Molecular characterisation of penicillin-resistant isolates of Streptococcus pneumoniae in Christchurch, New Zealand
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Streptococcus pneumoniae is an important cause of morbidity and mortality worldwide. Acquisition of antimicrobial resistance, particularly to β-lactams, by this pathogen has complicated treatment options. The prevalence of penicillin-resistant pneumococci in New Zealand has increased greatly since 1993. To investigate the population structure of resistant pneumococci, 200 isolates collected between 1997-2001 with reduced susceptibility to penicillin were obtained from a Christchurch clinical laboratory. Isolates were examined using DNA macro-restriction profiling (MRP) and analysis of pbp genes. Four major clonal lineages were identified, the largest and most homogenous containing 97 (48.5%) of the isolates. Two of the four major PFGE groups (MRPs 2 and 3), could tentatively be described as belonging to globally widespread epidemic clones; the Spain23F-1 clone and the France9v-3 clone respectively. To verify whether the New Zealand penicillin-resistant clones of S. pneumoniae had been recovered elsewhere in the world, 74 clinical isolates were chosen for analysis by multilocus sequence typing (MLST). These isolates were national samples, and represented pneumococci from multiple serotypes, multiple years, and from both invasive and non-invasive isolates. MLST is a technique based on nucleotide sequences of housekeeping genes. MLST permits strain identification by interrogation of a global database on the Internet. The isolates identified from throughout New Zealand were mainly found to belong to well-described international clonal lineages. The globally widespread Spain23F-1 and Spain9V-3 clones were represented by 14 and 11 isolates, respectively. The most prolific group of antibiotic-resistant pneumococci in New Zealand were derived from a recently described Taiwan19F-14 clone, and represented by 26 isolates. Although the Taiwan19F-14 clone seemed genetically homogenous as assessed by MRP, the isolates varied in phenotype as observed by the range of β-lactam MICs within the clonal population. Examination of β-lactam resistance in New Zealand variant of the Taiwan19F-14 clone was performed by pbp gene RFLP and DNA sequence analysis. Resistance was imparted at the molecular level by mosaic pbp genes. It appears that horizontal gene transfer from other resistant S. pneumoniae isolates has led to current pbp alleles observed in the Taiwan19F-14 clone. The molecular mechanism of erythromycin resistance was investigated in 150 pneumococcal isolates. On hundred and forty-one isolates (94.0%) had high-level erythromycin resistance (MIC ≥ 256 μg/ml); the remaining 9 (6.0%) isolates had MICs between 2-8 μg/ml. PCR detection of the macrolide resistance determinants was performed on all 150 isolates. The mef(A) gene was detected in 105 (70.0%) isolates and the erm(B) gene in 142 (94.7%) isolates. Both the mef(A) and erm(B) genes were detected in 97 (64.7%) isolates. The mef(A) erm(B) genotype was associated with the New Zealand variant of the Taiwan19F-14 clone.