Antimicrobial resistance mechanisms of linezolid resistant staphylococci and enterococci collected in Gauteng, South Africa

Abstract

Staphylococci and enterococci are key human pathogens responsible for infections associated with healthcare settings. Linezolid is crucial for managing multidrug-resistant (MDR) infections. The monitoring of resistance to antimicrobial agents is a global effort. Linezolid has two surveillance programs which endeavour to monitor linezolid resistance, namely: Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) and Linezolid Experience and Accurate Determination of Resistance (LEADER). In contrast to the ZAAPS and LEADER surveillance programs, linezolid resistance data is lacking in South Africa. This study aimed to determine linezolid resistance 23S ribosomal ribonucleic acid (rRNA) gene mutations and the acquired chloramphenicol-florfenicol resistance (cfr) gene of staphylococci and enterococci obtained from public and private hospitals in Gauteng, South Africa. A total of 79 staphylococcal isolates (43 Staphylococcus capitis, 27 Staphylococcus epidermidis and nine Staphylococcus haemolyticus) and 32 enterococcal isolates (28 Enterococcus faecalis and four Enterococcus faecium) were obtained for investigation. Initial linezolid susceptibility was evaluated using the VITEK® 2 automated system (bioMérieux, France). Staphylococcal and enterococcal isolates showing intermediate resistant and resistant according to the 2019 Clinical and Laboratory Standards Institute (CLSI) guidelines were selected for this study. The minimum inhibitory concentration (MIC) values were confirmed using the ETEST® (bioMérieux, France). Confirmatory identification multiplex polymerase chain reaction (M-PCR) assays and cfr gene detection by polymerase chain reaction (PCR) was used, followed by evaluation of relatedness using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) of seven S. epidermidis isolates and three S. capitis isolates determined the 23S rRNA gene mutations and confirmed the presence of the cfr gene. The ETEST® (bioMérieux, France) MIC values of the staphylococcal isolates ranged between 8 μg/mL and > 256 μg/mL and the enterococcal isolates MIC values ranged between 2 μg/mL and 4 μg/mL. All the staphylococcal isolates were resistant to linezolid and the enterococcal isolates showed susceptibility and intermediate resistance, according to the 2019 CLSI guidelines. The cfr gene was found in eight S. epidermidis isolates. The S. capitis isolates and S. haemolyticus isolates were all cfr negative. The dominant sequence type (ST) among the S. epidermidis isolates was ST23 (n = 4), followed by ST2 (n = 2) and ST22 (n = 1), all of which are clinically relevant STs having been extensively reported among nosocomial infections. Several 23S rRNA gene mutations were observed in this study among the S. epidermidis isolates and the S. capitis isolates. Known and previously reported mutations found in this study were C2190T, G2603T and C2561T among S. epidermidis and S. capitis. However, several unknown and previously unreported 23S rRNA gene mutations were observed among S. capitis, namely: T2157A, T2346C, C2287G, A2295G, A2296G, C2302G, A2305G, C2308G and A2314C. The S. capitis isolate that showed all previously unreported 23S rRNA mutations had a significantly higher MIC value, thus indicating that 23S rRNA gene mutations are a significant contributing factor in linezolid resistance. These novel 23S rRNA gene mutations are not previously reported in the literature and are therefore important for future research. The PFGE results showed diversity among the staphylococcal isolates between hospitals, suggesting a wide spread of the strains. Linezolid resistance is concerning for antimicrobial management efforts and the data generated from this study provides valuable information regarding the prevalence of linezolid resistant strains circulating in the Gauteng region of South Africa.