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

Molecular approach for detection and phylogenetic classification of Bartonella species

AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

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

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°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

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 &beta;-naphthylamide dihydrochloride (PA&beta;N) 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 &micro;g/mL and 10 &micro;g/mL, respectively, significantly&nbsp;reduced the CO minimal inhibitory concentration (MIC) of B. intermedia, supporting an efflux pump mechanism. In contrast, VRP, PA&beta;N and RSP did not restore CO susceptibility. A time kill assay showed a bactericidal effect of the CO&ndash;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&ndash;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&ndash;CCCP may open a new field to identify new treatments to restore antibiotic efficacy in humans

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

International audienceBrucella 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 β-naphthylamide dihydrochloride (PAβN) 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, PAβN 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