Rapid detection of extra-intestinal pathogenic Escherichia coli multi-locus sequence type 127 using a specific PCR assay.

PURPOSE: Members of the ST127 uropathogenic E. coli (UPEC) clone have a high virulence potential and are also highly virulent in insect infection models. However, strains of this lineage are reported in relatively low numbers in many studies. ST127 strains are also usually widely susceptible to antibiotics and, consequently, their true prevalence may be under-recognized as they will be eradicated during empirical therapy. A genuine concern is the possibility that members of this highly virulent lineage will acquire resistance, leading to a more serious threat. The aim of this study was to design and validate a PCR assay specific to ST127. METHODOLOGY: Genomic sequences obtained from various UPEC isolates from the leading clones were used in comparative genomic analyses to allow identification of highly discriminatory sequences specific to E. coli ST127. The fliC (flagellin) and a homologue of the upaG (autotransporter adhesin) gene were identified as meeting our criteria and were used to develop a multiplex PCR assay. A total of 143 UPEC isolates representing 99 different MLST clones from three locations (North West and South West England and Riyadh, Saudi Arabia) were used to validate the PCR assay. RESULTS: The multiplex PCR readily identified all 29 E. coli ST127 isolates but, equally importantly, produced no false positives with representatives of any of the other 98 STs tested. CONCLUSION: We report the design and validation of a specific multiplex PCR for the rapid and reliable identification of ST127, which can be used for enhanced surveillance for this high-risk clone.

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 world's 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.

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.

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.

Long-term infection with Helicobacter felis and inactivation of the tumour suppressor gene p53 cumulatively enhance the gastric mutation frequency in Big Blue transgenic mice.

The aims of this study were to determine whether colonization with Helicobacter felis resulted in the accumulation of mutations within murine gastric tissue and whether the degree of genetic damage was increased by p53 deficiency. Female C57BL/6 mice carrying either the lambda/lacI transgene (Big Blue transgenic mice) or the lambda/lacI transgene and deficient in one allele of the p53 tumour suppressor gene (TSG-p53/Big Blue) were inoculated with H felis. Seven months after inoculation, mutations in the target lacI gene were assessed using the Big Blue transgenic mutagenesis assay system in these animals and in controls. There was an approximately two-fold increase in lacI mutations in gastric mucosa harvested from mice infected with H felis and also from non-infected mice heterozygous for the p53 allele relative to wild-type mice. The mutation frequency in mice infected with H felis and deficient in one allele of p53 was increased approximately three-fold. Active gastric inflammation was significantly greater in H felis-infected p53 hemizygous mice compared with H felis p53 wild-type mice. Gastric epithelial proliferation was similarly increased with infection in both of these latter groups of mice. In infected mice, there was a significant correlation between the mutation frequency and the degree of active gastric inflammation. These data suggest a synergistic action between infection with H felis and p53 deficiency in the accumulation of mutations within gastric tissue. Active neutrophil infiltration in gastric Helicobacter infection may contribute to the increased levels of mutation observed.

NapA protects Helicobacter pylori from oxidative stress damage, and its production is influenced by the ferric uptake regulator.

The Helicobacter pylori protein NapA has been identified as a homologue of the Escherichia coli protein Dps. It is shown in this study that, like Dps, NapA is produced maximally in stationary phase cells and contributes to the ability of H. pylori to survive under oxidative stress conditions. Moreover, NapA co-localizes with the nuclear material, suggesting that it can interact with DNA in vivo. Furthermore, it is demonstrated that repression of NapA production by iron starvation was not so pronounced in a H. pylori fur mutant, suggesting that the ferric uptake regulator (Fur) is involved in napA regulation, and a potential fur box by which this control could be mediated is identified. This finding is consistent with the regulation of iron-binding proteins by Fur and also the modulation of Fur during oxidative stress, thus allowing NapA levels to be increased in the environmental conditions under which its ability to protect DNA from attack by toxic free radicals is most beneficial to the cell.

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

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.

Monotherapy with mastic does not eradicate Helicobacter pylori infection from mice.

OBJECTIVE: To determine the ability of mastic monotherapy to eradicate Helicobacter pylori infection from mice. MATERIALS AND METHODS: The susceptibility of H. pylori SS1 to mastic was assessed by broth dilution determination of the MIC and MBC. Mice were inoculated intragastrically with either a suspension of H. pylori SS1 (n = 70) or brain-heart infusion broth alone (n = 10). Mice were given antimicrobial chemotherapy 4 weeks after infection and were administered the mouse equivalent of either 2 g of mastic twice daily for 7 days or a triple therapy regimen containing the mouse equivalent of 400 mg of metronidazole, 250 mg of clarithromycin and 20 mg of omeprazole twice daily for 7 days. Mice were killed either immediately or 1 month after the completion of treatment, and their stomachs cultured for H. pylori. RESULTS: The mastic MIC and MBC of H. pylori SS1 were 7.80 and 31.25 mg/L, respectively. The triple therapy regimen eradicated infection from 19 of 20 SS1-infected mice. Mastic failed to eradicate infection from any of the 18 SS1-infected mice (P < 0.001) and there was no signifi- cant reduction in gastric bacterial load in mice treated with this regimen. CONCLUSION: Despite reported beneficial effects in ulcer patients and the good in vitro activity of mastic against H. pylori, this compound is unable to eradicate H. pylori infection from mice.

Production of the RdxA protein in metronidazole-susceptible and -resistant isolates of Helicobacter pylori cultured from treated mice.

The objective of this study was to use immunoblotting with RdxA antisera to examine the production of the RdxA protein in mouse-derived metronidazole-susceptible and -resistant isolates of Helicobacter pylori. A 24 kDa immunoreactive band corresponding to RdxA was observed in all 15 metronidazole-susceptible and five of 50 metronidazole-resistant isolates. The rdxA gene of these five isolates contained missense mutations and transformation experiments confirmed that these mutations were associated with inactivation of the rdxA gene. No RdxA protein was produced in the other 45 metronidazole-resistant strains, including one in which the nucleotide sequence of the rdxA gene was unchanged. These results demonstrate a high correlation between production of the RdxA protein and susceptibility of H. pylori to metronidazole. Testing for the absence of the RdxA protein identifies the majority of strains that will respond poorly to metronidazole-containing eradication regimens.

Metronidazole resistance in Helicobacter pylori.

Modern triple drug regimens are highly effective for treating Helicobacter pylori infection, but bacterial resistance to one of the most effective antibiotics, metronidazole, is a serious and increasing problem. The activity of metronidazole in H. pylori is dependent on reduction of its nitro moiety to highly reactive compounds that cause DNA strand breakage. The acquisition of resistance is highly associated with mutational inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase. Recent evidence has suggested that inactivation of frxA (NADPH flavin oxidoreductase), fdxB (ferredoxin-like protein) and possibly other reductase-encoding genes may also contribute to the resistant phenotype. Improved understanding of the mechanisms of metronidazole resistance in H. pylori is essential for the development and validation of biopsy-based tests for detection of resistance in clinical practice.