The interrelationship between cytotoxin-associated gene A (CagA), vacuolating cytotoxin (VacA), and Helicobacter pylori-related diseases was investigated in 155 H. pylori-infected patients. Four (7%) of 60 subjects had mixed cagA+ and cagA- H. pylori infections. The H. pylori isolates from 98.3% of 121 patients with anti-CagA antibodies were cagA+. The occurrence of cagA+ H. pylori among 76 patients with peptic ulcer disease (PUD) was higher (93.4%) than among 79 patients with functional dyspepsia (FD; 64.6%) (odds ratio [OR] = 7.80; P < .001). VacA+ isolates were isolated from 56.6% of the PUD patients and 35.4% of the FD patients (OR = 2.37; P = .0132). For type I (cagA+VacA+) isolates, these numbers were 56.6% and 31.6%, respectively (P = .003). Only 4% of the 71 VacA+ isolates were cagA-. In addition, 37% of the patients with PUD were infected with cagA+VacA- H. pylori. Chi 2 results did not improve when VacA was entered into the model in the presence of cagA, indicating that only cagA is associated with PUD.

ObjectivesListeria monocytogenes is a food-borne pathogen that can cause meningitis. The listerial genotype ST6 has been linked to increasing rates of unfavourable outcome over time. We investigated listerial genetic variation and the relation with clinical outcome in meningitis.MethodsWe sequenced 96 isolates from adults with listerial meningitis included in two prospective nationwide cohort studies by whole genome sequencing, and evaluated associations between bacterial genetic variation and clinical outcome. We validated these results by screening listerial genotypes of 445 cerebrospinal fluid and blood isolates from patients over a 30-year period from the Dutch national surveillance cohort.ResultsWe identified a bacteriophage, phiLMST6 co-occurring with a novel plasmid, pLMST6, in ST6 isolates to be associated with unfavourable outcome in patients (p 2.83e-05). The plasmid carries a benzalkonium chloride tolerance gene, emrC, conferring decreased susceptibility to disinfectants used in the food-processing industry. Isolates harbouring emrC were growth inhibited at higher levels of benzalkonium chloride (median 60 mg/L versus 15 mg/L; p <0.001), and had higher MICs for amoxicillin and gentamicin compared with isolates without emrC (both p <0.001). Transformation of pLMST6 into naive strains led to benzalkonium chloride tolerance and higher MICs for gentamicin.ConclusionsThese results show that a novel plasmid, carrying the efflux transporter emrC, is associated with increased incidence of ST6 listerial meningitis in the Netherlands. Suggesting increased disease severity, our findings warrant consideration of disinfectants used in the food-processing industry that select for resistance mechanisms and may, inadvertently, lead to increased risk of poor disease outcome.

No abstract

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease.

Helicobacter pylori isolates in the Western world possess the cytotoxin-associated gene A (cagA). cagA-positive H. pylori is found to be associated with peptic ulcer disease (PUD) and gastric adenocarcinoma. To investigate the cagA status of H. pylori isolates from Chinese patients with PUD and chronic gastritis (CG), H. pylori populations from 83 patients, 48 with PUD and 35 with CG, were assessed by two different cagA-specific PCRs, Southern blotting, and colony hybridization. The combined results from PCR, Southern blotting, and colony hybridization indicate a prevalence of cagA-positive H. pylori isolates of 98% (47 of 48) among Chinese PUD patients and 100% (35 of 35) among Chinese CG patients. Amplification with primer sets 1 and 2 yielded 52 and 95% of the 82 cagA-positive Chinese H. pylori, respectively. In contrast, the sensitivity of cagA-specific PCR for cagA-positive H. pylori isolates from Dutch patients with primer set 1 was 92% (112 of 122) and that with primer set 2 was 91% (50 of 55). The prevalence of cagA-positive H. pylori populations in Chinese patients with PUD and CG is almost universally high. Therefore, cagA cannot be used as a marker for the presence of PUD in Chinese patients. Our data further suggest that allelic variation in cagA may exist and that distinct H. pylori genotypes may circulate in China and Western Europe.

The two-partner secretion (TPS) pathway is widespread among gram-negative bacteria and facilitates the secretion of very large and often virulence-related proteins. TPS systems consist of a secreted TpsA protein and a TpsB protein involved in TpsA transport across the outer membrane. Sequenced Neisseria meningitidis genomes contain up to five TpsA-and two TpsB-encoding genes. Here, we investigated the distribution of TPS-related open reading frames in a collection of disease isolates. Three distinct TPS systems were identified among meningococci. System 1 was ubiquitous, while systems 2 and 3 were significantly more prevalent among isolates of hyperinvasive clonal complexes than among isolates of poorly invasive clonal complexes. In laboratory cultures, systems 1 and 2 were expressed. However, several sera from patients recovering from disseminated meningococcal disease recognized the TpsAs of systems 2 and 3, indicating the expression of these systems during infection. Furthermore, we showed that the major secreted TpsAs of systems 1 and 2 depend on their cognate TpsBs for transport across the outer membrane and that the system 1 TpsAs undergo processing. Together, our data indicate that TPS systems may contribute to the virulence of N. meningitidis.

Nucleotide sequence accession numbers. The nucleotide sequences of cagA and glmM have been deposited in the GenBank database under accession no. AJ252963 to AJ252986 and AJ252987 to AJ253010, respectively. Genomic Analysis Reveals Variation between Mycobacterium tuberculosis H37Rv and the Attenuated M. tuberculosis H37Ra Strain

Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous H. pylori population, resistant H. pylori colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible H. pylori colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous H. pylori populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary H. pylori population is used to test for clarithromycin susceptibility.