SCV'S: FORMATION AND CHARACTERISATION IN STAPHYLOCOCCUS SP

Staphylococcus aureus is the most common cause of hospital-acquired infection and contributes significantly to patient morbidity and mortality. The ability of S. aureus to switch to an alternative phenotype in the presence of antimicrobial agents is clearly favourable. One of these alternatives are small colony variants (SCVs). The novel phenotypes include changes to colony morphology, antibiotic susceptibility, haemolytic activity and many other physiological activities. It is now recognised that SCVs have a deficiency in electron transport, owing to mutations affecting its efficacy. This study investigated SCVs in various ways. In the evolution component changes (mutations) occurring sequentially in successive cycling (15 cycles), were identified. In this experiment selection was made for sequentially SCV mutants and wild type revertants. Two sequenced clinical MRSA strains COL and N315 were chosen so changes in sequence in SCVs and wild type revertants could be compared. Selection for SCVs was made independently for triclosan and gentamicin for both strains. The final SCV and WT strains isolated were compared physiologically and genetically and showed differences in frequency, biochemical profiles, pigment production, haemolysis, catalase, coagulase, levels of intracellular ATP and phage yield. The genomic sequence of the final 4 cycle isolates (SCV15) showed numerous and diverse mutations occurred COL and N315 SCVs. Over 70 mutations were found and 33 were determined as historic mutations and the rest were termed novel mutations. The novel mutations occurred during the cycling process. The historic mutations occurred prior to the experiment and these mutations were acquired during growth in laboratory culture. Only one mutation was found to be common between COL and N315 and this was in the fabI gene. These data indicate mutations occurring in ~1.3% of the genome (~ 40 Kb) can generate mutants with the SCV phenotype. Susceptibility to phage 80α and transduction of S. aureus wild type and their SCVs 1-3 was studied. Wild type strain of S. aureus and SCV3 both yielded a high number of lysogens (~68%) the remaining being resistant mutants. SCV 1 and SCV 2 provided a much lower proportion of lysogens (4-10%). There was no obvious relationship between cellular ATP levels and lysogen formation. Consequently the frequency of lysogen formation (or that of resistance mutants) cannot be related to energy status. Transduction of ciprofloxacin resistance (grlA) was observed into COL wild type at a 5-10-fold higher frequency than into SCV1. Transduction of rifampicin resistance (rpoB) into SCVs was reduced almost 10-fold. As transduction was significantly decreased into SCVs it is hypothesised this process was influenced by ATP levels. The data thus suggests that SCV strains will be less efficient in gene exchange by transduction in vivo. Three SCVs previously isolated from S. aureus COL on the basis of different growth rate were further studied. Results clearly support the hypothesis that there is a physiological diversity in SCV populations. Sensitivity of S. aureus wild type and SCVs strains to various antimicrobial was determined. The SCV strains were more sensitive to some antibiotics and heavy metals than the wild type strain.