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Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

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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.

No MeSH data available.


Related in: MedlinePlus

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.
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f2: 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.

Mentions: The first antibiotic to generate inhibitory halo 0 mm was RIF, with only 4 passages to obtain confluent growth (strain 57.19). However, overall, the antibiotic requiring the least number of passages to generate confluent growth was CIP with a mean of 10.6 passages. On the other hand, CHL required >37.3 passages (Table 1). Thin growth was observed inside the halo recorded during the process of mutant selection of 57.20Azm. Thus, after the initial 35 passages, most antibiotic-resistant mutants showed confluent growth in the presence of the antibiotic disc (inhibitory halo 0 mm) except for two out of three mutants selected with CHL, which presented inhibitory halos of 18 mm (57.18Chl-35) and 32 mm (57.20Chl-35). After 5 additional serial passages (total: 40 passages), 57.20Chl-40 achieved a halo of 0 mm, while 57.18Chl-40 remained with an inhibitory halo of 18 mm (Fig. 2).


Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants
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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035977&req=5

f2: 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.
Mentions: The first antibiotic to generate inhibitory halo 0 mm was RIF, with only 4 passages to obtain confluent growth (strain 57.19). However, overall, the antibiotic requiring the least number of passages to generate confluent growth was CIP with a mean of 10.6 passages. On the other hand, CHL required >37.3 passages (Table 1). Thin growth was observed inside the halo recorded during the process of mutant selection of 57.20Azm. Thus, after the initial 35 passages, most antibiotic-resistant mutants showed confluent growth in the presence of the antibiotic disc (inhibitory halo 0 mm) except for two out of three mutants selected with CHL, which presented inhibitory halos of 18 mm (57.18Chl-35) and 32 mm (57.20Chl-35). After 5 additional serial passages (total: 40 passages), 57.20Chl-40 achieved a halo of 0 mm, while 57.18Chl-40 remained with an inhibitory halo of 18 mm (Fig. 2).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus