Quantification and prevalence of Salmonella in beef cattle presenting at slaughter

Aims: A survey to determine the prevalence and numbers of Salmonella in beef cattle presented for slaughter at abattoirs across Australia was conducted between September 2002 and January 2003. Methods and Results: Automated immunomagnetic separation (AIMS) was used for detection and isolation of Salmonella enriched from cattle faeces. Salmonella were enumerated from positive samples using a combination of the Most Probable Number (MPN) technique and AIMS. A total of 310 faecal samples were tested, 155 were from lot-fed cattle and 155 from grass-fed cattle. Salmonella spp. were isolated from 21 (6.8%) of the cattle and the prevalence amongst grass-fed cattle (4.5%) was not significantly different to that found in lot-fed cattle (9%). Counts of Salmonella in positive faeces varied from

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

Long-term persistence of multi-drug-resistant Salmonella enterica serovar Newport in two dairy herds

Objective - To evaluate the association between maintaining joint hospital and maternity pens;and persistence of multi-drug-resistant (MDR) Salmonella enterica serovar Newport on 2 dairy farms. Design - Observational study. Sample Population - Feces and environmental samples from 2 dairy herds. Procedure - Herds were monitored for fecal shedding of S enterica Newport after outbreaks of clinical disease. Fecal and environmental samples were collected approximately monthly from pens housing sick cows and calving cows and from pens containing lactating cows. Cattle shedding the organism were tested serially on subsequent visits to determine carrier status. One farm was resampled after initiation of interventional procedures, including separation of hospital and maternity pens. Isolates were characterized via serotyping, determination of antimicrobial resistance phenotype, detection of the CMY-2 gene, and DNA fingerprinting. Results - The prevalence (32.4% and 33.3% on farms A and B, respectively) of isolating Salmonella from samples from joint hospital-maternity pens was significantly higher than the prevalence in samples from pens housing preparturient cows (0.8%, both farms) and postparturient cows on Farm B (8.8%). Multi-drug-resistant Salmonella Newport was isolated in high numbers from bedding material, feed refusals, lagoon slurry, and milk filters. One cow excreted the organism for 190 days. Interventional procedures yielded significant reductions in the prevalences of isolating the organism from fecal and environmental samples. Most isolates were of the C2 serogroup and were resistant to third-generation cephalosporins. Conclusions and Clinical Relevance - Management practices may be effective at reducing the persistence of MDR Salmonella spp in dairy herds, thus mitigating animal and public health risk.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (> 75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.

Antibiotic and biocide resistance in Salmonella enterica and Escherichia coli 0157

Bacterial resistance to antibiotics and biocides is a prevalent problem, which may be exacerbated by the commonplace and often unnecessary inclusion of biocides into domestic products. Addition of antimicrobials, to domestic disinfectants has raised concern about promoting microbial resistance and potential cross-resistance to therapeutic antibiotics. This study investigated the potential for resistance in Salmonella enterica serovars Enteritidis, Typhimurium, Virchow and Escherichia call 0157 to commonly used biocides, to identify mechanisms underlying resistance and whether these provided cross-resistance to antibiotics. Salmonella enterica and E. coli 0157 strains were serially exposed to sub-inhibitory. concentrations of erythromycin (ERY), benzalkonium chloride (BKC), chlorhexidine hydrochloride (CHX)and triclosan (TLN). Once resistance was achieved permeability changes in the outer membrane, including LPS, cell surface charge and hydrophobicityand the presence of,an active efflux were investigated as possible resistance candidates. Thin layer chromatography (TLC) and Gas chromatography (GC) were carried out to examine fatty acid and lipid changes in E. coli 0157 isolates with reduced susceptibility to TLN. Cross-resistance was studied by the Stoke's method and standard microdilution assays. Examination of the outer membrane proteins and LPS did not reveal any significant changes between parent and resistant strains. The hydrophobicity of the cells increased as the cells were passaged and became less. susceptible. An active efflux system was the most likely mechanism of resistance in all strains tested and a fab1 mutation was associated with E. coli 0157 resistant to TLN isolates. In all isolates investigated the resistance was stable for over 30 passages in biocide-free media. A high degree of cross-resistance was obtained in TLN-resjstant Escherichia coli 0157 strains, which repeatedly exerted decreased susceptibility to various antimicrobials, including chloramphenicol, erythromycin, imipenem, tetracycline and trimethoprirn:, as well as to various biocides. The results of this laboratory-based investigation suggest that it is possible for microorganisms to become resistant to biocides when repeatedly exposed to sublethal concentrations. This may be especially the case in the domestic environment where administration of biocides is poorly controlled. Eventually it could lead to the undesirable situation of resident strains becoming resistant to disinfection and cross resistant to other antimicrobials.

Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential of Salmonella enterica and Escherichia coli O157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for both S. enterica and E. coli O157. Cross-resistance in adaptively resistant S. enterica varied with the serotype; Salmonella enterica serovar Enteritidis expressed cross-resistance to chloramphenicol, whereas Salmonella enterica serovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistant Salmonella enterica serovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistant Salmonella serovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms. E. coli O157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.

Investigating the prevalence of Salmonella in dogs within the Midlands region of the United Kingdom

Background - The intimate relationship between dogs and their owners has the potential to increase the risk of human exposure to bacterial pathogens. Over the past 40 years, there have been several reports on transmission of salmonellae from dogs to humans. This study therefore aimed to determine the prevalence of Salmonella in the faeces of dogs from the Midlands region of the United Kingdom to assess exposure risk and potential for zoonotic transmission. Results - A total of 436 apparently healthy dogs without diarrhoea from households (n = 126), rescue centres (n = 96), boarding kennels (n = 43), retired greyhound kennels (n = 39) and a pet nutrition facility (n = 132) were investigated for Salmonella shedding. Faecal samples were processed by an enrichment culture based method. The faeces from one dog (0.23 %; 95 % confidence limit 0.006 %, 1.27 %) was positive for Salmonella. The species was S. enterica subspecies arizonae. Conclusion - This study showed that the prevalence of Salmonella from faeces from apparently healthy dogs from a variety of housing conditions is low; however, Salmonella shedding was still identified.

Exploring the biological basis for Salmonella persistence in food manufacturing environments

The persistence of Salmonella spp. in low moisture foods is a challenge for the food industry as despite control strategies already in place, notable outbreaks still occur. The aim of this study was to characterise isolates of Salmonella, known to be persistent in the food manufacturing environment, by comparing their microbiological characteristics with a panel of matched clinical and veterinary isolates. The gross morphology of the challenge panel was phenotypically characterised in terms of cellular size, shape and motility. In all the parameters measured, the factory isolates were indistinguishable from the human, clinical and veterinary strains. Further detailed metabolic profiling was undertaken using the biolog Microbial ID system. Multivariate analysis of the metabolic microarray revealed differences in metabolism of the factory isolate of S.Montevideo, based on its upregulated ability to utilise glucose and the sugar alcohol groups. The remainder of the serotype-matched isolates were metabolically indistinguishable. Temperature and humidity are known to influence bacterial survival and through environmental monitoring experimental parameters were defined. The results revealed Salmonella survival on stainless steel was affected by environmental temperatures that may be experienced in a food processing environment; with higher survival rates (D25=35.4) at temperatures at 25°C and lower humidity levels of 15% RH, however a rapid decline in cell count (D10=3.4) with lower temperatures of 10°C and higher humidity of 70% RH. Several resident factories strains survived in higher numbers on stainless steel (D25=29.69) compared to serotype matched clinical and veterinary isolates (D25=22.98). Factory isolates of Salmonella did not show an enhanced growth rate in comparison to serotype matched solates grown in Luria broth, Nutrient broth and M9 minimal media indicating that as an independent factor, growth was unlikely to be a major factor driving Salmonella persistence. Using a live / dead stain coupled with fluorescence microscopy revealed that when no longer culturable, isolates of S.Schwarzengrund entered into a viable nonculturable state. The biofilm forming capacity of the panel was characterised and revealed that all were able to form biofilms. None of the factory isolates showed an enhanced capability to form biofilms in comparison to serotype-matched isolates. In disinfection studies, planktonic cells were more susceptible to disinfectants than cells in biofilm and all the disinfectants tested were successful in reducing bacterial load. Contact time was one of the most important factors for reducing bacterial populations in a biofilm. The genomes of eight strains were sequenced. At the nucleotide and amino acid level the food factory isolates were similar to those of isolates from other environments; no major genomic rearrangements were observed, supporting the conclusions of the phenotypic and metabolic analysis. In conclusion, having investigated a variety of morphological, biochemical and genomic factors, it is unlikely that the persistence of Salmonella in the food manufacturing environment is attributable to a single phenotypic, metabolic or genomic factor. Whilst a combination of microbiological factors may be involved it is also possible that strain persistence in the factory environment is a consequence of failure to apply established hygiene management principles.

Características de virulência, resistência e diversidade genética de sorovares de Salmonella com impacto na saúde pública, isolados de frangos de corte no Brasil /

Salmonella Enteritidis, S. Typhimurium and S. Infantis are often associated with cases of human infections worldwide and is transmitted through consumption of contaminated food, particularly those of animal origin, especially chicken meat. This thesis was fractionated into three chapters, the first one relating to general considerations about the topics discussed in the following chapters. The second chapter aimed to evaluate virulence characteristics, antimicrobial resistance and the genetic similarity of 51 strains of S. Infantis isolated in samples of poultry origin from an industry located in the state of São Paulo, Brazil, during the 2009 to 2010 period. The third chapter aimed to analyze 111 strains of S. Enteritidis, 45 of Salmonella Typhimurium and 31 of Salmonella Typhimurium monophasic variant I 4, [5], 12:i:- isolated from chicken carcasses in different brazilian slaughterhouses from 2009 to 2011, and to estimate the risk to human health, based on the presence of virulence genes and antimicrobial resistance, correlating to the pathogenicity profiles (antimicrobial resistance and presence of virulence and resistance genes) with the genetic profile (ribogroup) of the isolates. To evaluate the antimicrobial susceptibility was performed the disk diffusion test for all serotypes of Salmonella, and exclusively to S. Enteritidis and S. Typhimurium, was also verified the minimum inhibitory concentration for ciprofloxacin and ceftazidime antibiotics. The presence of virulence genes invA (invasion), lpfA (fimbriae-adhesion), agfA (fimbriae-biofilm) and sefA (fimbriae-adhesion) were evaluated by PCR. The strains that showed resistance to antibiotics of β-lactams class were evaluated for the presence of resistance genes blaTEM, blaSHV, blaCTX-M and blaAmpC. For resistant strains to quinolones and fluoroquinolones antibiotics classes were searched the qnrA and qnrS genes. The phylogenetic relationship among the isolates was determined by RAPD method for S. Infantis strains, and by ribotyping technique to S. Enteritidis and S. Typhimurium.

Étude de la distribution de Salmonella spp. dans les tissus chez le porc suite à une infection naturelle et expérimentale

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.