Occurrence of Virulence and Resistance Genes in Salmonella in Cloacae of Slaughtered Chickens and Ducks at Pluck Shops in Trinidad.

ABSTRACT: The study used PCR to determine the molecular basis of the antibiotic resistance and virulence profiles of isolates of Salmonella by targeting genes encoding for carriage and persistence in the poultry. Of a total 1,503 cecal samples collected from poultry, 91 (6.1%) were positive for Salmonella. Ten different serotypes were detected from Salmonella isolates. The study was also conducted to determine the occurrence of 13 virulence and 12 resistance genes in isolates of Salmonella. All 46 isolates of Salmonella tested were positive for one or more of the 12 virulence genes detected, ranging from 0.0% (viaB) to 100.0% (invA, mgtB, pipA, and spi4D) (P < 0.05). Occurrence of virulence genes varied significantly (P < 0.05) by serotype but not by animal species. Only 4 (33.3%) of 12 resistance genes assayed were detected: strA, ampC, cmy2, and qnrB. Overall, the occurrence of detected resistance genes was 71.7% (33 of 46), and 87.1 and 40.0% of the isolates from chickens and ducks, respectively, were positive (P = 0.0009). The occurrence of resistance genes ranged from 2.2% (cmy2) to 50.0% (qnrB) in isolates positive for resistance gene. The findings provide evidence that poultry from "pluck shops" are colonized by Salmonella pathogens that harbor virulence and antimicrobial resistance genes; this may have clinical and therapeutic consequences, if the genes detected are expressed. Although there is a need for prudent use of antimicrobial agents in poultry production systems, there should be constant monitoring for the prevalence of resistance in Salmonella isolates using phenotypic methods. The importance of monitoring the occurrence of resistance genes in the pathogens in Trinidad cannot be ignored.

Prevalence, Serovars, and Antimicrobial Resistance of Salmonella in Cecal Samples of Chickens Slaughtered in Pluck Shops in Trinidad.

The aim of the study was to determine the prevalence and zonal distribution of Salmonella serotypes in poultry and to determine the antimicrobial resistance profile of Salmonella isolates. A total of 1,503 cecal samples of poultry were randomly collected from 33 pluck shops across Trinidad. Isolation and identification of Salmonella followed standard methods, and the disk diffusion method was used to determine resistance of isolates to 14 antimicrobial agents. Ninety-one (6.1%) of the 1,503 samples collected from four zones were positive for Salmonella. The frequency of isolation of Salmonella from chicken ceca (6.5%) was higher than that detected in duck ceca (5.1%), but the difference was not statistically significant (P > 0.05). Ten serotypes were detected, with Salmonella Molade, Salmonella enterica subsp. enterica I, and Salmonella Typhimurium the most prevalent at 56.0, 11.0, and 8.8%, respectively. The highest frequency of isolation of Salmonella was recorded in the northeast zone (59.3%). All 91 isolates exhibited resistance to at least 1 of the 14 antimicrobial agents. The highest frequency of resistance was exhibited to ampicillin (51.0%), kanamycin (49.5%), and streptomycin (37.4%). A total of 22 resistance patterns were exhibited by the 91 isolates of Salmonella, and 13 isolates (14.3%) exhibited multiple drug resistance. The results emphasize the need to implement hygienic practices to reduce the levels of contamination at poultry pluck shops and the need for prudent use of antimicrobial agents in the poultry production system in Trinidad.

Prevalence of methicillin resistant Staphylococcus aureus (MRSA) in pigs and workers at abattoirs in Trinidad and Tobago.

INTRODUCTION: Methicillin resistant Staphylococcus aureus (MRSA), a major cause of zoonotic infections, has emerged globally in livestock, particularly pigs. People with occupational contact with food producing animals are at high risk of colonization. The aim of this study was to determine the prevalence of MRSA in pigs and abattoir workers throughout Trinidad and Tobago as well as their resistance to other antimicrobial agents. METHODOLOGY: Nasal and skin behind the ear swabs from pigs and nasal swabs from humans were enriched in Mueller Hinton broth with 6.5% sodium chloride, followed by phenol red mannitol broth with 75 mg/L aztreonam and 5 mg/L ceftizoxime. The enriched sample was then plated on both CHROMagar MRSA and Brilliance MRSA. All incubation was at 37ºC for approximately 24 h. Suspect MRSA isolates were confirmed as MRSA using the Penicillin-Binding Protein (PBP2a) test kit and polymerase chain reaction (PCR) to detect the mecA gene. Resistance of the S. aureus and MRSA isolates to 16 antimicrobial agents was determined using the disc diffusion method. RESULTS: Of the 929 pigs and 44 humans sampled, MRSA strains were isolated at a frequency of 0.9% (8/929) and 2.3% (1/44) respectively. All isolates exhibited resistance to one or more of the 16 antimicrobial agents. CONCLUSIONS: The study demonstrated that pigs and workers at slaughter houses in Trinidad and Tobago harbour multidrug resistance S. aureus and MRSA. This is of public health significance as occupational exposure of humans can lead to an increased risk of infection and therapeutic failure.

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in broilers and workers at 'pluck shops' in Trinidad.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a cause of zoonotic infections in many countries. People with occupational contact with food animal production are at risk of colonization. The aim of this study was to determine the prevalence of MRSA and their frequency of resistance to other antimicrobial agents from broilers and workers at the 'pluck shops' in Trinidad. For isolation of MRSA, choanal, cloacal and pharyngeal swabs taken from broilers and nasal swabs from humans were enriched then plated on CHROMagar MRSA and Brilliance MRSA. MRSA was confirmed using the PBP2a test kit, resistance to oxacillin and cefoxitin and polymerase chain reaction (PCR) for the mecA gene. Antimicrobial resistance of the MRSA isolates to 16 antimicrobial agents was determined using the disc diffusion method. Of the 287 broilers and 47 humans sampled, MRSA was isolated at a frequency of 2 (0.7%) and 0 (0.0%) respectively. All the MRSA isolates exhibited resistance to one or more of the 16 antimicrobial agents. The study demonstrated that broilers at 'pluck shops' in Trinidad harbor MRSA. This is the first isolation of MRSA from poultry in Trinidad, West Indies, and this finding is of public health significance since occupational exposure of humans can lead to increased risk of acquiring MRSA infections.

Detection of a CTX-M group 2 beta-lactamase gene in a Klebsiella pneumoniae isolate from a tertiary care hospital, Trinidad and Tobago.

BACKGROUND: Identification of the prevalence and spread of ESBL-mediated antibiotic resistance is essential especially in the hospital setting. It is for this reason, more and more studies are highlighting the importance of complementing phenotypic ESBL-detection techniques with molecular techniques in order to understand the basis and extent of this form of resistance among clinically evolved bacterial populations, especially those belonging to the Enterobacteriaceae family. However, in Trinidad and Tobago and other Caribbean countries, very little is known regarding ESBL detection rates and/or the prevalence of genes conferring ESBL resistance. METHODOLOGY: Sixty-six Klebsiella pneumoniae isolates from clinical specimens phenotypically identified by the Microscan Walkaway-96 System as potential ESBL-producers were analysed in this study. Screening and confirmation of these isolates as ESBL producers was done by the Clinical and Laboratory Standards Institute (CLSI) approved methods. Polymerase chain reaction amplification of beta-lactamase genes bla TEM, bla SHV, bla CTX-M1, bla CTX-M2 and bla AmpC was performed to identify mechanisms of ß-lactam resistance. RESULTS: ESBL-producing K. pneumoniae was confirmed in 78.8% (41/52) from isolates collected from a variety of sources during the period, April-July 2015. bla SHV (84.8%) and bla CTX-M (46.9%) were the predominant ß-lactamase genes identified. A single K. pneumoniae isolate possessed a bla CTX-M group 2 beta-lactamase gene. RAPD analysis identified a number of epidemiologically related isolates. However, current isolates were unrelated to isolates from previous years. CONCLUSION: This study revealed that among K. pneumoniae isolates exhibiting extended spectrum ß-lactam resistance, there was a high prevalence of bla SHV and bla CTX-M genes. This result highlights the need for a reliable epidemiological apparatus that involves the molecular characterisation of ESBL resistance.

AEROBIC MICROFLORA AND ENDOPARASITES OF FRESHLY SHOT WILD AGOUTI (DASYPROCTA LEPORINA) IN TRINIDAD, WEST INDIES.

The agouti ( Dasyprocta leporina ) is a New World wild rodent hunted for its meat in Trinidad and other Latin American countries. Studies on agouti under captive conditions have yielded some data on health-related aspects, but relatively very little is known about their wild counterparts. The environment of the agouti can influence the microflora and parasites harbored by the animals, which may contain zoonotic pathogens. Here, the microflora found on the nasal mucosa and sections of the intestinal tract and endoparasites of freshly shot agouti from various areas of Trinidad are described. Staphylococcus epidermidis , S. intermedius , Bacillus spp., Enterobacter spp. and Escherichia coli comprised the majority of bacteria isolated from the nasal mucosa whereas Escherichia coli , Streptococcus viridans, Bacillus spp. and Klebsiella pneumoniae were predominant in all sections of the intestinal tract. The fungi Aspergillus fumigatus , Aspergillus spp., Candida spp., Penicillium spp., and Mucor spp. were only isolated from the nasal cavity but not in any section of the intestinal tract. The parasites Strongyloides spp., Ascaridia spp., a hookworm, a trematode, and Trichuris spp. were detected at variable frequencies in each of the sections of the intestines (small intestine, large intestine, caecum), whereas Eimeria spp. were found in all sections (76.9%, 10 of 13 agoutis). These wild agoutis were presumably healthy at the time of death and represent animals that hunters may encounter. Some of the detected pathogens and parasites have the potential to cause opportunistic infections or infestations, especially in immune-compromised hosts.

First identification of methicillin-resistant Staphylococcus pseudintermedius strains among coagulase-positive staphylococci isolated from dogs with otitis externa in Trinidad, West Indies.

BACKGROUND: Otitis externa is a common inflammatory ear disease in dogs caused by a variety of pathogens, and coagulase-positive staphylococci are frequently isolated from such infections. OBJECTIVE: To identify antimicrobial susceptibility profiles and methicillin-resistant strains among coagulase-positive staphylococci isolated from otitis externa in dogs. METHODS: A cross-sectional study was performed over 2 years on 114 client-owned dogs presented to the Veterinary Teaching Hospital with a primary complaint of ear infections. Swabs were obtained from both ears and cultured for staphylococci which were subsequently confirmed as coagulase-positive using rabbit plasma. Antimicrobial susceptibility assays were assessed on all isolates followed by subsequent genetic analysis for species identification and detection of the mecA gene. RESULTS: Sixty-five coagulase-positive staphylococci were isolated from 114 client-owned dogs. The isolates exhibited resistance against neomycin (58.5%), streptomycin (49.2%), penicillin (49.2%), polymyxin B (44.6%), tetracycline (36.9%), sulphamethoxazole/trimethoprim (33.8%), kanamycin (33.8%), doxycycline (32.3%), norfloxacin (23.1%), amoxicillin/clavulanate (20%), ciprofloxacin (20%), enrofloxacin (18.5%), gentamicin (16.9%), and cephalothin (9.2%). Forty (61.5%) of the isolates were resistant to at least three or more antimicrobials and 10 were sensitive to all. Using a multiplex polymerase chain reaction assay based on species-specific regions of the thermonuclease (nuc) gene, 38/65 (58.5%) isolates were classified as Staphylococcus aureus, 23/65 (35.4%) as S. pseudintermedius, 2/65 (3.1%) as S. intermedius, and 2/65 (3.1%) as S. schleiferi. Analysis for the mecA gene revealed two positive isolates of S. pseudintermedius which were oxacillin-resistant, representing a first report of such organisms in the Caribbean. CONCLUSION: Despite the relatively high prevalence of multidrug-resistant coagulase-positive staphylococci in Trinidad, these are largely susceptible to gentamicin consistent with use in clinical practice. The first detection of methicillin-resistant S. pseudintermedius (MRSP) in dogs is likely to have implications on the treatment options for otitis externa in dogs and potential public health significance.

Incidence of Nontyphoidal Salmonella in Food-Producing Animals, Animal Feed, and the Associated Environment in South Africa, 2012-2014.

BACKGROUND: Nontyphoidal salmonellosis continues to pose a global threat to human health, primarily by causing food-borne illnesses, and food-producing animals are the principal reservoirs of many pathogenic serovars. To identify key control points and generate information that may enable future estimation of the transmission routes between the environment, animals, and humans, we examined data on Salmonella isolates in South Africa. METHODS: Samples were obtained from livestock and poultry on farms, meat at abattoirs, raw materials at feed mills, animal feed, and environmental sources (eg, poultry houses, abattoirs, feed mills, water) from 2012 to 2014 in compliance with each establishment's protocols conforming to International Organization for Standardization (ISO) (ISO/TS 17728, ISO 18593:2004 and ISO 17604:2003) standards. Isolation and serotyping of Salmonella were performed according to the scope of accreditation of the respective laboratories conforming to ISO/IEC 17025:2005 standard techniques. RESULTS: Salmonella was isolated from 9031 of 180 298 (5.0%) samples, and these isolates were distributed among 188 different serovars. Salmonella Enteritidis was the most frequent isolate, with 1944 of 180 298 (21.5%) originating from poultry on farms, poultry meat, and poultry houses, followed by Salmonella Havana, with 677 of 180 298 (7.5%), mostly from environmental samples. Serovars that are uncommonly associated with human disease (Salmonella Idikan, Salmonella Salford, and Salmonella Brancaster) were isolated at higher frequencies than Salmonella Typhimurium, a common cause of human illness. Environmental samples accounted for 3869 of 9031 (42.8%) samples positive for Salmonella. CONCLUSIONS: We describe the frequent isolation of Salmonella of a wide variety of serovars, from an array of animal feeds, food animals, and food animal environment. As prevention of human salmonellosis requires the effective control of Salmonella in food animals, these data can be used to facilitate Salmonella control in food animals and thereby prevent human infections.

Challenges of Sanitary Compliance Related to Trade in Products of Animal Origin in Southern Africa.

Irrespective of the existence of potentially pathogenic organisms carried by animals, foods of animal origin remain the prime nutrition of humans world-wide. As such, food safety continues to be a global concern primarily to safeguard public health and to promote international trade. Application of integrated risk-based quality assurance procedures on-farm and at slaughterhouses plays a crucial role in controlling hazards associated with foods of animal origin. In the present paper we examine safety assurance systems and associated value chains for foods of animal origin based on historical audit results of some Southern African countries with thriving export trade in animal products, mainly to identify areas for improvement. Among the key deficiencies identified were: i) failure to keep pace with scientific advances related to the ever-changing food supply chain; ii) lack of effective national and regional intervention strategies to curtail pathogen transmission and evolution, notably the zoonotic Shiga toxin-producing Escherichia coli; and iii) a lack of effective methods to reduce contamination of foods of wildlife origin. The introduction of foods of wildlife origin for domestic consumption and export markets seriously compounds already existing conflicts in legislation governing food supply and safety. This analysis identifies gaps required to improve the safety of foods of wildlife origin.

A cyclophosphamide-sensitive cell compartment is essential for homologous protection conferred by licensed vaccines for the control of avian pathogenic Escherichia coli in chickens.

Avian pathogenic Escherichia coli (APEC) exert substantial economic costs on poultry producers worldwide. Vaccination is an attractive method of control, but the immunological basis of protection is poorly understood. Here, we examine the effect of intramuscular injection of cyclophosphamide or saline on homologous protection induced by licensed inactivated or live-attenuated APEC O78 vaccines in chickens. In saline-treated birds, both vaccines induced significant APEC-specific IgY and protection against homologous challenge, as evidenced by enumeration of tissue-associated bacteria and analysis of pathology. In cyclophosphamide-treated birds, B cells were severely depleted whereas percentages of circulating CD4- and CD8-positive T cells were normal as detected by flow cytometry. Further, such birds did not produce APEC-specific IgY and were as susceptible to challenge as age-matched unvaccinated controls. The data indicate that homologous protection conferred by licensed APEC vaccines strictly requires a cyclophosphamide-sensitive cell population that includes B cells.

Immune responses associated with homologous protection conferred by commercial vaccines for control of avian pathogenic Escherichia coli in turkeys.

Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.

Analysis of immune responses induced by avian pathogenic Escherichia coli infection in turkeys and their association with resistance to homologous re-challenge.

Avian pathogenic Escherichia coli (APEC) cause severe respiratory and systemic disease in poultry yet the nature and consequences of host immune responses to infection are poorly understood. Here, we describe a turkey sub-acute respiratory challenge model and cytokine, cell-mediated and humoral responses associated with protection against homologous re-challenge. Intra-airsac inoculation of turkeys with 105 colony-forming units of APEC O78:H9 strain χ7122nalR induced transient and mild clinical signs of colibacillosis followed by clearance of the bacteria from the lungs and visceral organs. Upon re-challenge with 107 χ7122nalR, primed birds were solidly protected against clinical signs and exhibited negligible bacterial loads in visceral organs, whereas age-matched control birds exhibited high lesion scores and bacterial loads in the organs. Levels of mRNA for signature cytokines suggested induction of a Th1 response in the lung, whereas a distinct anti-inflammatory cytokine profile was detected in the liver. Proliferative responses of splenocytes to either Concanavalin A or soluble χ7122nalR antigens were negligible prior to clearance of bacteria, but APEC-specific responses were significantly elevated at later time intervals and at re-challenge relative to control birds. Primary infection also induced significantly elevated χ7122nalR-specific serum IgY and bile IgA responses which were bactericidal against χ7122nalR and an isogenic Δrfb mutant. Bactericidal activity was observed in the presence of immune, but not heat-inactivated immune serum, indicating that the antibodies can fix complement and are not directed solely at the lipopolysaccharide O-antigen. Such data inform the rational design of strategies to control a recalcitrant endemic disease of poultry.

Investigating the contributing factors to postmortem pH changes in springbok, eland, red hartebeest and kudu edible offal.

The objective of the study was to assess pH measurements between offal organs of different species and the association between pH taken 4 h post-slaughter and different predictor variables in the liver and lungs. A linear regression analysis was conducted on selected variables to identify the main predictors and their interactions affecting the pH of meat 4 h post-slaughter. In an increasing order of magnitude during winter, the pH achieved at 16 h - 36 h post-slaughter in springbok heart, liver, spleen, kidney and lungs was significantly (p < 0.05) higher than pH 6.0. The pH attained in springbok carcasses was (p < 0.05) below 6.0, whilst no significant differences were observed from the regulatory reference (pH 6.0) in the heart. There was a positive association between the pH of game meat 4 h post-slaughter and liver congestion. The pH of game meat 4 h post-slaughter increased by 0.11 units (p < 0.05) per millilitre increase in liver congestion and decreased by 0.04 units (p< 0.05) per minute increase in the shooting-to-bleeding interval, irrespective of the species. The lack of a statistically significant association between some selected variables and pH changes in this study suggested that either the factors may have a small effect which is only detectable with large data-sets and/or the effect may be modified by other unidentified factors. As some of the offal organs had final pH readings above 6.0, alternative measures are required to inactivate certain endogenous pathogens in edible wild game offal sourced from endemic areas.

Sequencing and functional annotation of avian pathogenic Escherichia coli serogroup O78 strains reveal the evolution of E. coli lineages pathogenic for poultry via distinct mechanisms.

Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes.

Zoonoses: a potential obstacle to the growing wildlife industry of Namibia.

Zoonoses, which account for approximately 75% of emerging human infectious diseases worldwide, pose a re-emerging threat to public health. With an ever-increasing interrelationship between humans, livestock and wildlife species, the threat to human health will rise to unprecedented levels. Wildlife species contribute to the majority of emerging diseases; therefore, there is an urgent need to define control systems of zoonoses of wildlife origin but very little information exists. In this review, we examine prevalent zoonotic infections reported in Namibia between 1990 and 2009 and assess their potential impact on the growing wildlife industry. A wide spectrum of zoonotic diseases was confirmed in both livestock and wildlife species, with rabies and anthrax cases being over-represented and also showing the widest species distribution. Whilst vaccination and ante-mortem inspection against these diseases may curb infected livestock species from entering the human food chain, such practices are difficult to implement in free-ranging wildlife species. In this context, there is a need to improve existing control measures and/or develop novel and better interventional strategies to reduce the threat of this re-emerging global problem. This review provides the basis for initiating a multidisciplinary evidence-based approach to control zoonoses in countries with thriving wildlife and game farming.

Retrospective application of transposon-directed insertion site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in cattle.

Massively parallel sequencing of transposon-flanking regions assigned the genotype and fitness score to 91% of Escherichia coli O157:H7 mutants previously screened in cattle by signature-tagged mutagenesis (STM). The method obviates the limitations of STM and markedly extended the functional annotation of the prototype E. coli O157:H7 genome without further animal use.

Role of two-component sensory systems of Salmonella enterica serovar Dublin in the pathogenesis of systemic salmonellosis in cattle.

Salmonella enterica serovar Dublin (S. Dublin) is associated with enteritis, typhoid and abortion in cattle. Infections are acquired by the oral route, and the bacteria transit through varied anatomical and cellular niches to elicit systemic disease. S. Dublin must therefore sense and respond to diverse extrinsic stimuli to control gene expression in a spatial and temporal manner. Two-component systems (TCSs) play key roles in such processes, and typically contain a membrane-associated sensor kinase (SK) that modifies a cognate response regulator. Analysis of the genome sequence of S. Dublin identified 31 conserved SK genes. Each SK gene was separately disrupted by lambda Red recombinase-mediated insertion of transposons harbouring unique sequence tags. Calves were challenged with a pool of the mutants together with control strains of defined virulence by the oral and intravenous routes. Quantification of tagged mutants in output pools derived from various tissues and cannulated lymphatic vessels allowed the assignment of spatial roles for each SK following oral inoculation or when the intestinal barrier was bypassed by intravenous delivery. Mutant phenotypes were also assigned in cultured intestinal epithelial cells. Mutants with insertions in barA, envZ, phoQ, ssrA or qseC were significantly negatively selected at all enteric and systemic sites sampled after oral dosing. Mutants lacking baeS, dpiB or citA were negatively selected at some but not all sites. After intravenous inoculation, only barA and phoQ mutants were significantly under-represented at systemic sites. The novel role of baeS in intestinal colonization was confirmed by oral co-infection studies, with a mutant exhibiting modest but significant attenuation at a number of enteric sites. This is the first systematic analysis of the role of all Salmonella TCSs in a highly relevant model of enteric fever. Spatial roles were assigned to eight S. Dublin SKs, but most were not essential for intestinal or systemic infection of the target host.

Efa-1/LifA mediates intestinal colonization of calves by enterohaemorrhagic Escherichia coli O26 : H- in a manner independent of glycosyltransferase and cysteine protease motifs or effects on type III secretion.

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of animal and zoonotic pathogens of worldwide importance. Our previous research established that intestinal colonization of calves by EHEC serotypes O5 : H- and O111 : H- requires EHEC factor for adherence (Efa-1), also known as lymphostatin (LifA). Towards an understanding of the mode of action of Efa-1/LifA, chromosomal in-frame deletions of predicted glycosyltransferase (DXD) and cysteine protease (CHD) motifs were created in a Deltastx1 derivative of EHEC O26 : H-. The magnitude and duration of faecal excretion of EHEC O26 : H- were significantly reduced by null mutation of efa-1/lifA, but were not impaired by DeltaDXD or DeltaCHD mutations, in contrast to observations made with truncated Efa-1/LifA mutants of Citrobacter rodentium in mice. Although C. rodentium Efa-1/LifA influences the induction of colonic hyperplasia in mice, EHEC O26 : H- Efa-1/LifA was not required for fluid accumulation or neutrophil recruitment in bovine ileal loops. In contrast to observations with EHEC O5 : H- or O111 : H- mutants, inactivation of efa-1/lifA in EHEC O26 : H- did not significantly affect adherence or secretion of type III secreted proteins that play pivotal roles in calf colonization. Lymphostatin activity could not be reliably demonstrated in lysates of EHEC O26 : H-; however, deletion of the glycosyltransferase and cysteine protease motifs in Efa-1/LifA from enteropathogenic E. coli O127 : H6 abolished lymphostatin activity. Our data uncouple the role of Efa-1/LifA in calf colonization from effects on type III secretion and reinforce the potential for pathotype- and serotype-specific phenotypes.

Norepinephrine augments Salmonella enterica-induced enteritis in a manner associated with increased net replication but independent of the putative adrenergic sensor kinases QseC and QseE.

Stress has long been correlated with susceptibility to microbial infection. One explanation for this phenomenon is the ability of pathogens to sense and respond to host stress-related catecholamines, such as norepinephrine (NE). In Gram-negative enteric pathogens, it has been proposed that NE may facilitate growth by mediating iron supply, or it may alter gene expression by activating adrenergic sensor kinases. The aim of this work was to investigate the relative importance of these processes in a model in which NE alters the outcome of Salmonella enterica serovar Typhimurium infection. A bovine ligated ileal loop model was used to study the effect of NE on enteritis induced by S. Typhimurium and on the bacterial in vivo replication rate. Mutants lacking putative adrenergic receptor genes were assessed in the loop model, in a calf intestinal colonization model, and in vitro. S. Typhimurium-induced enteritis was significantly enhanced by addition of 5 mM NE. This effect was associated with increased net bacterial replication in the same model. Exogenous ferric iron also stimulated bacterial replication in the medium used but not transcription of enteritis-associated loci. The putative adrenergic sensors QseC and QseE were not required for NE-enhanced enteritis, intestinal colonization of calves, or NE-dependent growth in iron-restricted medium and did not influence expression or secretion of enteritis-associated virulence factors. Our findings support a role for stress-related catecholamines in modulating the virulence of enteric bacterial pathogens in vivo but suggest that bacterial adrenergic sensors may not be the vital link in such interkingdom signaling in Salmonella.

Interactions of typical and atypical enteropathogenic Escherichia coli strains with the calf intestinal mucosa ex vivo.

Enteropathogenic Escherichia coli (EPEC) can be found in healthy and diarrheic cattle; however, little is known about the role of attaching and effacing (A/E) lesion formation in colonization of bovine intestinal mucosa by such strains. We show that typical and atypical EPEC induce A/E lesions on calf intestinal explants independently of Tir tyrosine phosphorylation and TccP. Our data support the existence of conserved Tir- and TccP-independent mechanisms of A/E lesion formation in a range of hosts and reinforce the zoonotic potential of EPEC in cattle.