Michael F. Loughlin

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes

BackgroundThe Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East.ResultsThis scheme uses 7 loci; atp D, fus A, gln S, glt B, gyr B, inf B, and pps. There were 12 sequence types (ST) identified in C. sakazakii, and 3 in C. malonaticus. A third (22/60) of C. sakazakii strains were in ST4, which had almost equal numbers of clinical and infant formula isolates from 1951 to 2008. ST8 may represent a particularly virulent grouping of C. sakazakii as 7/8 strains were clinical in origin which had been isolated between 1977 - 2006, from four countries. C. malonaticus divided into three STs. The previous Cronobacter biotyping scheme did not clearly correspond with STs nor with species.ConclusionIn conclusion, MLST is a more robust means of identifying and discriminating between C. sakazakii and C. malonaticus than biotyping. The MLST database for these organisms is available online at http://pubmlst.org/cronobacter/.

Global regulation of virulence and the stress response by CsrA in the highly adapted human gastric pathogen Helicobacter pylori

Although successful and persistent colonization of the gastric mucosa depends on the ability to respond to changing environmental conditions and co‐ordinate the expression of virulence factors during the course of infection, Helicobacter pylori possesses relatively few transcriptional regulators. We therefore investigated the contribution of the regulatory protein CsrA to global gene regulation in this important human pathogen. CsrA was necessary for full motility and survival of H. pylori under conditions of oxidative stress. Loss of csrA expression deregulated the oxidant‐induced transcriptional responses of napA and ahpC, the acid induction of napA, cagA, vacA, the urease operon, and fur, as well as the heat shock responses of napA, groESL and hspR. Although the level of napA transcript was higher in the csrA mutant, its stability was similar in the wild‐type and mutant strains, and less NapA protein was produced in the mutant strain. Finally, H. pylori strains deficient in the production of CsrA were significantly attenuated for virulence in a mouse model of infection. This work provides evidence that CsrA has a broad role in regulating the physiology of H. pylori in response to environmental stimuli, and may be important in facilitating adaptation to the different environments encountered during colonization of the gastric mucosa. Furthermore, CsrA appears to mediate its effects in H. pylori at the post‐transcriptional level by influencing the processing and translation of target transcripts, with minimal effect on the stability of the target mRNAs.

Helicobacter pylori mutants defective in RuvC Holliday junction resolvase display reduced macrophage survival and spontaneous clearance from the murine gastric mucosa

ABSTRACT Homologous recombination contributes to the extraordinary genetic diversity of Helicobacter pylori and may be critical for surface antigen expression and adaptation to environmental challenges within the stomach. We generated isogenic, nonpolar H. pylori ruvC mutants to investigate the function of RuvC, a Holliday junction endonuclease that resolves recombinant joints into nicked duplex products. Inactivation of ruvC reduced the frequency of homologous recombination of H. pylori between 17- and 45-fold and increased sensitivity to DNA-damaging agents and the antimicrobial agents levofloxacin and metronidazole. The H. pylori ruvC mutants were more susceptible to oxidative stress and exhibited reduced survival within macrophages. Experiments with the H. pylori SS1 mouse model revealed that the 50% infective dose of the ruvC mutant was approximately 100-fold higher than that of the wild-type SS1 strain. Although the ruvC mutant was able to establish colonization with bacterial loads that were initially similar to those of the parental SS1 strain, infection was spontaneously cleared from the murine gastric mucosa over periods that varied from 36 to 67 days. These results demonstrate that, in this infection model, RuvC is essential for continued survival of H. pylori in vivo and raises the possibility that inactivation of ruvC might be of value in an attenuated vaccine strain.

Biofilm formation on enteral feeding tubes by Cronobacter sakazakii, Salmonella serovars and other Enterobacteriaceae

WHO (2007) recommended that to reduce microbial risks, powdered infant formula should be reconstituted with water at temperatures >70 degrees C, and that such feeds should be used within 2h of preparation. However, this recommendation does not consider the use of enteral feeding tubes which can be in place for more than 48h and can be loci for bacterial attachment. This study determined the extent to which 29 strains of Cronobacter sakazakii, Salmonella serovars, other Enterobacteriaceae and Acinetobacter spp. can adhere and grow on enteral feeding tubes composed of polyvinyl chloride and polyurethane. The study also included silver-impregnated tubing which was expected to have antibacterial activity. Bacterial biofilm formation by members of the Enterobacteriaceae was ca. 10(5)-10(6) cfu/cm after 24h. Negligible biofilm was detected for Acinetobacter gensp. 13; ca. 10 cfu/cm, whereas Cr. sakazakii strain ATCC 12868 had the highest biofilm cell density of 10(7) cfu/cm. Biofilm formation did not correlate with capsule production, and was not inhibited on silver-impregnated tubing. Bacteria grew in the tube lumen to cell densities of 10(7)cfu/ml within 8h, and 10(9)cfu/ml within 24h. It is plausible that in vivo the biofilm will both inoculate subsequent routine feeds and as the biofilm ages, clumps of cells will be shed which may survive passage through the neonates stomach. Therefore biofilm formation on enteral feeding tubes constitutes a risk factor for susceptible neonates.

Host Adaptation and Immune Modulation Are Mediated by Homologous Recombination in Helicobacter pylori

Rearrangement of genomic DNA via homologous recombination provides an alternative mechanism of gene regulation that is essential for successful colonization of the gastric mucosa by Helicobacter pylori. Inoculation of outbred mice with the H. pylori SS1 wild-type strain elicited a T helper (Th) 2 response and established a persistent infection. In contrast, inoculation with an isogenic H. pylori strain defective for homologous recombination elicited a Th1-mediated immune response and clearance of infection within 70 days. We, therefore, demonstrate that recombination is critical for mediating persistence of a microbial pathogen through the induction of ineffective immune responses.

Helicobacter pylori mutants defective in the clpP ATP-dependant protease and the chaperone clpA display reduced macrophage and murine survival.

The ATP-dependent caseinolytic proteases (Clp) are important in resistance against environmental stresses, antibiotic treatments and host immune defences for a number of pathogenic bacteria. ClpP is the proteolytic subunit, whilst ClpA acts both as a chaperone and as an ATPase driving the degradation of damaged or mis-made proteins. The gastric pathogen Helicobacter pylori infects approximately half of the worlds population and can cause gastric or duodenal ulcers, gastric malignancies and mucosa-associated lymphoid tissue lymphomas. The conditions of its in vivo environment expose the organism to host immune cells and upon treatment, antibiotics, conditions likely to cause protein damage. We generated isogenic nonpolar mutants in strain SS1 of clpP and clpA and double mutants with both genes inactivated. Such mutants showed increased sensitivity to antibacterials causing protein damage and/or oxidative stress, in addition to a reduced survival in human macrophages. In the mouse infection model the double mutant SS1 clpAP lacked all ability to colonize the murine host. This suggests that the ability to recover from protein damage is of key importance in the pathogenesis of this organism.

Campylobacter jejuni activates NF-κB independently of TLR2, TLR4, Nod1 and Nod2 receptors ☆

Campylobacter jejuni activates the host transcription factor NF-kappaB that regulates the expression of a number of genes involved in the inflammatory response to bacterial infection. Signaling pathways leading to NF-kappaB by pathogens and/or their products include transmembrane Toll-like receptors (TLRs) and intracellular receptors nucleotide-binding oligomerization domain proteins (Nods). This study was carried out to investigate the role of TLRs (TLR2 and TLR4) and Nods (Nod1 and Nod2) receptors in mediating NF-kappaB activation by C. jejuni. By means of transfecting receptors/molecules under study and measuring reporter gene activity, NF-kappaB activation and subsequent cytokine production by live, heat-killed C. jejuni, or boiled cell extract (BCE) were observed in a range of tissue culture cell lines. This activation is reduced upon transfection of cells with the dominant negative versions (DNV) of TLR-adaptor molecule MyD88. NF-kappaB activation was observed to be augmented in cell lines transfected with TLR2, Nod1, and Nod2 but not with TLR4. Additionally, NF-kappaB activation by C. jejuni was observed to be independent of Nod1 and Nod2 in cells transfected with DNV of these receptors. NF-kappaB activation pathway by C. jejuni may represent a novel mechanism utilising unknown receptors up-regulated by yet to be characterized active component(s). To our knowledge, such observations have not been previously reported for C. jejuni or any other food-borne pathogen.

Novel therapeutic targets in Helicobacter pylori

The failure of current regimens to treat the gastric pathogen Helicobacter pylori is a growing problem. Responsible for gastritis and peptic ulcer disease, and designated as a Class 1 carcinogen, its presence in up to 90% of the population of the developing world makes its treatment a primary concern. The use of genomic, proteomic and transcriptomic data to determine essential gene products as targets for novel therapeutic agents is of key interest in this research. This review describes how such data can be obtained, evaluated and eventually used as a basis for the development of both vaccine and novel anti-helicobacter agents. It indicates both past successes and possible new avenues to exploit the increased availability of such data, whilst also examining the limitations of such approaches.

Induction of a chemoattractant transcriptional response by a Campylobacter jejuni boiled cell extract in colonocytes

BackgroundCampylobacter jejuni, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of Campylobacter jejuni NCTC 11168.ResultsRNA was extracted from the human colonocyte line HCA-7 (clone 29) after incubation for 6 hours with Campylobacter jejuni boiled cell extract and was used to probe the Affymetrix Human Genome U133A array. Genes differentially affected by Campylobacter jejuni boiled cell extract were identified using the Significance Score algorithm of the Bioconductor software suite and further analyzed using the Ingenuity Pathway Analysis program. The chemokines CCL20, CXCL3, CXCL2, Interleukin 8, CXCL1 and CXCL6 comprised 6 of the 10 most highly up-regulated genes, all with Significance Scores ≥ 10. Members of the Tumor Necrosis Factor α/Nuclear Factor-κB super-family were also significantly up-regulated and involved in the most significantly regulated signalling pathways (Death receptor, Interleukin 6, Interleukin 10, Toll like receptor, Peroxisome Proliferator Activated Receptor-γ and apoptosis). Ingenuity Pathway Analysis also identified the most affected functional gene networks such as cell movement, gene expression and cell death. In contrast, down-regulated genes were predominantly concerned with structural and metabolic functions.ConclusionA boiled cell extract of Campylobacter jejuni has components that can directly switch the phenotype of colonic epithelial cells from one of resting metabolism to a pro-inflammatory one, particularly characterized by increased expression of genes for leukocyte chemoattractant molecules.

Using ‘omic’ technology to target Helicobacter pylori

Helicobacter pylori is a causative agent for ulcers, gastritis and cancer of the stomach. Its colonisation of approximately half the worlds population makes it a serious health risk and the increase in resistance to antibiotic therapies means that the search for new treatments to abrogate the disease is of growing importance. Since the publication of the complete DNA sequence data for H. pylori, it has been possible to examine the global pattern for different aspects of the bacteria, such as its proteome, genome and transcriptome and use this information to address the problem of H. pylori associated disease. This review examines the use of techniques collected together under the term ‘omics’ in order to target the disease caused by H. pylori and suggests where these technologies may lead in the future.