Diversity of Multidrug Efflux Genes and Phenotypic Evaluation of the In vitro Resistance Dynamics of Clinical Staphylococcus Aureus Isolates Using Methicillin; a Model β-lactam

Abstract

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) across the world often leave clinicians with little or no choice of treatment options. The multi-drug efflux (MDE) genes are bacterial survival mechanisms responsible for the pumping out of antibiotics and other biocides from the cytoplasm. Whilst effort is being made in the development of antibiotic adjuvants such as efflux pumps inhibitors, information is needed on the diversity of these MDEs in the circulating S. aureus and on the growth dynamics of the clinical isolates in response to antibiotics is not regularly examined.

Methods: Here, we evaluated the diversity of MDEs in cinical S. aureus recovered in a tertiary academic hospital, Pretoria, South African hospital using PCR and also employed visual minimum inhibitory concentration and quantitative analysis of spectrophometric measurements of bacterial growth in the presence of a model β lactam antibiotic (methicillin), to phenotypically elucidate the resistance pattern of these isolates in response to methicillin.

Results: Three major distribution patterns of MDEs were observed in the clinical isolates evaluated. Moreover, norA, nor B and tet38 were present in 98.9% of the isolates while other MDE were present in different proportions ranging from 40 to 98.6% of the isolates. In addition, S. aureus isolates, be it of MRSA or MSSA genotype did not habour the same set of MDEs despite being recovered from the same hospital setting. Finally, we showed that MSSA displayed phenotypic resistance to methicilllin despite the non-detection of the mecA resistance gene.

Conclusions: Our data suggest that the growth of S. aureus may be enhanced by β lactams (methicillin) and that MSSA may also display resistance to methicillin and perhaps other β lactam antibiotics. The high prevalence of MDEs suggestive of resistance to a broad spectrum of biocides and fluoroquinolones are particularly disturbing.

Keywords: Methicillin-resistant Staphylococcus aureus (MRSA); Multi-drug efflux (MDE) genes; Quantitative analysis; antibiotic adjuvants; β lactam antibiotic.

Fig. (1)

Heatmap matrix showing a visual representation of the distribution pattern of the 11 MDEs in MRSA and MSSA isolates. The blue reagions indicate the set of isolates in which an MDE was not detected while the red regions indicate the set of isolates in which the MDEs were detected by PCR. A similarity in the distribution pattern of qacA/B and smr; norA and norB can be seen.

Fig. (2)

A: A dendrogram showing the co-clustering of clinical MRSA and MSSA isolates. B: Cluster-dependent heterogenic growth response to methicillin based on the general trend of the contribution of each isolates in a cluster. The error bar shows standard error from the mean. Groups 1 and 2 show exclusive clustering of MRSA while Groups 3 and 4 show co-clustering of MSSA and MRSA isolates. Samples ID with SA are MSSA and those with TA are MRSA.

Fig. (3)

Homogenous response pattern to methicillin by MRSA and MSSA. The lines linking each methicillin concentration indicate polynomial trends of the bacterial growth. Isolates IDs with TA indicate MRSA while SA indicate MSSA.