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 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.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification

Objectives: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-ToIC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. Methods: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. Results: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. Conclusions: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Genome scale reconstruction of a Salmonella metabolic model comparison of similarity and differences with a commensal Escherichia coli strain

Salmonella are closely related to commensal Escherichia coli but have gained virulence factors enabling them to behave as enteric pathogens. Less well studied are the similarities and differences that exist between the metabolic properties of these organisms that may contribute toward niche adaptation of Salmonella pathogens. To address this, we have constructed a genome scale Salmonella metabolic model (iMA945). The model comprises 945 open reading frames or genes, 1964 reactions, and 1036 metabolites. There was significant overlap with genes present in E. coli MG1655 model iAF1260. In silico growth predictions were simulated using the model on different carbon, nitrogen, phosphorous, and sulfur sources. These were compared with substrate utilization data gathered from high throughput phenotyping microarrays revealing good agreement. Of the compounds tested, the majority were utilizable by both Salmonella and E. coli. Nevertheless a number of differences were identified both between Salmonella and E. coli and also within the Salmonella strains included. These differences provide valuable insight into differences between a commensal and a closely related pathogen and within different pathogenic strains opening new avenues for future explorations.

Triclosan resistance in Salmonella enterica serovar Typhimurium

Objectives: The aim of this study was to characterize the mechanisms of resistance to triclosan in Salmonella enterica serovar Typhimurium. Methods: Mutants resistant to triclosan were selected from nine S. enterica serovar Typhimurium strains. Mutants were characterized by genotyping, mutagenesis and complementation of fabI and analysis of efflux activity. Fitness of triclosan-resistant mutants was determined in vitro and in vivo. Results: Three distinct resistance phenotypes were observed: low- (LoT), medium- (MeT) and high-level (HiT) with MICs of 4-8, 16-32 and > 32 mg/L of triclosan, respectively, for inhibition. The genotype of fabI did not correlate with triclosan MIC. Artificial overexpression and mutagenesis of fabI in SL1344 each resulted in low-level triclosan resistance, indicating that FabI alone does not mediate high-level triclosan resistance in Salmonella Typhimurium. Active efflux of triclosan via AcrAB-TolC confers intrinsic resistance to triclosan as inactivation of acrB and tolC in wild-type strains and the triclosan-resistant mutants led to large decreases in triclosan resistance, which were reversed by complementation. Exemplars of each phenotype were evaluated for fitness in vivo; no fitness cost was seen and mutants colonized and persisted in chickens throughout a 28 day competitive index experiment. Conclusions: These data show that triclosan resistance can occur via distinct pathways in salmonella and that mutants selected after single exposure to triclosan are fit enough to compete with wild-type strains.

Proteomic analysis of triclosan resistance in Salmonella enterica serovar Typhimurium

Objectives: The aim of this study was to determine and compare the proteomes of three triclosan-resistant mutants of Salmonella enterica serovar Typhimurium in order to identify proteins involved in triclosan resistance. Methods: The proteomes of three distinct but isogenic triclosan-resistant mutants were determined using two-dimensional liquid chromatography mass separation. Bioinformatics was then used to identify and quantify tryptic peptides in order to determine protein expression. Results: Proteomic analysis of the triclosan-resistant mutants identified a common set of proteins involved in production of pyruvate or fatty acid with differential expression in all mutants, but also demonstrated specific patterns of expression associated with each phenotype. Conclusions: These data show that triclosan resistance can occur via distinct pathways in Salmonella, and demonstrate a novel triclosan resistance network that is likely to have relevance to other pathogenic bacteria subject to triclosan exposure and may provide new targets for development of antimicrobial agents.

Fitness and dissemination of disinfectant-selected multiple-antibiotic-resistant (MAR) strains of Salmonella enterica serovar Typhimurium in chickens

Objectives: The aims of this study were to determine whether strains of Salmonella enterica serovar Typhimurium which had acquired low-level multiple antibiotic resistance (MAR) through repeated exposure to farm disinfectants were able to colonize and transmit between chicks as easily as the parent strain and, if such strains were less susceptible to fluoroquinolones, would high-level resistance be selected after fluoroquinolone treatment. Methods: Two mutants were compared with the isogenic parent. In the first experiment, day-old chicks were co-infected with both the parent and a mutant to determine their relative fitness. In the second experiment, parent and mutant strains (in separate groups of chicks) were assessed for their ability to transmit from infected (contact) to non-infected (naive) birds and with respect to their susceptibility to fluoroquinolone treatment. Birds were regularly monitored for the presence of Salmonella in caecal contents. Replica plating was used to monitor for the selection of antibiotic-resistant strains. Results: The parent strain was shown to be significantly fitter than the two mutants and was more rapidly disseminated to naive birds. Antibiotic treatment did not preferentially select for the two mutants or for resistant strains. Conclusions: The disinfectant-exposed strains, although MAR, were less fit, less able to disseminate than the parent strain and were not preferentially selected by therapeutic antibiotic treatment. As such, these strains are unlikely to present a greater problem than other salmonellae in chickens.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella entetica serovar typhimurium selected following exposure to disinfectants

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-To1C for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar. The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples. Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive. When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×105 copies per reaction (equals to 1.58×103 to 1.58×107 cfu/ml L. monocytogenes) were detectable. As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.

Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica

Aims: The aim of the study was to investigate how stresses like low pH, which may be encountered in farms or food preparation premises, shape populations of Salmonella enterica by the selection of stress-resistant variants. Methods and Results: Stationary-phase cultures of S. enterica serovar Enteritidis and serovar Typhimurium (one strain of each) were exposed to pH 2Æ5 for up to 4 h, followed by growth at pH 7 for 48 h. This process was repeated 15 times in two separate experiments, which increased the acid resistance of the three out of four populations we obtained, by three- to fourfold. Sustainable variants derived from the populations showed changes in colony morphology, expression of SEF17 fimbriae, growth, increased heat resistance and reduced virulence. Conclusions: The study demonstrates that low pH environments can select for populations of S. enterica with persistent phenotypic changes such as increased acid resistance and occasionally increased SEF17 expression and lower virulence. Significance and Impact of the Study: There is a common belief that increased acid resistance coincides with increased virulence. This study demonstrates for the first time that increased acid resistance often impairs virulence and affects the general phenotype of S. enterica.

Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N-nitroso compound formation attributed to red meat consumption

Red meat consumption causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC). The genotoxic effects of these ATNCs were investigated using two different Comet assay protocols to determine the genotoxicity of fecal water samples. Fecal water samples were obtained from two studies of a total of 21 individuals fed diets containing different amounts of red meat, protein, heme, and iron. The first protocol incubated the samples with HT-29 cells for 5 min at 4 degrees C, whereas the second protocol used a longer exposure time of 30 min and a higher incubation temperature of 37 degrees C. DNA strand breaks were quantified by the tail moment (DNA in the comet tail multiplied by the comet tail length). The results of the two Comet assay protocols were significantly correlated (r = 0.35, P = 0.003), however, only the second protocol resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect on fecal water genotoxicity by diet (P > 0.20), mean transit time (P = 0.588), or weight (P = 0.705). However, there was a highly significant effect of age (P = 0.019). There was no significant correlation between concentrations of ATNCs in fecal homogenates and fecal water genotoxicity (r = 0.04, P = 0.74). ATNC levels were lower in fecal water samples (272 microg/kg) compared to that of fecal homogenate samples (895 microg/kg) (P < 0.0001). Failure to find dietary effects on fecal water genotoxicity may therefore be attributed to individual variability and low levels of ATNCs in fecal water samples.