE and Southern hybridization. Whole-genome sequence-based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre-hrvest level. The findings from multi-tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre-harvest level is highly warranted in the future.

Salmonella Enteritidis is responsible for a significant proportion of foodborne salmonellosis in the United States and continues to be attributable to table eggs despite increased federal oversight. Technologies, including feed additives, continue to be evaluated for preharvest application and their potential food safety benefits. Diamond V Original XPC, a Saccharomyces cerevisiae fermentation-based postbiotic (SCFP), was evaluated for its effectiveness in reducing Salmonella Enteritidis (SE) colonization in young layer pullets. A total of 40 day-old Hy-Line W-36 layer pullets were equally divided and randomly assigned to one of two dietary treatments, with SCFP or without SCFP (PCON), and orally gavaged on day 28 with SE at 106 CFU/mL. Another 20 day-old pullets were fed the same control feed without SCFP and blank inoculated on day 28 with 1 mL of sterile phosphate-buffered saline to serve as a negative control. Qualitative and quantitative analyses of cecal contents for Salmonella were performed for all birds on day 32. The prevalence of SE in the ceca of all directly challenged birds was 100%; however, the SE concentration in birds fed SCFP diet (3.35 log CFU/g) was significantly lower (P < 0.0001) than that of the PCON birds not fed SCFP (4.49 log CFU/g). The proportion of birds with enumerable SE concentrations was lower in SCFP-fed pullets (57.9%) than in the PCON pullets (95.0%). These data suggest that inclusion of SCFP in the diet may aid in the reduction of SE within the ceca of commercial laying hens and could serve as an additional preharvest food safety hurdle. HIGHLIGHTS

Turkey cellulitis, also known as clostridial dermatitis is a significant cause of morbidity, mortality, and carcass condemnation at slaughter resulting in considerable losses for turkey producers. Here, we assessed the potential role of Avian Pathogenic Escherichia coli (APEC) in a cellulitis outbreak on a turkey farm in Iowa. Birds from one farm with a history of cellulitis and one farm with no history of disease (for comparison) were followed from the age of 10 weeks (before the outbreak) to 18 weeks (just prior to slaughter). E. coli recovered from the litter, from skin lesions of birds with cellulitis, and from systemic lesions of birds submitted for necropsy, were assessed. A total of 333 isolates were analyzed and screened for virulence-associated genes, antimicrobial resistance genes including heavy metal resistance, adhesins, invasins, and protectins, iron acquisition systems and their phylogenetic group through multiplex PCR. In addition, PCR was used to serogroup the isolates, and pulsed field gel electrophoresis (PFGE) was used to analyze a subset of strains from the farm environment (litter) and birds at 17 and 18 weeks of age when the cellulitis infection appeared to peak. Overall, E. coli isolates recovered from cellulitis lesions and systemic infection were identified as APEC, while a lower prevalence of E. coli recovered from the litter met the criteria of APEC-like. Direct comparison of E. coli isolates from the litter, lesions, and systemic strains using PFGE failed to find identical clones across all three sources reflecting the diversity of strains present in the poultry environment causing disease. This study highlights the role of APEC in turkey cellulitis and should not be overlooked as a significant contributor to the disease in turkeys.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections. Characteristically, these organisms exhibit strong resistance to the bactericidal action of host serum. Although numerous serum resistance factors in ExPEC have been identified, their regulatory mechanisms during in vivo infection remain largely unknown. Here, RNA sequencing analyses together with quantitative reverse-transcription PCR revealed that ExPEC genes involved in the biosynthesis of extracytoplasmic polysaccharides (ECPs) including K-capsule, lipopolysaccharide (LPS), colanic acid, peptidoglycan and Yjb exopolysaccharides were significantly upregulated in response to serum under low oxygen conditions and during bloodstream infection. The oxygen sensor FNR directly activated the expression of K-capsule and colanic acid and also indirectly modulated the expression of colanic acid, Yjb exopolysaccharides and peptidoglycan via the known Rcs regulatory system. The global regulator Fur directly or indirectly repressed the expression ofECP biosynthesis genes in iron replete media, whereas the low iron conditions in the bloodstream could relieve Fur repression. Using in vitro and animal models, FNR, Fur and the Rcs system were confirmed as contributing to ExPEC ECP production, serum resistance and virulence. Altogether, these findings indicated that the global regulators FNR andFur and the signaling transduction system Rcs coordinately regulated the expression of ECP biosynthesis genes leading to increased ExPEC serum resistance in response to low oxygen and low iron levels in the bloodstream.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bloodstream and other extraintestinal infections in human and animals. The greatest challenge encountered by ExPEC during an infection is posed by the host defense mechanisms, including lysozyme. ExPEC have developed diverse strategies to overcome this challenge. The aim of this study was to characterize the molecular mechanism of ExPEC resistance to lysozyme. For this, 15,000 transposon mutants of a lysozyme-resistant ExPEC strain NMEC38 were screened; 20 genes were identified as involved in ExPEC resistance to lysozyme—of which five were located in the gene cluster between galF and gnd, and were further confirmed to be involved in O-specific polysaccharide biosynthesis. The O-specific polysaccharide was able to inhibit the hydrolytic activity of lysozyme; it was also required by the complete lipopolysaccharide (LPS)-mediated protection of ExPEC against the bactericidal activity of lysozyme. The O-specific polysaccharide was further shown to be able to directly interact with lysozyme. Furthermore, LPS from ExPEC strains of different O serotypes was also able to inhibit the hydrolytic activity of lysozyme. Because of their cell surface localization and wide distribution in Gram-negative bacteria, O-specific polysaccharides appear to play a long-overlooked role in protecting bacteria against exogenous lysozyme.

Objective Extraintestinal Pathogenic E. coli (ExPEC), are responsible for host diseases such as Neonatal Meningitis Escherichia coli (NMEC), the second-leading cause of neonatal bacterial meningitis, Avian Pathogenic E. coli (APEC), a cause of extraintestinal disease in poultry, and Uropathogenic E. coli (UPEC), the most common cause of urinary tract infections. Virulence factors associated with NMEC include outer membrane protein A (OmpA) and type I fimbriae (FimH), which also occur in APEC and UPEC. OmpA contributes to NMEC’s ability to cross the blood–brain barrier, persist in the bloodstream and has been identified as a potential vaccine target for ExPEC, however the protein has amino acid variants, which may influence virulence of strains or alter vaccine efficacy. Although OmpA is present in virtually all E. coli, differences in its amino acid residues have yet to be surveyed in ExPEC. Results Here the ompA gene (n = 399) from ExPEC collections were sequenced and translated in silico. Twenty-five different OmpA polymorphism patterns were identified. Seven polymorphism patterns were significantly associated with an ExPEC subpathotype, but chromosomal history most likely accounts for most differences found. The differences in OmpA protein sequences suggest that OmpA may influence variation in virulence and host specificity within ExPEC subpathotypes.

Characterization of methicillin-resistant Staphylococcus (MRS) is a continuous challenge at diagnostic laboratories. The phenotypic methods present heterogeneous results and the occurrence of variants of mecA gene turned this goal even more challenging to achieve. The present study provided an accurate and highly discriminatory screening tool for MRS, improving its detection.

Colibacillosis caused by Avian Pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality and carcass condemnation to the poultry industry worldwide resulting in significant economic losses. We assessed a multiplex PCR for classifying diagnostic APEC and characterized these isolates using gene profile analysis.48 E. coli isolates collected between August and October 2018 through the Poultry Disease Research Center (PDRC) Diagnostic Laboratory were analyzed. Isolates were screened using multiplex PCR targeting genes associated with APEC chromosomal and plasmid virulence. Isolates were assessed for relationship between gene profile and host tissue of origin. Overall, isolates met the criteria for definition as well-developed pathogens with more than 90 % of isolates positive for the genes iroN, ompTp and hlyF; 78 % were positive for aerJ and 67 % for iss. A significantly lower prevalence was observed for cvaC, etsB, ireA and papC (range 5–36 %). When overall gene prevalence was examined for tissue of isolation, we found that APEC from the ovary, bone marrow, pericardium and lung had higher average numbers of genes compared to isolates recovered from skin and yolk sac. Genes associated with the ColV virulence plasmid (iss, iroN, hlyF and ompTp) were detected in 43 of 48 isolates (89.5%) further confirming the ColV plasmid is the defining trait of the APEC subpathotype. The use of a multiplex panel to screen for APEC has shown good correlation with pathogenesis, and tissue source and correlates well with invasive strains. Path panel diagnostics is available through PDRC, providing significant value to APEC screening.

Aim The molecular typing and the susceptibility of Staphylococcus aureus strains of swine origin to antibiotics, oregano (Origanum vulgare L.) essential oil (EO) and Chilean blackberry maqui (Aristotelia chilensis (Molina) Stuntz) extract were determined. Methods and results Twenty S. aureus strains of swine origin were subjected to molecular typing, of which six strains were selected for antimicrobial susceptibility testing. The epsilon test (Etest) was used to determine the antibiotic susceptibility. The susceptibility to natural antimicrobials (NAs): oregano EO, maqui extract, thymol (Thy) and carvacrol (Carv), was carried out using the disk diffusion method. The S. aureus strains were genetically diverse. All strains were resistant to at least one class of antibiotic, and two strains were multidrug-resistant. The minimum inhibitory concentration of oregano EO, Thy and Carv was 0·01-0·04%. Maqui extract did not show antistaphylococcal activity. Conclusions Natural antimicrobials extracted from oregano have an inhibitory activity against S. aureus strains from swine origin, with no effect using maqui extract. Significance and impact of the study This study provides information about the characteristics of S. aureus strains of swine origin, and about the potential use of NAs from oregano to enhance the control of antibiotic-resistant S. aureus strains in the pork supply chain.

The fluoroquinolone-resistant sequence type 1193 (ST1193) of Escherichia coli, from the ST14 clonal complex (STc14) within phylogenetic group B2, has appeared recently as an important cause of extraintestinal disease in humans. Although this emerging lineage has been characterized to some extent using conventional methods, it has not been studied extensively at the genomic level. Here, we used whole-genome sequence analysis to compare 355 ST1193 isolates with 72 isolates from other STs within STc14. Using core genome phylogeny, the ST1193 isolates formed a tightly clustered clade with many genotypic similarities, unlike ST14 isolates. All ST1193 isolates possessed the same set of three chromosomal mutations conferring fluoroquinolone resistance, carried the fimH64 allele, and were lactose non-fermenting. Analysis revealed an evolutionary progression from K1 to K5 capsular types and acquisition of an F-type virulence plasmid, followed by changes in plasmid structure congruent with genome phylogeny. In contrast, the numerous identified antimicrobial resistance genes were distributed incongruently with the underlying phylogeny, suggesting frequent gain or loss of the corresponding resistance gene cassettes despite retention of the presumed carrier plasmids. Pangenome analysis revealed gains and losses of genetic loci occurring during the transition from ST14 to ST1193 and from the K1 to K5 capsular types. Using time-scaled phylogenetic analysis, we estimated that current ST1193 clades first emerged approximately 25 years ago. Overall, ST1193 appears to be a recently emerged clone in which both stepwise and mosaic evolution have contributed to epidemiologic success.

Background Rhodococcus equi (R. equi) infections are endemic in many horse facilities in the United States resulting significant economic loses annually. Currently, there is no commercial vaccine available and the emergence of isolates that are resistant to the current treatment and prophylaxis using antibiotics prompts closer surveillance of this pathogen. Objective This study compares three different genotyping techniques, Pulsed Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and whole genome SNP-based phylogeny to determine the most accurate method to monitor the spread of macrolide-and-rifampin-resistant R. equi. Methods 16 macrolide and rifampin-resistant and 6 susceptible R. equi and their Illumina Miseq whole genome sequences were used in this study. The isolates were sub-typed by PFGE with VspI and a dendrogram based on their similarities generated. Additionally, three phylogenetic trees were constructed using CSI phylogeny on (i) whole genome sequences (WGS), (ii) in silico MLST sequences and (iii) MLST sequences obtained after PCR-amplification and Sanger sequencing. Results PFGE identified 18 different genetic profiles and grouped the 22 isolates into 3 clusters independently of their susceptibilities. The phylogenetic trees built from WGS and MLST data showed similar topology, separating the isolates into 2 major clades in accordance with their susceptibility profiles (susceptible and resistant). However, only the trees generated with next generation sequencing data could detect the clonality of the resistant isolates.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, a disease that affects poultry production worldwide and leads to multimillion-dollar losses annually. Here, we report the genome sequence of APEC O2-211, a sequence type 117 (ST117) strain isolated from a diseased chicken.

Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of neonatal bacterial meningitis worldwide. We report the genome sequence of the multidrug-resistant NMEC serotype O75:H5:K1 strain mcjchv-1, which resulted in an infant’s death., Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of neonatal bacterial meningitis worldwide. We report the genome sequence of the multidrug-resistant NMEC serotype O75:H5:K1 strain mcjchv-1, which resulted in an infant’s death. The O75 serogroup is rare among NMEC isolates; therefore, this strain is considered an emergent pathogen.

This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone.

Avian pathogenic Escherichia coli (APEC) is the etiologic agent of colibacillosis, an important cause of morbidity and mortality in poultry. Though, many virulence factors associated with APEC pathogenicity are known, their regulation remains unclear. FNR (fumarate and nitrate reduction) is a well-known global regulator that works as an oxygen sensor and has previously been described as a virulence regulator in bacterial pathogens. The goal of this study was to examine the role of FNR in the regulation of APEC virulence factors, such as Type I fimbriae, and processes such as adherence and invasion, type VI secretion, survival during oxidative stress, and growth in iron-restricted environments. To accomplish this goal, APEC O1, a well-characterized, highly virulent, and fully sequenced strain of APEC harboring multiple virulence mechanisms, some of which are plasmid-linked, was compared to its FNR mutant for expression of various virulence traits. Deletion of FNR was found to affect APEC O1's adherence, invasion and expression of ompT, a plasmid-encoded outer membrane protein, type I fimbriae, and aatA, encoding an autotransporter. Indeed, the fnr− mutant showed an 8-fold reduction in expression of type I fimbriae and a highly significant (P < 0.0001) reduction in expression of fimA, ompT (plasmid-borne), and aatA. FNR was also found to regulate expression of the type VI secretion system, affecting the expression of vgrG. Further, FNR was found to be important to APEC O1's growth in iron-deficient media and survival during oxidative stress with the mutant showing a 4-fold decrease in tolerance to oxidative stress, as compared to the wild type. Thus, our results suggest that FNR functions as an important regulator of APEC virulence.

The Clermont scheme has been used for subtyping of Escherichia coli since it was initially described in early 2000. Since then, researchers have used the scheme to type and sub-type commensal E. coli and pathogenic E. coli, such as extraintestinal pathogenic E. coli (ExPEC), and compare their phylogenetic assignment by pathogenicity, serogroup, distribution among ExPEC of different host species and complement of virulence and resistance traits. Here, we compare assignments of human and avian ExPEC and commensal E. coli using the old and revised Clermont schemes to determine if the new scheme provides a refined snapshot of isolate classification. 1,996 E. coli from human hosts and poultry, including 84 human neonatal meningitis E. coli isolates, 88 human vaginal E. coli, 696 human uropathogenic E. coli, 197 healthy human fecal E. coli, 452 avian pathogenic E. coli (APEC), 200 retail poultry E. coli, 80 crop and gizzard E. coli from healthy poultry at slaughter and 199 fecal E. coli from healthy birds at slaughter. All isolates were subject to phylogenetic analysis using the Clermont et al. (2000, 2013) schemes and compared to determine the effect of the new classification on strain designation. Most of the isolates’ strain designation remained where they were originally assigned. Greatest designation change occurred in APEC where 53.8% of isolates were reclassified; while classification rates among human strains ranged from 8 to 14%. However, some significant changes were observed for UPEC associated strains with significant (P < 0.05) designation changes observed from A to C and D to E or F phylogenetic types; a similar designation change was noted among NMEC for D to F designation change. Among the APEC significant designation changes were observed from A to C and D to E and F. These studies suggest that the new scheme provides a tighter and more meaningful definition of some ExPEC; while the new typing scheme has a significant impact on APEC classification. A comparison of phylogenetic group assignment by content of virulence, resistance, replicon and pathogenicity island genes in APEC suggests that insertion of pathogenicity islands into the genome appears to correlate closely with revised phylogenetic assignment.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTIs), which are some of the world's most common bacterial infections of humans. Here, we examined the role of FNR ( f umarate and n itrate r eduction), a well-known global regulator, in the pathogenesis of UPEC infections. We constructed an fnr deletion mutant of UPEC CFT073 and compared it to the wild type for changes in virulence, adherence, invasion, and expression of key virulence factors. Compared to the wild type, the fnr mutant was highly attenuated in the mouse model of human UTI and showed severe defects in adherence to and invasion of bladder and kidney epithelial cells. Our results showed that FNR regulates motility and multiple virulence factors, including expression of type I and P fimbriae, modulation of hemolysin expression, and expression of a novel pathogenicity island involved in α-ketoglutarate metabolism under anaerobic conditions. Our results demonstrate that FNR is a key global regulator of UPEC virulence and controls expression of important virulence factors that contribute to UPEC pathogenicity.

SUMMARY. Avian pathogenic Escherichia coli (APEC) infection is responsible for great economic losses to the poultry industry worldwide and there is increasing evidence of its zoonotic importance. In this study, 219 E.coli isolates from 84 poultry flocks in Egypt, including 153 APEC, 30 avian fecal E. coli (AFEC), and 36 environmental E. coli, were subjected to phylogenetic grouping and virulence genotyping. Additionally, 50 of these isolates (30 APEC from colisepticemia and 20 AFEC) were subjected to a moreextensive characterization which included serogrouping, antimicrobial susceptibility analysis, screening for seven intestinal E. coli virulence genes (stx1, stx2, eae, espP, KatP, hlyA ,a ndfliCh7), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), andin vivo virulence testing. More than 90% of the total APEC examined possessediroN,ompT,hlyF,iss ,a ndiutA, indicating that Egyptian APECs, like their counterparts from the United States, harbor plasmid pathogenicity islands (PAIs). The majority of APEC and AFEC were of phylogenetic groups A, B1, and D. For the 50-isolate subgroup, more than 70% of APEC and 80% of AFEC were multidrug resistant. Among the subgroup of APEC, MLST analysis identified 11 sequence types (ST) while seven STs were found among AFEC. Based on PFGE, the genetic relatedness of APEC and AFEC ranged from 50%–100% and clustered into four primary groups at 50% similarity. Two of the eight APEC strains tested in chickens were able to induce 25% mortality in 1-day-old chicks. APECs were distinguished from AFECs and environmental E. coli by their content of plasmid PAI genes, whereas APEC isolated from colisepticemia and AFEC were not distinguishable based on their antimicrobial resistance patterns, as both groups were multidrug resistant. Avian E. coli strains from broiler flocks in Egypt show similar sequence types to E. coli associated with human infection.

The objective of this study was to determine the prevalence and molecular typing of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in food-producing animals and retail meat in Fargo, North Dakota. A two-step enrichment followed by culture methods were used to isolate S. aureus from 167 nasal swabs from animals, 145 samples of retail raw meat, and 46 samples of deli meat. Positive isolates were subjected to multiplex polymerase chain reaction in order to identify the genes 16S rRNA, mecA, and Panton-Valentine Leukocidin. Pulsed-field gel electrophoresis and multilocus sequence typing were used for molecular typing of S. aureus strains. Antimicrobial susceptibility testing was carried out using the broth microdilution method. The overall prevalence of S. aureus was 37.2% (n = 133), with 34.7% (n = 58) of the animals positive for the organism, and the highest prevalence observed in pigs (50.0%) and sheep (40.6%) (p < 0.05); 47.6% (n = 69) of raw meat samples were positive, with the highest prevalence in chicken (67.6%) and pork (49.3%) (p < 0.05); and 13.0% (n = 6) of deli meat was positive. Five pork samples (7.0%) were positive for MRSA, of which three were ST398 and two were ST5. All exhibited penicillin resistance and four were multidrug resistant (MDR). The Panton-Valentine Leukocidin gene was not detected in any sample by multiplex polymerase chain reaction. The most common clones in sheep were ST398 and ST133, in pigs and pork both ST398 and ST9, and in chicken ST5. Most susceptible S. aureus strains were ST5 isolated from chicken. The MDR isolates were found in pigs, pork, and sheep. The presence of MRSA, MDR, and the subtype ST398 in the meat production chain and the genetic similarity between strains of porcine origin (meat and animals) suggest the possible contamination of meat during slaughtering and its potential transmission to humans. College of Agriculture, Food Systems and Natural Resources College, North Dakota State University College of Veterinary Medicine, Iowa State University

Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide threatening the use of one of the most important antimicrobials for treating human disease. Here, we examined a collection (n = 980) of Avian Pathogenic Escherichia coli (APEC) isolated from poultry with colibacillosis from the US and internationally for the presence of mcr-1 and mcr-2, genes known to encode colistin resistance. Included in the analysis was an additional set of avian fecal E. coli (AFEC) (n = 220) isolates from healthy birds for comparative analysis. The mcr-1 gene was detected in a total of 12 isolates recovered from diseased production birds from China and Egypt. No mcr genes were detected in the healthy fecal isolates. The full mcr-1 gene from positive isolates was sequenced using specifically designed primers and were compared with sequences currently described in NCBI. mcr-1 positive isolates were also assessed for phenotypic colistin resistance and extended spectrum beta lactam phenotypes and genotypes. This study has identified mcr-1 in APEC isolates dating back to at least 2010 and suggests that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced.

Pathogens associated with food production (livestock) animals come in many forms causing a multitude of disease for humans. For the purpose of this review, these infectious agents can be divided into three broad categories: those that are associated with bacterial disease, those that are associated with viruses, and those that are parasitic in nature. The goal of this chapter is to provide the reader with an overview of the most common pathogens that cause disease in humans through exposure via the food chain and the consequence of this exposure as well as risk and detection methods. We have also included a collection of unusual pathogens that although rare have still caused disease, and their recognition is warranted in light of emerging and reemerging diseases. These provide the reader an understanding of where the next big outbreak could occur. The influence of the global economy, the movement of people, and food makes understanding production animal-associated disease paramount to being able to address new diseases as they arise.

Avian-pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, a disease that affects all facets of poultry production worldwide, resulting in multimillion dollar losses annually. Here, we report the genome sequence of an APEC O18 sequence type 95 (ST95) strain associated with disease in a chicken.

Neonatal meningitis Escherichia coli (NMEC) is a common agent of neonatal bacterial meningitis, causing high neonatal mortality and neurologic sequelae in its victims. Here, we present the complete genome sequence of NMEC O18 (also known as NMEC 58), a highly virulent (O18ac:K1, ST416) strain.

Avian pathogenic Escherichia coli (APEC) is associated with colibacillosis in poultry. Here, we present the first complete sequence of an APEC strain of the O7:HNT serotype and ST73 sequence type, isolated from a broiler with cellulitis. Complete genomes of APEC with distinct genetic backgrounds may be useful for comparative analysis.

Neonatal meningitis Escherichia coli (NMEC) is one of the top causes of neonatal meningitis worldwide. Here, 85 NMEC and 204 fecal E. coli isolates from healthy humans (HFEC) were compared for possession of traits related to virulence, antimicrobial resistance, and plasmid content. This comparison was done to identify traits that typify NMEC and distinguish it from commensal strains to refine the definition of the NMEC subpathotype, identify traits that might contribute to NMEC pathogenesis, and facilitate choices of NMEC strains for future study. A large number of E. coli strains from both groups were untypeable, with the most common serogroups occurring among NMEC being O18, followed by O83, O7, O12, and O1. NMEC strains were more likely than HFEC strains to be assigned to the B2 phylogenetic group. Few NMEC or HFEC strains were resistant to antimicrobials. Genes that best discriminated between NMEC and HFEC strains and that were present in more than 50% of NMEC isolates were mainly from extraintestinal pathogenic E. coli genomic and plasmid pathogenicity islands. Several of these defining traits had not previously been associated with NMEC pathogenesis, are of unknown function, and are plasmid located. Several genes that had been previously associated with NMEC virulence did not dominate among the NMEC isolates. These data suggest that there is much about NMEC virulence that is unknown and that there are pitfalls to studying single NMEC isolates to represent the entire subpathotype.

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.

The aims of this study were to determine the ability of amplified fragment length polymorphism (AFLP) to differentiate Salmonella isolates from different units of swine production and to demonstrate the relatedness of Salmonella between farms and abattoirs by AFLP. Twenty-four farms in the midwestern United States were visited four times from 2006 to 2009. At each farm or abattoir visit, 30 fecal samples or 30 mesenteric lymph nodes were collected, respectively. A total of 220 Salmonella isolates were obtained, serotyped, and genotyped by multilocus sequence typing (MLST) and AFLP. These 220 isolates clustered into 21 serotypes, 18 MLST types, and 14 predominant AFLP clusters based on a genetic similarity threshold level of 60%. To assess genetic differentiation between farms, harvest cohorts, and pigs, analysis of molecular variance was conducted using AFLP data. The results showed 65.62% of overall genetic variation was attributed to variance among pigs, 27.21% to farms, and 7.17% to harvest cohorts. Variance components at the farm ( P = 0.003) and pig ( P = 0.001) levels were significant, but not at the harvest cohort level ( P = 0.079). A second analysis, a permutation test using AFLP data, indicated that on-farm and at-abattoir Salmonella from pigs of the same farms were more related than from different farms. Therefore, among the three subtyping methods, serotyping, MLST, and AFLP, AFLP was the method that was able to differentiate among Salmonella isolates from different farms and link contamination at the abattoir to the farm of origin.

Vaccine regimes and maternal antibody protection are important in ensuring the health of poultry and critically important in decreasing contamination of poultry products with foodborne pathogens that threaten human health. Here, we assessed the role of passive immunity on the resistance of progeny to early colonization when challenged with Salmonella Heidelberg. Two broiler breeder hen flocks which had received a Salmonella vaccine regime consisting of two live attenuated and two killed vaccines during rearing were selected for study. ELISA titers were used to assess antibody levels in the parent flocks, with one low- and one high- titer flock selected for study. Progeny chicks (1 day of age) were taken from each flock and challenged with Salmonella Heidelberg at low (103) or high doses (105) at 3 days of age. At 14 days postinoculation, all birds were euthanatized and their liver, spleen, and ceca collected for culture. ELISA analysis found Flock A (30-wk flock) demonstrated higher Salmonella antibody titers in the parents as well as yolk titers in the progeny, resulting in greater early protection from colonization by Salmonella Heidelberg while Salmonella colonization rates were higher in the progeny of the older parent Flock (B) that demonstrated lower antibody titers in the parents and the yolks of the progeny. These results suggest that as the breeder hens' protective antibodies wane with age; the maternal antibody protection in their progeny also becomes less effective in preventing cecal colonization by Salmonella early in life, which has the potential for affecting the health of the bird and contamination of meat products destined for the consumer.

This paper describes the application of gas-phase Fourier transform infrared (FTIR) spectroscopy for its ability to discriminate between Salmonella enterica typhimurium (Salmonella typhimurium) contaminated packaged beef samples and uncontaminated samples. A suitable headspace sampling system was used to deliver the headspace volatiles from the packaged meat to the FTIR gas cell. FTIR spectral signatures collected on headspace volatiles of meat packages were used to classify the meat samples based on their S. typhimurium populations. The most informative wavenumbers (features) were selected using three univariate and one multivariate feature selection algorithms. The selected wavenumbers were then used to develop the statistical discriminant models and validated using bootstrapping. It was found that sequential forward selection provided the highest estimated classification accuracy of 99% and mean estimated classification accuracy of 95% (validated using linear discriminant analysis and bootstrapping technique). The results support the use of gas phase FTIR to discriminate S. typhimurium contaminated beef samples.

Aims: This study assessed the effects of the therapeutic use of Tylan® in a large-scale turkey production facility on the selection of macrolide-resistant Campylobacter. Methods and Results: A flock of production turkeys (c. 30 000 birds) was followed from brooding to slaughter, and the effects of macrolide application was assessed in one half of the flock from finishing stage to final product and compared against the control barn where no macrolide was used. Overall, Campylobacter prevalence in turkeys was almost 100% by 4 weeks of age. When Campylobacter prevalence was assessed in relation to treatment, high levels of macrolide resistance were evident in this group following treatment, with Campylobacter coli becoming the dominant strain type. Over time, and in the absence of a selection agent, the population of resistant strains decreased suggesting that there was a fitness cost associated with macrolide resistance carriage and persistence. Macrolide resistance was detected in the control barn at a very low level (four isolates recovered during the study), suggesting that the creation or selection of macrolide-resistant Campylobacter was correlated with the treatment regime used. Molecular analysis of a selection of macrolide-resistant Campylobacter recovered was assessed using PCR, RFLP and sequence analysis of the 23S rRNA. The majority of isolates displaying high-level macrolide resistance (>256 μg ml−1) possessed an A2075G transition mutation in the 23S rRNA and the CmeABC efflux pump. Conclusions: These studies suggest that macrolide resistance can be promoted through the application of treatment during the grow-out phase and once established in a production facility has the potential to persist and be transferred to final product. Significance and Impact of the Study: The study highlights the prudent use of antimicrobials in treatment of disease in poultry. Of significance is the presence of macrolide-resistant Campylobacter in poultry production and finished product as a consequence of macrolide usage.

Escherichia coli strains causing avian colibacillosis and human neonatal meningitis, urinary tract infections, and septicemia are collectively known as extraintestinal pathogenic E. coli (ExPEC). Characterization of ExPEC strains using various typing techniques has shown that they harbor many similarities, despite their isolation from different host species, leading to the hypothesis that ExPEC may have zoonotic potential. The present study examined a subset of ExPEC strains: neonatal meningitis E. coli (NMEC) strains and avian-pathogenic E. coli (APEC) strains belonging to the O18 serogroup. The study found that they were not easily differentiated on the basis of multilocus sequence typing, phylogenetic typing, or carriage of large virulence plasmids. Among the APEC strains examined, one strain was found to be an outlier, based on the results of these typing methods, and demonstrated reduced virulence in murine and avian pathogenicity models. Some of the APEC strains tested in a rat model of human neonatal meningitis were able to cause meningitis, demonstrating APEC's ability to cause disease in mammals, lending support to the hypothesis that APEC strains have zoonotic potential. In addition, some NMEC strains were able to cause avian colisepticemia, providing further support for this hypothesis. However, not all of the NMEC and APEC strains tested were able to cause disease in avian and murine hosts, despite the apparent similarities in their known virulence attributes. Thus, it appears that a subset of NMEC and APEC strains harbors zoonotic potential, while other strains do not, suggesting that unknown mechanisms underlie host specificity in some ExPEC strains.

ColV plasmids of extraintestinal pathogenic Escherichia coli (ExPEC) encode a variety of fitness and virulence factors and have long been associated with septicemia and avian colibacillosis. These plasmids are found significantly more often in ExPEC, including ExPEC associated with human neonatal meningitis and avian colibacillosis, than in commensal E. coli . Here we describe pAPEC-O103-ColBM, a hybrid RepFIIA/FIB plasmid harboring components of the ColV pathogenicity island and a multidrug resistance (MDR)-encoding island. This plasmid is mobilizable and confers the ability to cause septicemia in chickens, the ability to cause bacteremia resulting in meningitis in the rat model of human disease, and the ability to resist the killing effects of multiple antimicrobial agents and human serum. The results of a sequence analysis of this and other ColV plasmids supported previous findings which indicated that these plasmid types arose from a RepFIIA/FIB plasmid backbone on multiple occasions. Comparisons of pAPEC-O103-ColBM with other sequenced ColV and ColBM plasmids indicated that there is a core repertoire of virulence genes that might contribute to the ability of some ExPEC strains to cause high-level bacteremia and meningitis in a rat model. Examination of a neonatal meningitis E. coli (NMEC) population revealed that approximately 58% of the isolates examined harbored ColV-type plasmids and that 26% of these plasmids had genetic contents similar to that of pAPEC-O103-ColBM. The linkage of the ability to confer MDR and the ability contribute to multiple forms of human and animal disease on a single plasmid presents further challenges for preventing and treating ExPEC infections.

Autotransporters (AT) are widespread in Gram-negative bacteria, and many of them are involved in virulence. An open reading frame (APECO1_O1CoBM96) encoding a novel AT was located in the pathogenicity island of avian pathogenic Escherichia coli (APEC) O1's virulence plasmid, pAPEC-O1-ColBM. This 3.5-kb APEC autotransporter gene ( aatA ) is predicted to encode a 123.7-kDa protein with a 25-amino-acid signal peptide, an 857-amino-acid passenger domain, and a 284-amino-acid β domain. The three-dimensional structure of AatA was also predicted by the threading method using the I-TASSER online server and then was refined using four-body contact potentials. Molecular analysis of AatA revealed that it is translocated to the cell surface, where it elicits antibody production in infected chickens. Gene prevalence analysis indicated that aatA is strongly associated with E. coli from avian sources but not with E. coli isolated from human hosts. Also, AatA was shown to enhance adhesion of APEC to chicken embryo fibroblast cells and to contribute to APEC virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) are major players in human urinary tract infections, neonatal bacterial meningitis, and sepsis. Recently, it has been suggested that there might be a zoonotic component to these infections. To determine whether the E. coli contaminating retail poultry are possible extraintestinal pathogens, and to ascertain the source of these contaminants, they were assessed for their genetic similarities to E. coli incriminated in colibacillosis (avian pathogenic E. coli [APEC]), E. coli isolated from multiple locations of apparently healthy birds at slaughter, and human ExPEC. It was anticipated that the retail poultry isolates would most closely resemble avian fecal E. coli since only apparently healthy birds are slaughtered, and fecal contamination of carcasses is the presumed source of meat contamination. Surprisingly, this supposition proved incorrect, as the retail poultry isolates exhibited gene profiles more similar to APEC than to fecal isolates. These isolates contained a number of ExPEC-associated genes, including those associated with ColV virulence plasmids, and many belonged to the B2 phylogenetic group, known to be virulent in human hosts. Additionally, E. coli isolated from the crops and gizzards of apparently healthy birds at slaughter also contained a higher proportion of ExPEC-associated genes than did the avian fecal isolates examined. Such similarities suggest that the widely held beliefs about the sources of poultry contamination may need to be reassessed. Also, the presence of ExPEC-like clones on retail poultry meat means that we cannot yet rule out poultry as a source of ExPEC human disease.

Campylobacter jejuni isolates (n = 340) were collected from nine turkey flocks in three rotations (A, B, and C) at a newly established turkey production facility in North Dakota and at processing. Samples were collected at weeks 1, 4, 9, and 18, as well as at two stages on the processing line at the processing plant. Campylobacter was not isolated from the first flocks in the rotations (A1, B1, and C1), but was detected at week 18 in the second flock groupings and at week 9 in the third flock groupings. The cumulative increase in Campylobacter prevalence observed in each subsequent rotation was attributed to flock rotation through the brooder barn, in which each flock was housed for 4 weeks before moving to a finishing barn; the brooder was the only common building shared by all flocks in each grouping (A, B, and C). C. jejuni isolates recovered were analyzed for the presence of selected virulence genes; 100% of the isolates tested were positive for the flaA, pldA, and cadF genes; 99.7% of the isolates were positive for the cdtB, cdtC, and ciaB genes. The prevalence of the cdtA and cjp05 genes was much lower at 11.2 and 67.5%, respectively. Results of this study indicate flock rotation may increase Campylobacter prevalence; molecular characterization provided information about Campylobacter from a new turkey production facility.

Aim: To determine the phenotypic and genotypic antimicrobial susceptibility profiles of Escherichia coli from bison carcasses. Methods and Materials: The antimicrobial resistance of 138 E. coli isolates recovered from processed bison carcasses was determined by using the National Antimicrobial Resistance Monitoring System panels, polymerase chain reaction assays, plasmid analysis and conjugation studies. Results: Resistance to 14 of the 16 antimicrobials was observed. Twenty-three (16·7%) isolates displayed resistance to at least one antimicrobial agent. The most prevalent resistances were to tetracycline (13·0%), sulfamethoxazole (7·9%) and streptomycin (5·8%). No resistance was observed to amikacin and ciprofloxacin. Further analysis of 23 antimicrobial-resistant E. coli isolates showed the presence of resistance genes corresponding to their phenotypic profiles. Results of conjugation studies carried out showed most isolates tested were able to transfer their resistance to recipients. Conclusion: This study indicated that multidrug-resistant E. coli isolates are present in bison. However, the resistance rate is lower than that reported in other meat species. Significance and Impact of the Study: The beneficial effects of antimicrobial-free feeding practice in bison may be promoting a reduction in the prevalence of antimicrobial resistance in commensal flora of bison.

Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae . Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli . IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC , cbi , and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations.