Abstract
The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.
Highlights
Bartonella bacilliformis is the causative agent of Carrion’s disease, a biphasic endemic illness of the Andean valleys
The development of the antibiotic-resistant mutants required approximately 18 months, 4 antibiotic-resistant mutants were obtained from each parental strain, one for each antibiotic included in the study
To the best of our knowledge only three in vitro studies has been developed to date[17,18,19], and none has determined the stability of the antibiotic resistance selected or the role of efflux pump overexpression
Summary
Bartonella bacilliformis is the causative agent of Carrion’s disease, a biphasic endemic illness of the Andean valleys. In the acute stage (the so-called Oroya fever) severe haemolytic anaemia is present, resulting in 40–85% of deaths in untreated people and decreases to around 10% if correctly treated[1,2,3]. In this stage the presence of concomitant infections such as bloodstream Salmonella infections, among others[1,4,5,6], are frequent due to the temporal immunosuppression induced by B. bacilliformis[7]. These characteristics are extended to other members of the Bartonella genus[13]
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