Abstract:
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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-beta-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 Delta62-65 (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. |