Ariel Imre

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.

Virulence potential of five major pathogenicity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens

BackgroundSalmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host.ResultsThe mutant lacking all 5 major SPIs was still capable of colonising the caecum while colonisation of the liver and spleen was dependent on the presence of both SPI-1 and SPI-2. In contrast, the absence of SPI-3, SPI-4 or SPI-5 individually did not influence virulence of S. Enteritidis for chickens, but collectively they contributed to the colonisation of the spleen. Proinflammatory signalling and heterophil infiltration was dependent on intact SPI-1 only and not on other SPIs.ConclusionsSPI-1 and SPI-2 are the two most important pathogenicity islands of Salmonella Enteritidis required for the colonisation of systemic sites in chickens.

A flagellated motile Salmonella Gallinarum mutant (SG Fla+) elicits a pro-inflammatory response from avian epithelial cells and macrophages and is less virulent to chickens

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SG) is a non-flagellated bacterium which causes fowl typhoid, a systemic disease associated with high mortality in birds. It has been suggested that the absence of flagella in SG is advantageous in the early stages of systemic infection through absence of TLR-5 activation. In order to investigate this hypothesis in more detail a flagellated and motile SG mutant (SG Fla(+)) was constructed. The presence of flagella increased invasiveness for chicken kidney cells (CKC) while its presence did not alter survival in HD11 macrophages. SG Fla(+) induced higher levels of CXCLi2, IL-6 and iNOS mRNA expression in CKC than the SG parent strain. The expression of genes responsible for immune response mediators in infected HD11 macrophages were not related to the presence of flagella. Mortality rates were lower in birds challenged with SG Fla(+) when compared with the SG parent. SG Fla(+) was recovered from caecal contents which showed pathological changes suggestive of inflammation and suggested increased colonization ability.

Gene expression analysis of Salmonella enterica SPI in macrophages indicates differences between serovars that induce systemic disease from those normally causing enteritis

Global gene expression of the invasive Salmonella serovars S. Enteritidis and S. Typhimurium, and the less-invasive S. Infantis and S. Hadar was studied during infection of a chicken macrophage cell line. Major functional gene groups responsible for intracellular physiological changes were regulated similarly in all four serovars. However, SPI1 and SPI4 genes of S. Enteritidis and S. Typhimurium were strongly repressed in the macrophages whereas S. Infantis, S. Hadar and other similar serovars maintained up-regulation of these gene sets. This phenomenon may explain some of the biological differences between invasive and non-invasive Salmonella serovars.

Site-directed (IS30-FljA) transposon mutagenesis system to produce nonflagellated mutants of Salmonella Enteritidis

Site-directed integration/mutagenesis systems are used to carry out targeted transpositions on DNA. The well-characterized IS30-element and its transposase have numerous advantages that predestine it to be a good candidate for such applications. In order to generate nonflagellated mutants of Salmonella Enteritidis, a new site-directed mutagenesis system has been developed and applied. The system was constructed based on the assumption that the DNA-binding FljA component of the fusion transposase would bind to its target (the operator of fliC), and as a consequence, insertions could be concentrated in the flagellin operon. The system consists of two components: one expresses the fusion transposase and the other is an integration donor plasmid harbouring the (IS30)(2) reactive structure. The application of this site-directed mutagenesis system on a strain of S. Enteritidis 11 (SE11) resulted in several nonmotile mutants with fliD insertion that could serve as negatively markered vaccine candidates. Analysis of less motile mutants generated by the fusion transposase revealed further hot spot sequences preferred by the fusion construct.

Molecular and Pathogenic Characterization of Salmonella enterica Serovar Bovismorbificans Strains of Animal, Environmental, Food, and Human Origin in Hungary

In this study, we characterized 110 strains of Salmonella enterica serovar Bovismorbificans contaminating environment, animals, food of animal origin, and human, to assess their significance along the food chain in Hungary. Additionally, five strains from Germany were tested for comparative purposes. Characterization involved antibiotic susceptibility testing, class 1 integron detection by polymerase chain reaction, plasmid profiling, virulotyping (using virulence gene-specific polymerase chain reactions), and pulsed-field gel electrophoresis. Pathogenic potential of selected strains was tested in orally infected 1-day-old specific pathogen-free chicks. Eighty-two percent of the strains were susceptible to the 16 antibiotics tested, and none of them had class 1 integron. A multidrug-resistant human isolate harbored a bla(SHV5)-type extended-spectrum beta-lactamase gene, first reported in this serotype. All the strains possessed avrA, ssaQ, mgtC, spi4, and sopB genes indicating the presence of Salmonella pathogenicity islands 1-5, respectively, missed the phage-related genes sopE and gipA, but retained the phage-related gene sodC1. An approximately 90 kb large plasmid was characteristic to 80% of the strains, all of which carried the spvC gene. In vivo colonization testing of four selected strains in 1-day-old chicks resulted in significantly reduced liver and spleen colonization ability as compared with the Salmonella Enteritidis control strain, whereas their caecal colonization ability differed less from that of Salmonella Enteritidis. Pulsed-field gel electrophoresis data revealed the dominance of two pulsotypes (C2 and C5) without any specific temporal, geographical, and/or source-related linkages. The results show that Salmonella Bovismorbificans studied here are less invasive than Salmonella Enteritidis, but they may colonize and persist in several animal species and successfully contaminate meat products of different animal origin in Hungary.

Vaccine potential of a nonflagellated, virulence-plasmid-cured (fliD–, pSEVΔ) mutant of Salmonella Enteritidis for chickens

The aim of these studies was to assess residual virulence and early protective capacity of a negatively markered live attenuated vaccine candidate Salmonella Enteritidis mutant against a highly virulent S. Enteritidis strain using a dayold chicken model. Nonflagellated FliD negative mutants of Salmonella Enteritidis 11 (SE11) with and without the virulence plasmid proved to be sufficiently attenuated (limited invasiveness in vitro/in vivo) without reduced ability to colonise chicken gut. The early protective activity of a nonflagellated, virulence-plasmidcured (fliD-, pSEVΔ) mutant against organ invasion, caecal colonisation and faecal shedding by the highly virulent challenge strain S. Enteritidis 147 Nal(R) proved to be effective and safe. The innate and adaptive immunity was demonstrable during the first four weeks of life, and the serological response was clearly distinguishable from the response induced by the wild parental strain. In conclusion, we provided data for the first time about a virulence-plasmid-cured, nonflagellated mutant of S. Enteritidis to serve as a basis for development of a negatively markered potential live oral vaccine against virulent S. Enteritidis in chicken.