Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Inhibition of Salmonella Typhimurium by tannins in vitro

Given the recent EU ban of antibiotics to promote the growth of farm animals, alternative approaches are needed for animal production systems. Tannins, which are already commercially marketed for animal nutrition, have bacteriostatic and bactericidal properties against pathogenic bacteria. The aim of this study was to investigate the inhibitory effect of various tannins against Salmonella Typhimurium (SL1344nal(r)) to identify potentially effective feed additives. Different sources of condensed and hydrolysable tannins were tested at concentrations between I and 6 mg ml(-1). The tannins tested were either commercial preparations or isolated from such preparations or from plants using Sephadex LH-20 based column chromatography. Some, but not all, of the tannins significantly decreased bacterial growth compared to tannin-free selenite cystine broth following incubation for 24 h at 37 degrees C. Gallotannins were especially effective and tara achieved 1.28 log(10) reductions after 24 hours. Antibacterial activity was also confirmed with inhibition zone diameters in a disc diffusion test. The experiments demonstrated that tannins may have potential as feed additives for reducing Salmonella infections in farm animals.

Spatial and temporal patterns in antimicrobial resistance of Salmonella Typhimurium in cattle in England and Wales

Salmonella is the second most commonly reported human foodborne pathogen in England and Wales, and antimicrobial-resistant strains of Salmonella are an increasing problem in both human and veterinary medicine. In this work we used a generalized linear spatial model to estimate the spatial and temporal patterns of antimicrobial resistance in Salmonella Typhimurium in England and Wales. Of the antimicrobials considered we found a common peak in the probability that an S. Typhimurium incident will show resistance to a given antimicrobial in late spring and in mid to late autumn; however, for one of the antimicrobials (streptomycin) there was a sharp drop, over the last 18 months of the period of investigation, in the probability of resistance. We also found a higher probability of resistance in North Wales which is consistent across the antimicrobials considered. This information contributes to our understanding of the epidemiology of antimicrobial resistance in Salmonella.

What makes a Salmonella strain epidemic? An expert opinion workshop

An expert opinion workshop was held on the subject of the cause, identification and control of new and emerging Salmonella strains. Experts were invited to complete questionnaires, contribute to structured discussions and take part in cluster group tasks. Outputs of the workshop included that, with current surveillance methods, it might take up to 2.5 years from the first introduction of a new strain into the UK livestock population to its identification as a human epidemic strain. In order to reduce the time to detection and provide more effective control options, several recommendations were made, including better back-tracing of human cases to their source, which would require more effective communication between those responsible for human and veterinary surveillance.

Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain

To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain. Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates. Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1. Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.

Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

Phenotype MicroArray (TM) in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium

The Phenotype MicroArray (TM) (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of d-tagatose, d-galactonic acid gamma-lactone and l-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.

Virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

A mixture containing galactoollgosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41 .vertical bar 71 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno (R) may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno (R) was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-1 6E cells, the presence of similar to 2 mM Bimuno) significantly reduced the invasion of S. Typhimuriurn SL1 344nal(r) (p < 0.0001). In the murine ligated ileal gut loops, the presence of Bimuno (R) prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse mocel, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, < 0.0001, respectively). Collectively, the results indicate that Bimuno (R) significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized L-histidine, L-glutamine, L-proline, L-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 degrees C than at 28 degrees C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.

Periplasmic adaptor protein AcrA has a distinct role in the antibiotic resistance and virulence of Salmonella enterica serovar Typhimurium

Objectives: AcrA can function as the periplasmic adaptor protein (PAP) in several RND tripartite efflux pumps, of which AcrAB-TolC is considered the most important. This system confers innate multiple antibiotic resistance. Disruption of acrB or tolC impairs the ability of Salmonella Typhimurium to colonize and persist in the host. The aim of this study was to investigate the role of AcrA alone in multidrug resistance and pathogenicity. Methods: The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was measured. Results: Following disruption of acrA, RT-PCR and western blotting confirmed that acrB/AcrB was still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also required for adherence to, and invasion of, tissue culture cells. Conclusions: Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph. These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.