Abstract:
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of
nosocomial infections worldwide. It is an ESKAPE pathogen and is known for causing
difficult-to-treat infections due to its antibiotic resistance. In addition to its antibiotic
resistance, this bacterium has a large arsenal of virulence factors that allow this pathogen to
cause disease and to evade the host immune system. An increase in the number of reports of
MRSA isolates from the burn unit and neonatal wards from various hospitals across the
Gauteng province prompted this study. The aim of this study was to molecularly characterise
the MRSA isolates associated with outbreaks in the burn and neonatal wards at four hospitals
in Gauteng, South Africa using multiplex polymerase chain reaction (M-PCR) assays, pulsedfield
gel electrophoresis (PFGE) and whole genome sequencing (WGS). The study also aimed
to determine the antibiotic and virulence gene profiles associated with these MRSA strains.
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To identify MRSA, a M-PCR assay was performed to confirm the presence of the
Staphylococcus 16S rRNA gene, the S. aureus-specific nuclease (nuc) gene and the
methicillin A (mecA) gene that confers resistance to beta-lactam antibiotics. The isolates were
also screened for the Panton-Valentine Leukocidin (pvl) gene, which encodes a pore forming
toxin associated with severe disease. All 85 isolates were confirmed to be MRSA and none of
the isolates were pvl-positive. Susceptibility testing of the MRSA isolates revealed that the
isolates were resistant to antibiotics such as penicillin (100%), cloxacillin (100%), gentamicin
(98%), clindamycin (97%), erythromycin (97%), ciprofloxacin (91%) and tetracycline (84%).
Susceptibility to vancomycin, teicoplanin, linezolid and fusidic acid was observed.
The dendrogram constructed based on the PFGE banding profiles revealed that the MRSA
isolates clustered into three major pulsotypes. The largest pulsotype, Pulsotype A, consisted
of 32 MRSA isolates that were recovered from burn and neonatal wards. Pulsotypes B and C
were made up of five isolates each and only consisted of isolates from the neonatal wards. All
three pulsotypes were composed of MRSA isolates from different hospitals, recovered
between 2015 and 2019. Five representative isolates were selected based on their clustering
and antibiotic resistance and sent for WGS. Three clones, ST239-MRSA-III, ST5-MRSA-I
and ST612-MRSA-IV were identified using WGS data. These clones were associated with
spa types t037, t045 and t1257 respectively. The clone ST239-MRSA-III-t037 was detected
in three different hospitals. The virulence factors detected in the five isolates included
staphylococcal enterotoxins A (SEA), SEB, SEG, SEK, SEN, SEO, and SEQ and the bicomponent
pore-forming leukocidins, gamma-hemolysin and leucocidin ED. The immune
evasion complex (IEC) genes identified were the staphylococcal complement inhibitor,
staphylokinase and SEA.
The movement of patients and healthcare workers between wards and hospitals may have
resulted in intra- and inter-hospital spread of MRSA. The study emphasises the importance of
having infection control programs in place and adhering to them. The importance of
continuous surveillance is also emphasised.
