Antibiotic Susceptibility of Biofilm Cells and Molecular Characterisation of Staphylococcus hominis Isolates from Blood

Abstract

Objectives: We aimed to characterise the staphylococcal cassette chromosome mec (SCCmec) type, genetic relatedness, biofilm formation and composition, icaADBC genes detection, icaD expression, and antibiotic susceptibility of planktonic and biofilm cells of Staphylococcus hominis isolates from blood.

Methods: The study included 67 S. hominis blood isolates. Methicillin resistance was evaluated with the cefoxitin disk test. mecA gene and SCCmec were detected by multiplex PCR. Genetic relatedness was determined by pulsed-field gel electrophoresis. Biofilm formation and composition were evaluated by staining with crystal violet and by detachment assay, respectively; and the biofilm index (BI) was determined. Detection and expression of icaADBC genes were performed by multiplex PCR and real-time PCR, respectively. Antibiotic susceptibilities of planktonic cells (minimum inhibitory concentration, MIC) and biofilm cells (minimum biofilm eradication concentration, MBEC) were determined by the broth dilution method.

Results: Eighty-five percent (57/67) of isolates were methicillin resistant and mecA positive. Of the mecA-positive isolates, 66.7% (38/57) carried a new putative SCCmec type. Four clones were detected, with two to five isolates each. Among all isolates, 91% (61/67) were categorised as strong biofilm producers. Biofilm biomass composition was heterogeneous (polysaccharides, proteins and DNA). All isolates presented the icaD gene, and 6.66% (1/15) isolates expressed icaD. This isolate presented the five genes of ica operon. Higher BI and MBEC values than the MIC values were observed for amikacin, vancomycin, linezolid, oxacillin, ciprofloxacin, and chloramphenicol.

Conclusions: S. hominis isolates were highly resistant to methicillin and other antimicrobials. Most of the detected SCCmec types were different than those described for S. aureus. Isolates indicated low clonality. The results indicate that S. hominis is a strong biofilm producer with an extracellular matrix with similar composition of proteins, DNA and N-acetylglucosamine; and presents high frequency and low expression of icaD gene. Biofilm production is associated with increased antibiotic resistance.

Fig 1. Pulsed-field gel electrophoresis dendrogram and biofilm production of S. hominis isolates.

¹Biofilm production level: OD <0.12 negative, 0.12–0.24 weak and >0.25 strong. Neg: negative. ²All: positive for icaR, icaA, icaD, icaB, and icaC. ³ND non-determinate. ⁴Similarity coefficients were generated from a similarity matrix calculated with the Jaccard coefficient using SPSS 22.0 software.

Fig 2. Biofilm detachment with NaIO4, proteinase K and DNase.

Biofilm detachment level of 64 biofilm producers S. hominis isolates after treatment with NaIO₄, proteinase K and DNase.

Fig 3. Increase BI mean and correlation with the differences in values observed between MIC and MBEC for all 67 isolates: no difference, one-fold increase, two-fold increase, three-fold increase.

*Indicates a correlation between increases in antibiotic resistance (between MBEC and MIC) and BI mean. p < 0.05 by both ANOVA and Chi-square tests.