Effect of Short-Term Antimicrobial Therapy on the Tolerance and Antibiotic Resistance of Multidrug-Resistant Staphylococcus capitis

Abstract

Background: Bacteria undergo adaptive mutation in the host. However, the specific effect of antimicrobial use on bacterial evolution and genome mutations related to bacterial survival within a patient is unclear.

Materials and methods: Three S. capitis strains were sequentially isolated from cerebrospinal fluid of a clinical inpatient. Antimicrobial susceptibility, growth rate, biofilm formation and whole blood survival of these strains were measured. Relative fitness was calculated. The virulence was examined in the Galleria mellonella model. Whole-genome sequencing and in silico analysis were performed to explore the genetic mechanisms of the changes in antimicrobial resistance phenotype. Hypothetical proteins are cloned, expressed and characterized by detection the susceptibility to gentamycin.

Results: The first isolate was susceptible to rifampin (MIC=0.25 μg/mL), resistant to gentamicin (MIC=16 μg/mL), while the later two isolates were resistant to rifampin (MIC >64 μg/mL), susceptible to gentamicin (MIC=4 μg/mL). For the latter two strains, compared to the first, frameshift mutation in a hypothetical protein encoding gene and base substitutions (in genes saeR, moaA and rpoB) were discovered. The mutation of rpoB gene caused rifampicin resistance. Mutations in saeR, moaA and hypothetical gene are associated with changes in other biological traits. Amino acid sequence-based structure and function identification of the hypothetical protein indicated that a mutation in the encoding gene might be associated with altered aminoglycoside susceptibility. Growth curve showed that the later two isolates grew faster than the first isolate with a positive fitness advantage of 13.5%, and 14.8%, accordingly. Biofilm form ability and whole blood survival of the derivative mutants were also enhanced. No significant differences of virulence in the G. mellonella model were observed.

Conclusion: We report here for the first time that short-term clinical antibiotic use was associated with resistance mutations, collateral sensitivity, and positive in vivo fitness advantages to S. capitis during infection.

Keywords: adaptive enhancement; collateral sensitivity; mutations; resistance; selective pressure.

Figure 1

Overview of the antibacterial regimen and strain isolation.

Figure 2

Predictive analysis of genes with unknown function. (A) Structure prediction of a hypothetical protein based on the amino acid sequence. (B) Structure of a homologous protein from the SWISS-MODEL database (SEQ ID NO: 5uvd.1.A). (C) Comparison of the proteins shown in panels (A and B) proteins. Blue, identical amino acids in both proteins; gray, different amino acids. (D) Possible cellular distribution of the hypothetical protein.

Figure 3

(A) Growth curve. (B) Biofilm formation. (C) In vivo infection of G. mellonella larvae. (D) survival of bacteria in whole blood *** indicated (p<0.001).