Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach.

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

Prioritisation of potential drug targets against Bartonella bacilliformis by an integrative in-silico approach

BACKGROUND Carrion's disease (CD) is a neglected biphasic illness caused by Bartonella bacilliformis, a Gram-negative bacteria found in the Andean valleys. The spread of resistant strains underlines the need for novel antimicrobials against B. bacilliformis and related bacterial pathogens. OBJECTIVE The main aim of this study was to integrate genomic-scale data to shortlist a set of proteins that could serve as attractive targets for new antimicrobial discovery to combat B. bacilliformis. METHODS We performed a multidimensional genomic scale analysis of potential and relevant targets which includes structural druggability, metabolic analysis and essentiality criteria to select proteins with attractive features for drug discovery. FINDINGS We shortlisted seventeen relevant proteins to develop new drugs against the causative agent of Carrion's disease. Particularly, the protein products of fabI, folA, aroA, trmFO, uppP and murE genes, meet an important number of desirable features that make them attractive targets for new drug development. This data compendium is freely available as a web server (http://target.sbg.qb.fcen.uba.ar/). MAIN CONCLUSION This work represents an effort to reduce the costs in the first phases of B. bacilliformis drug discovery.

Bartonella bacilliformis, endemic pathogen of the Andean region, is intrinsically resistant to quinolones.

OBJECTIVES: To analyze the sequence of the region involved in the development of quinolone resistance of the gyrA and parC genes in a series of Bartonella bacilliformis isolates recovered prior to the introduction of quinolones, as well as one clinical isolate recovered in the 1970s, establishing the susceptibility levels to nalidixic acid and ciprofloxacin. METHODS: Five B. bacilliformis were studied: four isolated before 1957, prior to the introduction of quinolones in clinical practice. The remaining strain was isolated in 1977. A fragment of the gyrA and parC genes was amplified and sequenced. Susceptibility to nalidixic acid and ciprofloxacin was established by the E-test method. RESULTS: All the strains were resistant to nalidixic acid (minimum inhibitory concentration (MIC) >256 mg/l). Three isolates presented decreased susceptibility to ciprofloxacin and two were highly resistant (MIC >32 mg/l). All the strains presented an Ala at position 91 of GyrA and position 85 of ParC. CONCLUSIONS: B. bacilliformis presents a constitutive resistance to quinolones, which may be related to the presence of Ala at position 91 of GyrA and 85 of ParC. These results advise against the current clinical guidelines recommending the use of ciprofloxacin to treat bartonellosis in some countries of the Andean area.

Heterogeneity of susceptibility to fluoroquinolones in Bartonella isolates from Australia reveals a natural mutation in gyrA.

OBJECTIVES: Bartonella sp. are intracellular bacteria associated with an increasing number of clinical manifestations but with few published data on in vitro susceptibility testing of antibiotics. Our objective was to evaluate in vitro antibiotic susceptibilities of 20 new Bartonella isolates from animals in Australia. METHODS: MICs were determined using Etest assay on Columbia agar supplemented with 5% horse blood. The presence of mutations in the quinolone-resistance-determining region (QRDR) of gyrA was searched for after PCR amplification and DNA sequencing using specific oligonucleotide primers. RESULTS: Bartonella isolates from Australia were susceptible to rifampicin, tetracyclines, beta-lactam and macrolide compounds but were resistant to vancomycin. We found heterogeneity of susceptibility for fluoroquinolones with ciprofloxacin being more effective (MICs from 0.06 to 0.5 mg/L) than ofloxacin (MICs from 0.5 to 4 mg/L). This heterogeneity was linked to a natural mutation Ser-83-->Ala (Escherichia coli numbering) in the QRDR. Surprisingly, this mutation was also present in the QRDR of Bartonella henselae, Bartonella quintana and Bartonella bacilliformis. CONCLUSIONS: Etest is a sensitive and reliable assay for evaluation of antibiotic susceptibility in the genus Bartonella. The higher sensitivity of this method allowed us to detect heterogeneity of susceptibility among fluoroquinolones that was associated with natural mutation in the QRDR of the DNA gyrase. Because a high level of resistance to fluoroquinolones due to a second mutation may be obtained easily in vitro, we believe that fluoroquinolone compounds should be avoided for the treatment of any Bartonella-related diseases.

Molecular mechanisms of resistance to antibiotics in Bartonella bacilliformis.

OBJECTIVES: Bartonella bacilliformis is the aetiological agent of Carrion's disease. Although ciprofloxacin, rifampicin and erythromycin have been successfully used in the treatment of the disease, failures and relapses have been reported. The objective of our study was to select in vitro mutants resistant to antibiotics in order to determine the frequency of mutations and to characterize the mechanism of resistance at the molecular level. METHODS: Antibiotic-resistant mutants were selected by serial passages of bacteria on blood agar plates containing antibiotics. Candidate genes involved in resistance were amplified and sequenced and compared in order to look at mutations associated with antibiotic resistance. RESULTS: Ciprofloxacin-, rifampicin- and erythromycin-resistant mutants were obtained after five, three and four passages, respectively. Conversely, no mutant was obtained with either gentamicin or doxycycline even after 16 passages. The ciprofloxacin mutant contained an amino acid change at position 87 (Asp --> Asn) in its quinolone resistance-determining region of the DNA gyrase protein, whereas the rifampicin-resistant strain had an amino acid change at position 531 (Ser --> Phe) in the rifampicin resistance-determining region of the rpoB gene. Similarly, the erythromycin-resistant mutant showed an A2058G mutation in the 23S rRNA gene. CONCLUSIONS: According with the current knowledge on the treatment of human bartonellosis, we believe that doxycycline in association with gentamicin may be the preferred regimen for the treatment of the acute and eruptive stages of Carrion's disease, but clinical trials are warranted to support our findings.