Jinhong Wang

Comprehensive Assignment of Roles for Salmonella Typhimurium Genes in Intestinal Colonization of Food-Producing Animals

Chickens, pigs, and cattle are key reservoirs of Salmonella enterica, a foodborne pathogen of worldwide importance. Though a decade has elapsed since publication of the first Salmonella genome, thousands of genes remain of hypothetical or unknown function, and the basis of colonization of reservoir hosts is ill-defined. Moreover, previous surveys of the role of Salmonella genes in vivo have focused on systemic virulence in murine typhoid models, and the genetic basis of intestinal persistence and thus zoonotic transmission have received little study. We therefore screened pools of random insertion mutants of S. enterica serovar Typhimurium in chickens, pigs, and cattle by transposon-directed insertion-site sequencing (TraDIS). The identity and relative fitness in each host of 7,702 mutants was simultaneously assigned by massively parallel sequencing of transposon-flanking regions. Phenotypes were assigned to 2,715 different genes, providing a phenotype–genotype map of unprecedented resolution. The data are self-consistent in that multiple independent mutations in a given gene or pathway were observed to exert a similar fitness cost. Phenotypes were further validated by screening defined null mutants in chickens. Our data indicate that a core set of genes is required for infection of all three host species, and smaller sets of genes may mediate persistence in specific hosts. By assigning roles to thousands of Salmonella genes in key reservoir hosts, our data facilitate systems approaches to understand pathogenesis and the rational design of novel cross-protective vaccines and inhibitors. Moreover, by simultaneously assigning the genotype and phenotype of over 90% of mutants screened in complex pools, our data establish TraDIS as a powerful tool to apply rich functional annotation to microbial genomes with minimal animal use.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry.

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium DeltaaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4log(10) colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium DeltaaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to DeltaspaS or DeltassaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the DeltaaroA mutant. Expression in the DeltaaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry.

Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis

Streptococcus suis causes disease in pigs worldwide and is increasingly implicated in zoonotic disease in East and South-East Asia. To understand the genetic basis of disease in S. suis, we study the genomes of 375 isolates with detailed clinical phenotypes from pigs and humans from the United Kingdom and Vietnam. Here, we show that isolates associated with disease contain substantially fewer genes than non-clinical isolates, but are more likely to encode virulence factors. Human disease isolates are limited to a single-virulent population, originating in the 1920, s when pig production was intensified, but no consistent genomic differences between pig and human isolates are observed. There is little geographical clustering of different S. suis subpopulations, and the bacterium undergoes high rates of recombination, implying that an increase in virulence anywhere in the world could have a global impact over a short timescale.

Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

ABSTRACT Haemophilus parasuis causes Glässers disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105 ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.

Whole genome investigation of a divergent clade of the pathogen Streptococcus suis.

Streptococcus suis is a major porcine and zoonotic pathogen responsible for significant economic losses in the pig industry and an increasing number of human cases. Multiple isolates of S. suis show marked genomic diversity. Here, we report the analysis of whole genome sequences of nine pig isolates that caused disease typical of S. suis and had phenotypic characteristics of S. suis, but their genomes were divergent from those of many other S. suis isolates. Comparison of protein sequences predicted from divergent genomes with those from normal S. suis reduced the size of core genome from 793 to only 397 genes. Divergence was clear if phylogenetic analysis was performed on reduced core genes and MLST alleles. Phylogenies based on certain other genes (16S rRNA, sodA, recN, and cpn60) did not show divergence for all isolates, suggesting recombination between some divergent isolates with normal S. suis for these genes. Indeed, there is evidence of recent recombination between the divergent and normal S. suis genomes for 249 of 397 core genes. In addition, phylogenetic analysis based on the 16S rRNA gene and 132 genes that were conserved between the divergent isolates and representatives of the broader Streptococcus genus showed that divergent isolates were more closely related to S. suis. Six out of nine divergent isolates possessed a S. suis-like capsule region with variation in capsular gene sequences but the remaining three did not have a discrete capsule locus. The majority (40/70), of virulence-associated genes in normal S. suis were present in the divergent genomes. Overall, the divergent isolates extend the current diversity of S. suis species but the phenotypic similarities and the large amount of gene exchange with normal S. suis gives insufficient evidence to assign these isolates to a new species or subspecies. Further, sampling and whole genome analysis of more isolates is warranted to understand the diversity of the species.

Discovery of Bacillus thuringiensis virulence genes using signature-tagged mutagenesis in an insect model of septicaemia

Transposon Tn917 was used to identify Bacillus thuringiensis genes required for virulence and survival in a Manduca sexta (tobacco hornworm) septicaemia model. Uniquely tagged transposons, n = 72, were constructed and used to generate 1152 insertion mutants. Sixteen pools of 72 mutants were screened in the infection model, and 12 virulence-attenuated mutants were unable to survive the infection. Analysis of the mutated DNA sequences implicated an arsR family transcriptional regulator, a histone-like DNA-binding protein, a transposon, and several sequences of unknown function in B. thuringiensis pathogenesis.

Generation of a Tn5 transposon library in Haemophilus parasuis and analysis by transposon-directed insertion-site sequencing (TraDIS)

Haemophilus parasuis is an important respiratory tract pathogen of swine and the etiological agent of Glässers disease. The molecular pathogenesis of H. parasuis is not well studied, mainly due to the lack of efficient tools for genetic manipulation of this bacterium. In this study we describe a Tn5-based random mutagenesis method for use in H. parasuis. A novel chloramphenicol-resistant Tn5 transposome was electroporated into the virulent H. parasuis serovar 5 strain 29755. High transposition efficiency of Tn5, up to 10(4) transformants/μg of transposon DNA, was obtained by modification of the Tn5 DNA in the H. parasuis strain HS071 and establishment of optimal electrotransformation conditions, and a library of approximately 10,500 mutants was constructed. Analysis of the library using transposon-directed insertion-site sequencing (TraDIS) revealed that the insertion of Tn5 was evenly distributed throughout the genome. 10,001 individual mutants were identified, with 1561 genes being disrupted (69.4% of the genome). This newly-developed, efficient mutagenesis approach will be a powerful tool for genetic manipulation of H. parasuis in order to study its physiology and pathogenesis.

Molecular characterization of virulence defects in Bacillus thuringiensis mutants

Sequence analysis of a virulence-attenuated Bacillus thuringiensis signature tagged mutant 6F8 led to the identification of an 18 182 bp locus encoding 29 potential protein-coding ORFs. Thirteen of the 29 putative ORFs were found to share extensive homology with genes on plasmid pE33L466 of the pathogenic Bacillus cereus E33L strain. Nine ORFs were not only found in a cluster, but also in the same gene order, in both organisms. A number of mobile elements, including a transposon Tn4430, a novel IS231 family insertion sequence ISBth4 and various phage-related proteins, were found flanking this conserved gene cluster. These features of the 6F8 locus suggested that it might have undergone several DNA insertions from different sources by horizontal gene transfer. Transcriptional analyses of the 6F8 locus revealed that ORFs 1-23 were cotranscribed as a single transcript. Null mutants were constructed to investigate the function of the sequences flanking the signature-tagged mutagenesis insertion sites. Competition assays performed with the wild-type and null mutants demonstrated that the Tn4430 transposon element plays an important role in the full virulence of B. thuringiensis during Manduca sexta infection. This study provides the first experimental evidence that a Tn4430 family transposon is directly associated with B. thuringiensis virulence.

Characterization of ISBth4, a functional new IS231 variant from Bacillus thuringiensis MEX312

A new insertion variant of IS231 family, designated ISBth4, was identified from Bacillus thuringiensis MEX312. ISBth4 is 2046bp in length and is delimited by two 17bp inverted repeats (IR) with one mismatch, flanked by two perfect 11bp direct repeats (DR). ISBth4 contains two open reading frames (ORFs), ORF1 and ORF2, which encode 72 and 473 amino acids, respectively. Multiple sequence alignments revealed that the potential transposase of ISBth4 contained five conserved domains N1, N2, N3, C1 and C2 that are similar to other IS231 elements; and the typical catalytic triad D(N2)-70-D(N3)-150-E(C1) and Y(2)R(3)E(6)K motifs as hallmarks of IS4 elements. Comparison of the amino acids of the potential ISBth4 transposase with those from other publicly available B. cereus group IS231 elements revealed a close similarity with ISBce7 (94% identity), ISBce5 (90%), IS231Y (89%) and ISBce8 (86%), and lower similarity to IS231N (49%), IS231M (48%) and ISBce12 (40%). Phylogenetic analysis of the evolutionary relationships between ISBth4 and the other IS231 elements showed that ISBth4 is more closely related to the IS231 sequences isolated from B. cereus strains than those from B. thuringiensis strains. In vivo transposition activity of ISBth4 was discovered in a mutant B18 from a MEX312 background, indicating that it is a functional insertion sequence in its B. thuringiensis natural host.

Patterns of antimicrobial resistance in Streptococcus suis isolates from pigs with or without streptococcal disease in England between 2009 and 2014

Highlights • Disease and non-disease associated isolates had different resistance profiles.• Antimicrobial resistance increased over time among clinical isolates.• Prevalence of isolates with resistance to multiple antimicrobials is increasing.• Combination of different approaches enhances the information obtained from the data.