Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

Genomic Epidemiology of a Protracted Hospital Outbreak Caused by a Toxin A-Negative Clostridium difficile Sublineage PCR Ribotype 017 Strain in London, England.

Clostridium difficile remains the leading cause of nosocomial diarrhea worldwide, which is largely considered to be due to the production of two potent toxins: TcdA and TcdB. However, PCR ribotype (RT) 017, one of five clonal lineages of human virulent C. difficile, lacks TcdA expression but causes widespread disease. Whole-genome sequencing was applied to 35 isolates from hospitalized patients with C. difficile infection (CDI) and two environmental ward isolates in London, England. The phylogenetic analysis of single nucleotide polymorphisms (SNPs) revealed a clonal cluster of temporally variable isolates from a single hospital ward at University Hospital Lewisham (UHL) that were distinct from other London hospital isolates. De novo assembled genomes revealed a 49-kbp putative conjugative transposon exclusive to this hospital clonal cluster which would not be revealed by current typing methodologies. This study identified three sublineages of C. difficile RT017 that are circulating in London. Similar to the notorious RT027 lineage, which has caused global outbreaks of CDI since 2001, the lineage of toxin-defective RT017 strains appears to be continually evolving. By utilization of WGS technologies to identify SNPs and the evolution of clonal strains, the transmission of outbreaks caused by near-identical isolates can be retraced and identified.

Molecular detection identified a type six secretion system in Campylobacter jejuni from various sources but not from human cases.

AIM: To determine the presence of the T6SS in Campylobacter jejuni from diverse sources. METHODS AND RESULTS: The recently identified type VI secretion system (T6SS) is a bacterial injection machinery that plays a role in virulence, symbiosis, bacterial interactions and environmental stress responses. This system has been recently discovered in the major enteric pathogen Camp. jejuni. In this study, we used multiplex PCR (mPCR), based on conserved genetic markers of the T6SS, to screen 366 Pakistani Camp. jejuni isolates from humans, poultry, cattle, wildlife or waste-water sources. We identified the T6SS in isolates from all of these sources except humans. The overall prevalence of the T6SS among the isolates was 17/366 (4·6%) and the T6SS positive isolates clustered into four different groups. Transcription of the T6SS genes, determined using RT-PCR, was observed in bacteria cultured at 37 or 42°C but not in 37°C cultures adjusted to pH3. CONCLUSIONS: Campylobacter jejuni isolates harbouring T6SS markers genes were identified in livestock and non-livestock sources but in this study we did not identify human diarrhoeal isolates which possessed the T6SS. We demonstrated down-regulation of T6SS in an acidic environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study questions the role of the T6SS in human diarrhoeal disease. Moreover this study did not identify a clear association of Camp. jejuni isolates harbouring T6SS with any of the niches tested. Our study highlights the need to establish the role of the T6SS in environmental survival or virulence.

Revisiting aminocoumarins for the treatment of melioidosis.

Burkholderia pseudomallei causes melioidosis, a potentially lethal disease that can establish both chronic and acute infections in humans. It is inherently recalcitrant to many antibiotics, there is a paucity of effective treatment options and there is no vaccine. In the present study, the efficacies of selected aminocoumarin compounds, DNA gyrase inhibitors that were discovered in the 1950s but are not in clinical use for the treatment of melioidosis were investigated. Clorobiocin and coumermycin were shown to be particularly effective in treating B. pseudomallei infection in vivo. A novel formulation with dl-tryptophan or l-tyrosine was shown to further enhance aminocoumarin potency in vivo. It was demonstrated that coumermycin has superior pharmacokinetic properties compared with novobiocin, and the coumermycin in l-tyrosine formulation can be used as an effective treatment for acute respiratory melioidosis in a murine model. Repurposing of existing approved antibiotics offers new resources in a challenging era of drug development and antimicrobial resistance.

The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

Genome sequence of the emerging pathogen Helicobacter canadensis.

We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.

Campylobacter jejuni cocultured with epithelial cells reduces surface capsular polysaccharide expression.

The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.

In vitro and in vivo properties of the bovine antimicrobial peptide, Bactenecin 5.

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.

Deciphering Campylobacter jejuni cell surface interactions from the genome sequence.

The completion of the Campylobacter jejuni genome sequence is a landmark in Campylobacter research. Discoveries directly arising from these data include the identification of a capsular polysaccharide, extensive capacity for phase variable gene expression and lipo-oligosaccharide structural phase variation. The recent identification of a unique system of general protein glycosylation in C. jejuni, a C. jejuni protein that is translocated into eukaryotic cells, and plasmid-encoded components of a putative type IV secretion system are likely to be significant in terms of the host-pathogen interaction.

Prevalence of Type VI Secretion System in Spanish Campylobacter jejuni Isolates.

Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole-genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS-positive C. jejuni strains.

Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K12.

Clostridium difficile toxin A was purified to homogeneity and was used to raise monospecific antiserum in rabbits. A gene bank of C. difficile DNA in Escherichia coli was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. Out of 4500 plaques screened with antitoxin A, 9 clones were positively identified. One of these clones lambda tA5 expressed a 235 kDa protein which exhibited a cytotonic effect on Chinese hamster ovary cells, and had the ability to haemagglutinate rabbit erythrocytes, both properties characteristic of toxin A. The size of the lambda tA5 insert DNA was 14.3 kb.

Nucleic acid immunization: concepts and techniques associated with third generation vaccines.

A radical change in vaccine methodology arrived nine years ago with the advent of nucleic acid immunization. Aspects such as plasmid design, gene selection, the use of immunostimulatory complexes and clinical trials are discussed in this review. Furthermore, concepts and protocols involved in the construction, evaluation and immunization of a DNA vaccine have been examined as new strategies to enhance this technology continues to grow.

Nucleotide sequence of Clostridium difficile toxin A gene fragment and detection of toxigenic strains by polymerase chain reaction.

A 1947 base pair (bp) fragment of the toxin A gene of Clostridium difficile was sequenced. A continuous open reading frame was found, which contained 4 distinct groups of repeat nucleotide sequence with 88 to 100% identity within each group. The arrangement of the groups (A, 81 bp, B, C and D, 63 bp) was ABCCCDABCDDABCCCDABCCDABCDABC. Based on nucleotide sequence data from the C repeat group, a pair of oligonucleotide primers were synthesised and used in the polymerase chain reaction (PCR) to amplify fragments from the toxin A gene. Several products of multiples of 63 bp length were amplified for all 33 toxigenic C. difficile strains tested in contrast to the 12 non-toxigenic strains tested which failed to amplify any product. This rapid technique is of potential use in the specific identification of toxigenic C. difficile strains in mixed culture and from clinical specimens.

Degenerate PCR primers for the amplification of fragments from genes encoding response regulators from a range of pathogenic bacteria.

Many bacterial responses to environmental stimuli are mediated by response regulators which coordinately regulate genes involved in particular adaptive responses. Degenerate oligonucleotide primers were used to amplify by the polymerase chain reaction (PCR), fragments from genes encoding eleven novel response regulators. Sequence and phylogenetic analysis revealed that phoB, phoP and creB gene fragments had been amplified from Yersinia enterocolitica and Yersinia pseudotuberculosis, and that a creB sequence had been amplified from Campylobacter jejuni. Four amplified fragments from C. jejuni, Listeria monocytogenes, Mycobacterium tuberculosis and Escherichia coli clearly came from response regulator genes, but were not closely related to any of the known genes. Mutagenesis of the newly identified genes should allow us to determine their function and the genes under their control.

A flagellar-specific ATPase (FliI) is necessary for flagellar export in Helicobacter pylori.

Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism. We have identified a gene in H. pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species. An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits. The majority (> 99%) of mutant cells were completely aflagellate. These results suggest that FliI is a novel ATPase involved in flagellar export in H. pylori.

Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis.

The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens. For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines. A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs. An isogenic Y. pestis htrA mutant was constructed using a reverse genetics approach. In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model. However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.

Identification, cloning and initial characterisation of FeuPQ in Brucella suis: a new sub-family of two-component regulatory systems.

To cause disease, Brucella species have to adapt to a range of different environments. Environmental sensing and adaptive responses in bacteria often involve the concerted action of a two-component regulatory system, consisting of sensor and response regulator components. Amplification and sequence analysis of response regulators from Brucella species identified a response regulator sequence with 96% similarity to Rhizobium leguminosarum FeuP. In R. leguminosarum, the FeuPQ two-component system is involved in the regulation of iron uptake. A Brucella suis feuP isogenic mutant was constructed but was not attenuated in the murine brucellosis model. The survival and multiplication of the mutant in macrophages was also unaffected. The FeuPQ regulon represents a newly characterised sub-family of response regulators.

Construction and characterisation of a Yersinia enterocolitica O:8 ompR mutant.

The ompR-envZ two-component regulatory system has been shown to contribute to virulence in a number of enteric bacterial pathogens. A Yersinia enterocolitica O:8 ompR homologue was amplified, cloned and sequenced, showing 99.2% homology to the Escherichia coli OmpR. An isogenic ompR mutant was constructed by reverse genetics-based methodology. The mutant was shown to have increased sensitivity to high osmolarity, high temperature and low pH stresses in vitro. In the murine yersiniosis model, the mutant was attenuated and offered partial protection against wild-type challenge.