Staphylococcus haemolyticus: a novel method for species identification based on the HSP60 gene and a model for vancomycin resistance
Creators: name: Lee, Soo Sen
Publisher: name: University of British Columbia
Year: 2009

description: S. haemolyticus is emerging as an important nosocomial pathogen. In spite of its increasing importance, the exact incidence of infections is still unknown due to the lack of accurate identification methods. Therefore, part of this thesis involves the evaluation of a novel identification method for S. haemolyticus based on the HSP60 gene. The HSP60 gene is ubiquitous and highly conserved among staphylococci, but still contains species-specific signature sequences which may be useful for species identification of staphylococci. To evaluate the specificity and sensitivity of the HSP60 method, HSP60 probes were generated from reference strains of S. haemolyticus (ATCC29970) and S. epidermidis (ATCC14990) for use in dot blot hybridization reactions with a collection of 66 consecutive bacteremic isolates of CNS, previously identified by the Microscan method and confirmed by BCCDC. The hybridization results were further compared to the API Staph method. The results were as follows: [Table] The HSP60 method was highly accurate for the species identification of both S. haemolyticus and S. epidermidis, while the Microscan method lacked specificity and the API method lacked sensitivity. Therefore the HSP60 gene is an excellent target for the species identification of S. haemolyticus. The second part of the thesis involved elucidating the mechanism of vancomycin resistance in S. haemolyticus, for which little is known. The incidence of vancomycin resistance was examined among the 66 isolates of CNS mentioned earlier. All but one isolate (B7786), a S. haemolyticus, was sensitive to vancomycin (MIC below 2 ug/ml). This isolate displayed intermediate resistance to vancomycin (MIC 4ug/ml). Population analysis of B7786, however, yielded a highly resistant and stable subpopulation (128G₅) (MIC 32 ug/ml; selection frequency 10⁻⁷). The resistant phenotype was characterized by comparing its biochemical, antibiotic, cytoplasmic and cell membrane proteins and vancomycin binding profiles with the parent isolate. While no significant differences were revealed in the biochemical and cytoplasmic protein profiles of I28G₅ compared to its parent, two membrane proteins, 44 kDa and 30 kDa, were observed to be downregulated and upregulated, respectively. The antibiotic profiles with ceftriazone, cefotaxime, tobramycin and amikacin were also dramatically decreased in I28G₅; pointing to an altered cell membrane. Furthermore, competition studies of I28G₅ with isoglutaminyl-D-alanine-D-alanine demonstrated a 16- fold increase in its vancomycin MIC with no effect on the parent; suggesting an altered resistant cell wall. Scatchard plots from saturation binding studies with ¹²⁵I⁻labeled vancomycin confirmed that I28G5 had an altered cell wall with 4-fold more receptors - than B7786 (23,790 nM vs 5,504nM per 10⁷ cells). However, only one type of receptors with similar affinity constants (Kd approximately 1 nM) for both the resistant phenotype and the parent were found. SEM and T EM revealed the reason for the greater number of receptors. The resistant cells had 60% larger diameters, four-fold thicker cell walls and poorly divided daughter cells with apparent missing splitting systems compared to the parent. Taken together, these data suggest that the resistant phenotype is associated with a defect in cell wall division, synthesis and turnover. We postulate that this defect results in an abnormally thickened cell wall with abundant cell wall material which can "mop" up vancomycin in the medium, reduce the its availability to the active sites