Carbonyl Cyanide 3-Chloro Phenyl Hydrazone (CCCP) Restores the Colistin Sensitivity in Brucella intermedia.

Brucella intermedia (formerly Ochrobactrum intermedium), a non-fermentative bacterium, has been isolated from animals and human clinical specimens. It is naturally resistant to polymyxins, including colistin (CO), and may cause opportunistic infections in humans. We isolated six Brucella intermedia strains from Senegalese monkey stool. In order to determine whether an efflux pump mechanism was involved in CO resistance in B. intermedia, we evaluated the effects of verapamil (VRP), reserpine (RSP), phe-arg ß-naphthylamide dihydrochloride (PAßN) and carbonyl cyanide 3-chloro phenyl hydrazone (CCCP), four efflux pump inhibitors, on these colistin-resistant strains. Using the broth microdilution method, a CO and CCCP combination of 2 µg/mL and 10 µg/mL, respectively, significantly reduced the CO minimal inhibitory concentration (MIC) of B. intermedia, supporting an efflux pump mechanism. In contrast, VRP, PAßN and RSP did not restore CO susceptibility. A time kill assay showed a bactericidal effect of the CO-CCCP combination. Genomic analysis revealed a potential implication in the CO resistance mechanism of some conserved efflux pumps, such as YejABEF, NorM and EmrAB, as previously reported in other bacteria. An inhibitory effect of the CO-CCCP combination was observed on biofilm formation using the crystal violet method. These results suggest that the intrinsic CO resistance in Brucella intermedia is linked to an efflux pump mechanism and that the synergistic effect of CO-CCCP may open a new field to identify new treatments to restore antibiotic efficacy in humans.

Streptococcus bouchesdurhonensis sp. nov. isolated from a bronchoalveolar lavage of a patient with pneumonia.

Strain Marseille-Q6994 was isolated from a 72-year-old patient with pneumonia from Bouches-du-Rhône department, in France. Cells were Gram positive, non-motile, catalase and oxidase-negative cocci. The major fatty acids were hexadecanoic (47.4%) and tetradecanoic acids (28.3%). 16S rRNA gene sequence comparison suggested that strain Marseille-Q6994 was affiliated to the Streptococcus genus. GroEL phylogenetic analysis separated strain Marseille-Q6994 in a distinct branch from the closely related Streptococcus-type strains with standing in nomenclature. Whole genome sequencing-based methods (OrthoAverage Nucleotide Identity, digital DNA-DNA hybridization and pangenome analysis) supported the classification of the strain into a novel species. Therefore, based on the phenotypic, genomic, and phylogenetic analyses, we propose the name Streptococcus bouchesdurhonensis sp. nov for which strain Marseille-Q6994T (CSUR Marseille-Q6994 = DSMZ 113892) is the type strain.

Helicobacter pylori induces expression and secretion of oncostatin M in macrophages in vitro.

BACKGROUND: Helicobacter pylori is pathogenic bacterium that is associated with several gastric diseases in humans. Disease is characterized by severe inflammatory responses is the stomach that are induced by various chemokines and cytokines. Previous reports indicated that some of these responses are mediated through Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. METHODS: We performed JAK/STAT specific microarrays to identify new components of this signaling pathway, which are affected by Helicobacter pylori infection of THP-1 cells. RESULTS: We found that expression and secretion of oncostatin M and of its receptor were strongly up-regulated by Helicobacter pylori. OSM secretion was independent of CagA, VacA or Type IV secretion system. Helicobacter pylori culture supernatant induced OSM secretion. CONCLUSION: The induction of the pleiotropic cytokine oncostatin M suggests a possible role in Helicobacter pylori-mediated inflammation and diseases.

Helicobacter pylori outer membrane vesicles induce expression and secretion of oncostatin M in AGS gastric cancer cells.

Helicobacter pylori, a human pathogen that colonizes the stomach of 50% of the world's population, is associated with gastritis, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. Diseases are characterized by severe inflammatory responses in the stomach that are induced by various chemokines and cytokines. Recently, oncostatin M (OSM), an IL-6 family cytokine, was detected in early gastric cancer biopsies. In this study, we showed that Helicobacter pylori induced secretion of OSM and overexpression of its type II receptor OSMRß (OSM/OSMRß) in a human gastric adenocarcinoma cell line (AGS) over 24 h of infection. Furthermore, we showed that the induction of OSM and OSMRß was carried out by heat-sensitive Helicobacter pylori outer membrane vesicle (OMV) protein. Collectively, our results established, for the first time, a direct relation between Helicobacter pylori OMVs and the OSM/OSMRß signaling axis.

Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity.

Detergent-resistant membranes of eukaryotic cells are enriched in many important cellular signalling molecules and frequently targeted by bacterial pathogens. To learn more about pathogenic mechanisms of Helicobacter pylori and to elucidate novel effects on host epithelial cells, we investigated how bacterial co-cultivation changes the protein composition of detergent-resistant membranes of gastric adenocarcinoma (AGS) tissue culture cells. Using iTRAQ (isobaric tags for relative and absolute quantification) analysis we identified several cellular proteins, which are potentially related to H. pylori virulence. One of the proteins, which showed a significant infection-dependent increase in detergent resistance, was the polarity-associated serine/threonine kinase MARK2 (EMK1/Par-1b). We demonstrate that H. pylori causes the recruitment of MARK2 from the cytosol to the plasma membrane, where it colocalizes with the bacteria and interacts with CagA. Using Mardin Darby Canine Kidney (MDCK) monolayers and a three-dimensional MDCK tissue culture model we showed that association of CagA with MARK2 not only causes disruption of apical junctions, but also inhibition of tubulogenesis and cell differentiation.

CagA of Helicobacter pylori alters the expression and cellular distribution of host proteins involved in cell signaling.

To expand our knowledge of Helicobacter pylori virulence mechanisms, we used iTRAQ (isobaric tagging reagents for relative and absolute quantification)-based proteomic analysis to investigate the effect of H. pylori on gastric AGS tissue culture cells. In particular, we were interested in finding out which effects of H. pylori were dependent on the cytotoxins CagA and VacA. Protein analysis was restricted to detergent-resistant membranes (DRMs), because both toxins were described previously to localize in lipid raft-like domains. Using H. pylori wild type and two isogenic mutants, DeltacagA and DeltavacA, we identified a total of 21 proteins that were either increased or decreased in the DRMs due to bacterial infection. The effect on three of these proteins, ezrin, syndecan-4 and Rab11-FIP1, were furthermore dependent on CagA. Because these proteins have been implicated in cell migration, adhesion and polarity, they might act as important mediators of CagA cytotoxicity.

Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm.

The definition of new species is currently based on polyphasic classification that includes both determination of phenotypic characteristics and DNA-DNA homology. However, none of these techniques is convenient for the rapid characterization of fastidious or non-culturable bacteria. Using sequences available in the GenBank database, we compared the similarities of gene fragments among the currently recognized Bartonella species. This comparison led to both the definition of similarity values that discriminated Bartonella at the species level and assessment of the relative discriminatory power of each gene examined. In this perspective, rpoB and gltA were found to be the most potent.

First molecular evidence of new Bartonella spp. in fleas and a tick from Peru.

Fleas, lice, and ticks collected in Peru in a suburban area of Cusco in November 1998 were tested by polymerase chain reaction for the presence of Bartonella DNA using primers amplifying a fragment of the intergenic spacer region (ITS) gene. Three new Bartonella genotypes were detected in Pulex fleas self-collected from the beds and clothes of schoolchildren and adults. A fourth new genotype was also detected from a tick found on a sheep in the same area. One of the genotypes is closely related to B. vinsoni subsp. berkhoffii, and the others seem to originate from unknown Bartonella species, whose medical importance has yet to be clarified.

Genetic classification and differentiation of Bartonella species based on comparison of partial ftsZ gene sequences.

Currently, 19 species are recognized in the genus Bartonella, 7 of which are involved in an increasing variety of human diseases. Development of molecular tools for detection, identification, and subtyping of strains and isolates has promoted research on Bartonella spp. We amplified and sequenced the portion of the ftsZ gene encoding the N-terminal region of the cell division protein for 13 Bartonella species: Bartonella alsatica, B. birtlesii, B. doshiae, B. elizabethae, B. grahami, B. koehlerae, B. schoenbuchensis, B. taylorii, B. tribocorum, Bartonella vinsonii subsp. arupensis, Bartonella vinsonii subsp. berkhoffii, Bartonella vinsonii subsp. vinsonii, and B. bovis Bermond et al.("B. weissii"). Phylogenetically derived trees revealed four statistically supported groups, indicating that sequencing of the ftsZ gene is a useful tool for identifying evolutionary relationships among Bartonella species. Furthermore, we amplified and sequenced the portion of the ftsZ gene encoding the C-terminal region of the protein for 4 B. bacilliformis isolates, 14 B. clarridgeiae isolates, 14 B. quintana isolates, and 30 B. henselae isolates that were obtained from different geographic regions, hosts, and clinical specimens. B. clarridgeiae and B. quintana sequences were highly conserved, while those of the four B. bacilliformis isolates differed from the type strain at 5 positions. Among B. henselae strains isolated from cats and patients, only two genotypes were detected: Houston and Marseille. Among 80 clinical samples we detected Bartonella spp. in 35 (43.75%) and found the assay to be comparable to that of a combined intergenic-spacer-region- and pap31-based PCR assay. Our results show the usefulness of the portion of the ftsZ gene encoding the C-terminal region for diagnosis of Bartonella infections. More samples should be tested to study its usefulness for epidemiological investigations.

Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum.

Bartonella endocarditis is a severe disease for which blood cultures frequently remain negative. We tested three PCR assays by using specimens of serum sampled early during the disease from 43 patients diagnosed in our laboratory as having Bartonella endocarditis on the basis of serological, culture, and/or valvular molecular detection. We tested a two-step nested PCR (TSN-PCR), a one-step nested PCR (OSN-PCR) with a regular thermal cycler, and a one-step nested PCR with the LightCycler (LCN-PCR). These assays were performed with primers derived from the riboflavin synthase-encoding gene ribC, never before amplified in our laboratory. Due to contamination of negative controls, the results of the TSN-PCR were not interpretable, and this technique was no longer considered. The LCN-PCR had a specificity of 100% and a sensitivity of 58.1%, higher than those of the OSN-PCR (18.6%; P < 0.01) and prolonged blood culturing (7.1%; P < 0.01). The LCN-PCR results correlated strictly with those of other direct diagnostic tests, when available, and identified the causative species for six patients previously diagnosed on the basis of serological analysis only. The efficacy of the LCN-PCR was not influenced by antibiotics (P = 0.96) but was altered by prolonged storage of serum specimens at -20 degrees C (P = 0.04). Overall, the LCN-PCR is specific and more sensitive than traditional methods (i.e., culturing and/or PCR with EDTA-treated blood). It can easily be applied to the diagnosis of patients with suspected Bartonella endocarditis, especially when only serum is available.

Genomic variation of Bartonella henselae strains detected in lymph nodes of patients with cat scratch disease.

Bartonella henselae is the primary agent of cat scratch disease (CSD). In order to study the genetic variation of B. henselae and the correlation of the various genotypes with epidemiological and clinical findings, two seminested, groEL- and pap31-based PCR assays were carried out with specimens from 273 patients. Amplicons were sequenced to determine the genotype of the causative Bartonella species. Compared to our reference intergenic spacer region-based PCR, the groEL- and pap31-based assays were 1.7 and 1.9 times more sensitive, respectively. All 107 positive patients were infected with B. henselae; neither Bartonella clarridgeiae nor other species were detected. Based on the groEL and pap31 sequences, B. henselae amplicons were classified into two genogroups, Marseille and Houston-1, and into four variants, Marseille, CAL-1, Houston-1, and a new variant, ZF-1. Patients infected with either one or the other genogroup did not exhibit different epidemiological or clinical characteristics. Our study highlights the genotypic heterogeneity of B. henselae in patients with CSD.

Phylogenetic classification of Bartonella species by comparing groEL sequences.

Bartonella is a bacterial genus classified in the alpha-Proteobacteria on the basis of 165 rDNA sequence comparison. The highly conserved heat-shock chaperonin protein, GroEL, has proved to be a valuable resolving tool to classify ten Bartonella species. The groEL gene was amplified and sequenced from ten Bartonella isolates: Bartonella alsatica, Bartonella vinsonii subsp. arupensis, Bartonella taylorii, Bartonella tribocorum, Bartonella birtlesii, Bartonella henselae Marseille (URLLY8), B. henselae (90-615), B. henselae (Fizz), B. henselae (CAL-1) and B. henselae (SA-2). Then, phylogenetic relationships were inferred between our isolates and eight other species and subspecies from the comparison of both 16S rDNA and groEL sequences using parsimony, neighbour-joining and maximum-likelihood methods. By using groEL sequences, the first reliable classification of most known Bartonella species and subspecies was established. Four strongly supported subgroups were distinguished: firstly, the two human pathogens B. henselae and Bartonella quintana; secondly, a cluster including four rodent isolates, Bartonella elizabethae, B. tribocorum, Bartonella grahamii and B. taylorii; thirdly, a cluster including the B. vinsonii subspecies (B. vinsonii subsp. vinsonii, arupensis and berkhoffii); and lastly, B. birtlesii and 'Bartonella weissi'. 'Bartonella washoensis', B. alsatica, Bartonella doshiae, Bartonella bacilliformis and Bartonella clarridgeiae did not reliably cluster with any other Bartonella species. In addition, the groEL gene was shown to be useful in subtyping six B. henselae isolates into three variants: Houston, Marseille and Fizz.

Improved culture from lymph nodes of patients with cat scratch disease and genotypic characterization of Bartonella henselae isolates in Australia.

Over a 4-year period we detected Bartonella henselae isolates in 104 of 297 specimens (35.1%) from Australian patients clinically suspected of having cat scratch disease by amplification of a fragment of the htrA gene. We isolated 17 B. henselae strains (20.5%) from the 83 PCR-positive human specimens available for culture. Our culture method was based on prolonged incubation in a moist atmosphere of blood agar to which hemin was added. We obtained more B. henselae isolates than the number of all other isolates from lymph nodes reported in the literature. In order to identify and study the genetic variation of Australian B. henselae isolates, we determined the sequence of a fragment of the pap31 gene from our 17 human isolates and also from 8 Australian cat isolates. Thirteen of the human B. henselae isolates belonged to the Houston genotype, variant Houston-1 (76.5%), and four belonged to the Marseille genotype, variant CAL-1 (23.5%). In contrast, seven cat isolates were classified as B. henselae Marseille, variant CAL-1 (87.5%), and one was classified as B. henselae Houston, variant Houston-1 (12.5%). Our study describes an efficient culture method for the diagnosis of cat scratch disease and contributes to the description of the genotypic distribution of B. henselae in Australia.

Genotypic characteristics of two serotypes of Bartonella henselae.

The study of 16S rRNA gene sequences of all isolates of Bartonella henselae obtained in our laboratory and others from human patients or cats has revealed two genotypes according to the sequence of the 16S rRNA gene. Two isolates of these genotypes have previously been related to two different serotypes, and lack of cross-protection of the two serotypes has been demonstrated in cats. We investigated the grouping of eight strains of B. henselae on the basis of 16S ribosomal DNA, 35-kDa protein, Pap 31 protein, and internal transcribed spacer (ITS) gene sequencing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles; and monoclonal antibody reactivity studies. Houston-1, 90-615, and SA2 strains showed the same patterns in SDS-PAGE, but they differed from the patterns of B. henselae isolates URBHLLY8, URBHLIE9, Cat6, Fizz, and CAL-1. Nine monoclonal antibodies derived from BALB/c mice immunized with B. henselae Houston-1 strain reacted only with strains Houston-1, 90-615, and SA2, and not with any other Bartonella strains. The two serogroups corresponded with two genotypes based on differences in the sequences of the genes encoding 16S rRNA, 35-kDa protein, and Pap 31 protein. Sequences of ITS genes were highly divergent among strains, as each had a unique sequence and the subdivision was not supported by DNA-DNA relatedness study. Study of 22 additional strains of B. henselae isolated from French bacteremic cats demonstrated that they all belong to one or the other of the proposed serotype or genotype.

Bartonella henselae in Ixodes ricinus ticks (Acari: Ixodida) removed from humans, Belluno province, Italy.

The potential role of ticks as vectors of Bartonella species has recently been suggested. In this study, we investigated the presence of Bartonella species in 271 ticks removed from humans in Belluno Province, Italy. By using primers derived from the 60-kDa heat shock protein gene sequences, Bartonella DNA was amplified and sequenced from four Ixodes ricinus ticks (1.48%). To confirm this finding, we performed amplification and partial sequencing of the pap31 protein and the cell division protein ftsZ encoding genes. This process allowed us to definitively identify B. henselae (genotype Houston-1) DNA in the four ticks. Detection of B. henselae in these ticks might represent a highly sensitive form of xenodiagnosis. B. henselae is the first human-infecting Bartonella identified from Ixodes ricinus, a common European tick and the vector of various tickborne pathogens. The role of ticks in the transmission of bartonellosis should be further investigated.