Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion.

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (approximately 14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells.

Identification of temperature regulated factors of Campylobacter jejuni and their potential roles in virulence.

Campylobacter jejuni is the major cause of bacterial gastroenteritis in man, while it is generally regarded as a commensal of the avian gut. Consumption and handling of contaminated poultry meat products are major risk factors for human infection. The body temperature in man (37 °C) and chickens (42 °C) differ markedly, and differential gene regulation and protein expression at different temperatures may in part explain the behaviour in the two hosts. We performed proteomics analyses with C. jejuni cells grown at 37 °C and 42 °C. Time-of-flight mass spectrometry (Q-Tof) analysis was carried out after samples were digested with the Filter-Aided Sample Preparation (FASP) method and peptides were fractionated by strong anion exchanges. Differentially regulated proteins were identified by Mascot and Scaffold analyses. Triple quadrupole (QQQ) mass spectrometer analysis confirmed that a total of 33 proteins were differentially regulated between 37 °C and 42 °C. Several upregulated proteins were selected for their corresponding gene knock-out mutants to be tested for their virulence in the Galleria mellonella model. To correlate with other tissue/animal models, the GADH mutant was selected for its reduced ability to colonize chickens. At 37 °C, the mutants of outer membrane protein Omp50 and Chaperone GroEL significantly increased virulence; while at 42 °C, the mutants of YceI, Omp50, and GADH reduced virulence against Galleria mellonella compared with the wild type strains. The results of current and previous studies indicate that GADH is a virulent factor in G. mellonella and a colonization factor in chickens. The workflow of this study may prove a new way to identify stress related virulent factors. The implications of these findings are discussed for pathogenesis in the model and other hosts.

Virulence Characterization of Salmonella enterica by a New Microarray: Detection and Evaluation of the Cytolethal Distending Toxin Gene Activity in the Unusual Host S. Typhimurium.

Salmonella enterica is a zoonotic foodborne pathogen that causes acute gastroenteritis in humans. We assessed the virulence potential of one-hundred and six Salmonella strains isolated from food animals and products. A high through-put virulence genes microarray demonstrated Salmonella Pathogenicity Islands (SPI) and adherence genes were highly conserved, while prophages and virulence plasmid genes were variably present. Isolates were grouped by serotype, and virulence plasmids separated S. Typhimurium in two clusters. Atypical microarray results lead to whole genome sequencing (WGS) of S. Infantis Sal147, which identified deletion of thirty-eight SPI-1 genes. Sal147 was unable to invade HeLa cells and showed reduced mortality in Galleria mellonella infection model, in comparison to a SPI-1 harbouring S. Infantis. Microarray and WGS of S. Typhimurium Sal199, established for the first time in S. Typhimurium presence of cdtB and other Typhi-related genes. Characterization of Sal199 showed cdtB genes were upstream of transposase IS911, and co-expressed with other Typhi-related genes. Cell cycle arrest, cytoplasmic distension, and nuclear enlargement were detected in HeLa cells infected by Sal199, but not with S. Typhimurium LT2. Increased mortality of Galleria was detected on infection with Sal199 compared to LT2. Thus, Salmonella isolates were rapidly characterized using a high through-put microarray; helping to identify unusual virulence features which were corroborated by further characterisation. This work demonstrates that the use of suitable screening methods for Salmonella virulence can help assess the potential risk associated with certain Salmonella to humans. Incorporation of such methodology into surveillance could help reduce the risk of emergence of epidemic Salmonella strains.

Galleria mellonella as an infection model for Campylobacter jejuni virulence.

Larvae of Galleria mellonella (Greater Wax Moth) have been shown to be susceptible to Campylobacter jejuni infection and our study characterizes this infection model. Following infection with C. jejuni human isolates, bacteria were visible in the haemocoel and gut of challenged larvae, and there was extensive damage to the gut. Bacteria were found in the extracellular and cell-associated fraction in the haemocoel, and it was shown that C. jejuni can survive in insect cells. Finally, we have used the model to screen a further 67 C. jejuni isolates belonging to different MLST types. Isolates belonging to ST257 were the most virulent in the Galleria model, whereas those belonging to ST21 were the least virulent.

Persistence of a wild type Escherichia coli and its multiple antibiotic-resistant (MAR) derivatives in the abattoir and on chilled pig carcasses.

The aim of this study was to evaluate the ability of an Escherichia coli with the multiple antibiotic resistance (MAR) phenotype to withstand the stresses of slaughter compared to an isogenic progenitor strain. A wild type E. coli isolate (345-2RifC) of porcine origin was used to derive 3 isogenic MAR mutants. Escherichia coli 345-2RifC and its MAR derivatives were inoculated into separate groups of pigs. Once colonisation was established, the pigs were slaughtered and persistence of the E. coli strains in the abattoir environment and on the pig carcasses was monitored and compared. No significant difference (P>0.05) was detected between the shedding of the different E. coli strains from the live pigs. Both the parent strain and its MAR derivatives persisted in the abattoir environment, however the parent strain was recovered from 6 of the 13 locations sampled while the MAR derivatives were recovered from 11 of 13 and the number of MAR E. coli recovered was 10-fold higher than the parent strain at half of the locations. The parent strain was not recovered from any of the 6 chilled carcasses whereas the MAR derivatives were recovered from 3 out of 5 (P<0.001). This study demonstrates that the expression of MAR in 345-2RifC increased its ability to survive the stresses of the slaughter and chilling processes. Therefore in E. coli, MAR can give a selective advantage, compared to non-MAR strains, for persistence on chilled carcasses thereby facilitating transit of these strains through the food chain.

Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni.

The contribution of gamma-glutamyl transpeptidase (GGT) to Campylobacter jejuni virulence and colonization of the avian gut has been investigated. The presence of the ggt gene in C. jejuni strains directly correlated with the expression of GGT activity as measured by cleavage and transfer of the gamma-glutamyl moiety. Inactivation of the monocistronic ggt gene in C. jejuni strain 81116 resulted in isogenic mutants with undetectable GGT activity; nevertheless, these mutants grew normally in vitro. However, the mutants had increased motility, a 5.4-fold higher invasion efficiency into INT407 cells in vitro and increased resistance to hydrogen peroxide stress. Moreover, the apoptosis-inducing activity of the ggt mutant was significantly lower than that of the parental strain. In vivo studies showed that, although GGT activity was not required for initial colonization of 1-day-old chicks, the enzyme was required for persistent colonization of the avian gut.

Host-associated genetic import in Campylobacter jejuni.

Host association of Campylobacter jejuni was analyzed by using multilocus sequence typing data for 713 isolates from chickens and bovids (cattle and sheep). Commonly used summary measures of genotypes (sequence type and clonal complex) showed poor accuracy, but a method using the full allelic profile showed 80% accuracy in distinguishing isolates from these 2 host groups. We explored the biologic basis of more accurate results with allelic profiles. Strains isolated from specific hosts have imported a substantial number of alleles while circulating in those host species. These results imply that 1) although Campylobacter moves frequently between hosts, most transmission is within species, and 2) lineages can acquire a host signature and potentially adapt to the host through recombination. Assignment using this signature enables improved prediction of source for pathogens that undergo frequent genetic recombination.

Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni.

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.