Strain characterisation, antibiotic resistance and Meca Gene analysis of Staphylococci
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Date
1999-12
Authors
Vorster, Alvera
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
S. aureus, is undoubtedly the most pathogenic of the Staphylococcus species,
having the ability to produce invasive and toxigenic infections. Historically, the
less virulent coagulase-negative staphylococci (ENS) were regarded as
clinically insignificant contaminants but they have become increasingly
implicated as opportunistic nosocomial pathogens. The increasing frequency of
methicillin and multiple-antibiotic resistance in staphylococci over the last four
decades has seriously compromised therapeutic options.
The study was designed to (a) identify and type staphylococcal species, (b)
undertake standardized antimicrobial susceptibility testing, and (c) determine the
prevalence of methicillin resistance in staphylococcal isolates.
Presumptive staphylococcal strains were isolated from the diagnostic
microbiology laboratories of Pelonomi (147 strains) and Universitas (144 strains)
hospitals. Subsequently, these strains were identified using conventional
biochemical methods. Species-specific PCR identification assays were
performed on selected ENS strains. Antimicrobial susceptibilities were
determined for 13 clinically available antibiotics on 144 staphylococcal isolates
and on selected strains for 5 developmental agents. RAPD and plasmid profiles
were generated to assess possible epidemiological strain relatedness. For the
detection of methicillin resistance in staphylococci the following methods were
used: (a) oxacillin MICs detecting phenotypic methicillin resistance levels (b) a
multiplex-PCR detecting the mecA gene, and (c) a slide agglutination test
(MASTALEX-MRSA) detecting PBP2' production.
The inclusion of bile-aesculin agar plates and a bacitracin susceptibility test into
the diagnostic laboratory protocol for the identification of staphylococci would
reduce misidentification of non-staphylococcal isolates by 12.8%. Colony
morphology in combination with the coagulase test could be instrumental in the
improved differentiation of S. aureus from CNS. Although expensive, when a
rapid and fairly comprehensive identification of CNS species is required, the
STAPH ID 32 API system was found to be satisfactory. Due to the apparent
inaccuracy of the PCR identification assay based on API, its use in the clinical
microbiology laboratory would be argued against; although if standardized and
expanded it could be considered for future incorporation in routine practice.
The presence of unique RAPD profiles for each specific Staphylococcus species
suggests RAPD profiling could offer a molecular identification technique for the
majority of commonly isolated CNS in the clinical microbiology laboratory. Good
typeability was observed for Primer I and III in CNS strains, however, for
S. aureus strains, poor typeability and discrimination was observed. It has been
found by other researchers that longer oligonucleotide primers (>10 bp in length)
are more efficient for S. aureus strain typing, but to the contrary in the present
study primers ERIC 1 and 2 were totally unsatisfactory. Combined susceptibility
data and plasmid profile analysis revealed strain relatedness in S. haemolyticus
isolates but RAPD Primers I and III indicated otherwise.
All staphylococcal strains isolated were vancomycin-susceptible. Of the
staphylococci isolated in the Universitas hospital, 34.3% were oxacillin-resistant.
Similarly, 30.1% staphylococci isolated in Pelinomi hospital were oxacillinresistant.
Resistance to ciprofloxacin, erythromycin, gentamicin and
clindamycin was found in 49% of oxacillin-resistant staphylococci. In
comparison to the other quinolones tested, moxifloxacin showed superior
activity against oxacillin-resistant CNS. The glycylcyclines, LY333328 and Q/D
may well be considered excellent alternatives to vancomycin for the treatment of
MRSA. Of the developmental agents investigated, linezolid showed consistent
in vitro activity against all staphylococci.
The inadequacy of a single diagnostic method for the detection of methicillin
resistance in staphylococci was evident when comparing (a) susceptibility data,
(b) multiplex-PCR for mecA gene detection, and (c) PBP2' detection. None of
these methods was seen to correlate with each other at the 100%-level. The
detection of PBP2' was rapid although, in comparison to mecA gene detection
and antimicrobial susceptibility tests, inaccurate for the identification of
methicillin resistance in staphylococci. DNA sequencing of a fragment of the
mecA gene in selected staphylococcal strains revealed minimal sequence
variation. This was an indication that variable levels of methicillin resistance in
staphylococci can be attributed to different mechanisms of methicillin resistance
or variations in the expression of the mecA gene, rather than mutations within
the gene itself. The low sequence variation observed in the mecA gene is
primarily responsible for initial assumptions of a clonal origin for methicillinresistance
in staphylococci. As of yet, pharmaceutical companies have failed to
produce an analogous antimicrobial agent to β-Iactam agents that would be able
to specifically target PBP2'. The development of such an agent would be
instrumental in the reduction of glycopeptide selection pressure. It is imperative
that correct identification, strain typing, susceptibility testing and methicillin
resistance detection is performed to direct therapy and epidemiologically
monitor methicillin-resistant strain types.
Description
Keywords
Staphylococcus -- Identification, Dissertation (M.Med.Sc. (Medical Microbiology))--University of the Free State, 1999