Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

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 Δ_62-65 (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.

Figure 1. Colony morphology.

The photograph shows B. bacilliformis presenting T1 colony morphology. The colony is characterised by a small, translucent round morphology, with a regular edge and a small halo. The colonies present a “bubble” in the center of the colony. The morphology was unstable and disappeared after reculture.

Figure 2. Evolution of disc diameter halo during serial passages.

(A) Azithromycin, (B) Chloramphenicol, (C) Ciprofloxacin, (D) Rifampicin. This figure demonstrates the ease with each mutant are selected for each antibiotic. The halo diameters (measured in mm) are reported every 5 passages or at the passage in which halo zero was obtained. In (B) is clearly visualised the difficulty with which resistance to chloramphenicol (CHL) is developed.

Figure 3. Hydrophobic and charge patterns associated with GyrA amino acid substitutions.

(A) Alterations in the hydrophobic pattern. Additionally to the amino acid substitutions detected (Ala-91 → Val and Asp-95 → Gly) the theoretical effect of the presence of Ser-91 also shows the gradual effect on the hydrophobic pattern related to the presence of Ser, Ala or Val at position 91. (B) Effect on the charge pattern. This graph only shows the effect of Asp-95 and Gly-95 since the presence of Val-91 does not result in charge pattern alterations. The (A) comprises the amino acid sequence from amino acids 84 to 99, while in (B) the amino acid sequences analysed are from amino acids 70 to 107.