The global transcriptomes of Salmonella enterica serovars Gallinarum, Dublin and Enteritidis in the avian host.

Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians, and at the same time predominately cause systemic infections in these hosts, are unknown. In the current study, we developed a surgical approach to study gene expression inside the peritoneal cavity of hens to shed light on this. Strains of the host specific S. Gallinarum, the cattle-adapted S. Dublin and the broad host range serovar, S. Enteritidis, were enclosed in semi-permeable tubes and surgically placed for 4 h in the peritoneal cavity of hens and for control in a minimal medium at 41.2 °C. Global gene-expression under these conditions was compared between serovars using tiled-micro arrays with probes representing the genome of S. Typhimurium, S. Dublin and S. Gallinarum. Among other genes, genes of SPI-13, SPI-14 and the macrophage survival gene mig-14 were specifically up-regulated in the host specific serovar, S. Gallinarum, and further studies into the role of these genes in host specific infection are highly indicated. Analysis of pathways and GO-terms, which were enriched in the host specific S. Gallinarum without being enriched in the two other serovars indicated that host specificity was characterized by a metabolic fine-tuning as well as unique expression of virulence associated pathways. The cattle adapted serovar S. Dublin differed from the two other serovars by a lack of up-regulation of genes encoded in the virulence associated pathogenicity island 2, and this may explain the inability of this serovar to cause disease in poultry.

The SPI-19 encoded type-six secretion-systems (T6SS) of Salmonella enterica serovars Gallinarum and Dublin play different roles during infection.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S. Gallinarum G9 and its ΔSPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 ΔSPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.

Identification of metabolic pathways essential for fitness of Salmonella Typhimurium in vivo.

Bacterial infections remain a threat to human and animal health worldwide, and there is an urgent need to find novel targets for intervention. In the current study we used a computer model of the metabolic network of Salmonella enterica serovar Typhimurium and identified pairs of reactions (cut sets) predicted to be required for growth in vivo. We termed such cut sets synthetic auxotrophic pairs. We tested whether these would reveal possible combined targets for new antibiotics by analyzing the performance of selected single and double mutants in systemic mouse infections. One hundred and two cut sets were identified. Sixty-three of these included only pathways encoded by fully annotated genes, and from this sub-set we selected five cut sets involved in amino acid or polyamine biosynthesis. One cut set (asnA/asnB) demonstrated redundancy in vitro and in vivo and showed that asparagine is essential for S. Typhimurium during infection. trpB/trpA as well as single mutants were attenuated for growth in vitro, while only the double mutant was a cut set in vivo, underlining previous observations that tryptophan is essential for successful outcome of infection. speB/speF,speC was not affected in vitro but was attenuated during infection showing that polyamines are essential for virulence apparently in a growth independent manner. The serA/glyA cut-set was found to be growth attenuated as predicted by the model. However, not only the double mutant, but also the glyA mutant, were found to be attenuated for virulence. This adds glycine production or conversion of glycine to THF to the list of essential reactions during infection. One pair (thrC/kbl) showed true redundancy in vitro but not in vivo demonstrating that threonine is available to the bacterium during infection. These data add to the existing knowledge of available nutrients in the intra-host environment, and have identified possible new targets for antibiotics.

Polyamines are essential for virulence in Salmonella enterica serovar Gallinarum despite evolutionary decay of polyamine biosynthesis genes.

Serovars of Salmonella enterica exhibit different host-specificities where some have broad host-ranges and others, like S. Gallinarum and S. Typhi, are host-specific for poultry and humans, respectively. With the recent availability of whole genome sequences it has been reported that host-specificity coincides with accumulation of pseudogenes, indicating adaptation of host-restricted serovars to their narrow niches. Polyamines are small cationic amines and in Salmonella they can be synthesized through two alternative pathways directly from l-ornithine to putrescine and from l-arginine via agmatine to putrescine. The first pathway is not active in S. Gallinarum and S. Typhi, and this prompted us to investigate the importance of polyamines for virulence in S. Gallinarum. Bioinformatic analysis of all sequenced genomes of Salmonella revealed that pseudogene formation of the speC gene was exclusive for S. Typhi and S. Gallinarum and happened through independent events. The remaining polyamine biosynthesis pathway was found to be essential for oral infection with S. Gallinarum since single and double mutants in speB and speE, encoding the pathways from agmatine to putrescine and from putrescine to spermidine, were attenuated. In contrast, speB was dispensable after intraperitoneal challenge, suggesting that putrescine was less important for the systemic phase of the disease. In support of this hypothesis, a ΔspeE;ΔpotCD mutant, unable to synthesize and import spermidine, but with retained ability to import and synthesize putrescine, was attenuated after intraperitoneal infection. We therefore conclude that polyamines are essential for virulence of S. Gallinarum. Furthermore, our results point to distinct roles for putrescine and spermidine during systemic infection.

Intestinal invasion of Salmonella enterica serovar Typhimurium in the avian host is dose dependent and does not depend on motility and chemotaxis.

Salmonella enterica serotype Typhimurium (S. Typhimurium) can invade in the intestine of the avian host, and knowledge on the mechanisms that govern this is potentially important for prevention of disease. This study investigated the invasion of S. Typhimurium in the avian host and to which extent it depended on motility and chemotaxis. Wild type and previously well-characterized transposon mutants in flagella genes fliC and fljB and in chemotaxis genes cheA, cheB and cheR were used as challenge strains in intestinal loop experiments. Invasion was shown to be dose dependent, but did not require functional flagella or chemotaxis genes. In support of the results from intestinal loop experiments, flagella and chemotaxis genes were not significantly important to the outcome of an oral infection. The results showed that S. Typhimurium invasion in the avian host was dose dependent and was not affected by the loss of flagella and chemotaxis genes.

Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation.

BACKGROUND: Klebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia. The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation. RESULTS: Isogenic fimbriae mutants of the clinical K. pneumoniae isolate C3091 were constructed, and their ability to form biofilm was investigated in a flow cell system by confocal scanning laser microscopy. The wild type strain was found to form characteristic biofilm and development of K. pneumoniae biofilm occurred primarily by clonal growth, not by recruitment of planktonic cells. Type 1 fimbriae did not influence biofilm formation and the expression of type 1 fimbriae was found to be down-regulated in biofilm forming cells. In contrast, expression of type 3 fimbriae was found to strongly promote biofilm formation. CONCLUSION: By use of well defined isogenic mutants we found that type 3 fimbriae, but not type 1 fimbriae, strongly promote biofilm formation in K. pneumoniae C3091. As the vast majority of clinical K. pneumoniae isolates express type 3 fimbriae, this fimbrial adhesin may play a significant role in development of catheter associated K. pneumoniae infections.

Screening for genes involved in Klebsiella pneumoniae biofilm formation using a fosmid library.

Klebsiella pneumoniae is a well-known opportunistic pathogen, often causing catheter-associated urinary tract infections. Biofilm formation on the catheter surfaces is an important step in the development of these infections. To identify the genes involved in the ability of K. pneumoniae to form a biofilm on abiotic surfaces, a novel strategy was used. A clone library was constructed by cloning the entire K. pneumoniae genome of the clinical isolate C3091 into a fosmid vector and the clone library was expressed in Escherichia coli. A total of 1152 clones were screened for enhanced biofilm formation compared with the E. coli parent strain using a biofilm microtiter plate assay. Nine clones with significantly enhanced biofilm formation were identified, subjected to random Tn5 transposon mutagenesis, screened for biofilm deficiency and the biofilm-promoting genes identified. Five of the clones contained the type 3 fimbriae gene cluster, a well-known K. pneumoniae virulence factor and biofilm promoter. Thus, the effectiveness of our approach was confirmed. Furthermore, genes encoding cell surface proteins and proteins involved in metabolism, none of them previously associated with biofilm formation in K. pneumoniae, were identified by our screening method. In conclusion, the use of fosmid libraries is an effective high throughput screening method to identify the genes involved in biofilm formation.

Silver-palladium surfaces inhibit biofilm formation.

Undesired biofilm formation is a major concern in many areas. In the present study, we investigated biofilm-inhibiting properties of a silver-palladium surface that kills bacteria by generating microelectric fields and electrochemical redox processes. For evaluation of the biofilm inhibition efficacy and study of the biofilm inhibition mechanism, the silver-sensitive Escherichia coli J53 and the silver-resistant E. coli J53[pMG101] strains were used as model organisms, and batch and flow chamber setups were used as model systems. In the case of the silver-sensitive strain, the silver-palladium surfaces killed the bacteria and prevented biofilm formation under conditions of low or high bacterial load. In the case of the silver-resistant strain, the silver-palladium surfaces killed surface-associated bacteria and prevented biofilm formation under conditions of low bacterial load, whereas under conditions of high bacterial load, biofilm formation occurred upon a layer of surface-associated dead bacteria.