Phenotypic and genotypic characterisation of altered penicillin-binding protein 3 (PBP3) mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus

Haemophilus influenzae is a significant opportunistic pathogen that causes a range of respiratory infections, including community-acquired pneumonia (CAP), acute exacerbations of chronic obstructive pulmonary disease (COPD) and acute otitis media (AOM). These infections frequently require antibiotic therapy for management, with antibiotics of the ˜í‚â§-lactam class such as amoxicillin, cefaclor, and amoxicillin-clavulanate historically used as first line therapies. However, the efficacy of these antibiotics is currently threatened by the increasing prevalence of ˜í‚â§-lactam resistance mediated by specific mutations in the ftsI gene that produce an N526K substitution in the encoded penicillin-binding protein 3 (PBP3), a protein that is the target of these antibiotics. This type of resistance, termed ˜í‚â§-lactamase-negative ampicillin-resistance (BLNAR), is difficult to detect in the diagnostic laboratory, as many BLNAR isolates do not actually show an ampicillin resistant phenotype using standard susceptibility testing methods. As a result genotypic testing methods are being increasingly adopted for BLNAR detection. Furthermore, the recent recognition of Haemophilus haemolyticus, a close non-pathogenic relative of H. influenzae, in diagnostic specimens from the respiratory tract, has compounded the issue. This is because H. haemolyticus isolates are frequently mis-identified as H. influenzae, which further complicates the role of the diagnostic laboratory in guiding antibiotic therapy for infections involving H. influenzae. A working strain collection comprising a total of 393 Haemophilus isolates was established and used for all the subsequent studies conducted in this thesis. Isolates were taken from; 1) the University of Tasmania (UTAS) culture collection (n=44), isolates was unreliable (sensitivity 84%; specificity 26%). Similar findings were reported for the sensitivity and specificity of the Nakamura et al. (2009) primers for detecting the N526K substitution in BLNAR isolates by amplification (sensitivity 96%; specificity 26%). The poor performance of these PCR primers was attributed to the fact that the N526K substitution can be encoded by an AAT-AAG codon change at base pair (bp) position 1576-1578 of ftsI, as well as the recently described AAT-AAA codon change. As the PCR primers investigated were designed for detection of the N526K substitution encoded by the AAG SNP only, they failed to detect N526K encoded by AAA. A search of ftsI gene sequences available on GenBank revealed that the AAT to AAG or AAA codon changes occurred with equal prevalence in N526K-positive BLNAR isolates, suggesting that the prevalence of BLNAR isolates would go under-reported when these PCR algorithms were used for BLNAR detection. Little was known about the ˜í‚â§-lactam resistance profile of H. haemolyticus, a respiratory commensal commonly mis-identified as H. influenzae in the diagnostic laboratory. As a result, Chapter 4 examined the phenotypic and genotypic characteristics of ˜í‚â§-lactam resistance mechanisms in a large collection of H. haemolyticus and H. influenzae isolates, collected during the GROMIT study using a well defined patient population. The prevalence and mechanisms of ˜í‚â§-lactam resistance were identified to be similar for both bacterial species, with 13.1% of H. haemolyticus, and 15.7% of H. influenzae isolates harbouring the TEM-1 ˜í‚â§-lactamase (with the same replicon and promoter types commonly reported in H. influenzae identified in H. haemolyticus), whilst 31.0% of both H. haemolyticus and H. influenzae isolates were positive for the N526K BLNAR-defining substitution. Further analysis of the ftsI gene encoding PBP3 in these N526K-positive BLNAR isolates revealed that some of the commonly recognized BLNAR-associated substitutions in H. influenzae form part of the baseline PBP3 sequence in H. haemolyticus. This suggests that respiratory isolates of H. haemolyticus might possibly represent a significant reservoir for ˜í‚â§-lactam resistance determinants in co-localised H. influenzae. Examination of the ftsI gene sequences of the H. haemolyticus isolates from Chapter 4 revealed some differences that might interfere with the SNP-based PCR assays of Hasegawa et al. (2003) that were previously evaluated in Chapter 3 using only H. influenzae isolates. When evaluated against a panel of susceptible (N526) and resistant (N526K) isolates of H. influenzae and H. haemolyticus in Chapter 5, the primer set (PBP3-S) designed to amplify N526-positive (BLNAS) isolates performed well for the identification of susceptible H. influenzae isolates. However it failed to amplify any H. haemolyticus isolates, irrespective of their N526/N526K status, due to a species-specific sequence variation in the forward primer-binding region. The discovery of this PCR limitation is significant, as these primers are frequently used in respiratory surveillance studies where H. haemolyticus is often mis-identified as H. influenzae, and will result in the mis-categorisation of susceptible H. haemolyticus isolates as low-BLNAR isolates of H. influenzae. A new PCR primer set was therefore developed to overcome this limitation and was 100% sensitive and specific for the separation of N526 isolates (by amplification) from N526K-positive isolates (which fail to amplify) of both species. This is an important new tool for the surveillance of the N526K-positive BLNAR genotype in XV-dependent Haemophilus species commonly encountered in the diagnostic laboratory. Chapter 6 explores the main observation made in Chapter 4, that some of the BLNAR-associated substitutions reported in N526K-positive isolates of NTHi, appear to form part of the baseline PBP3 genotype in susceptible isolates of H. haemolyticus. The ftsI gene sequences from 100 clinical isolates, including susceptible (N526) and resistant (N526K) H. influenzae and H. haemolyticus isolates, were examined using a range of bioinformatic approaches for evidence of inter-species recombination events. Mosaic ftsI gene sequences were identified in 33% of the isolates tested and shown to represent inter-species recombination events. All recombination events occurred in N526K-positive isolates of either species and frequently resulted in the horizontal transfer of only partial ftsI gene fragments. There was no evidence to support the horizontal transfer of the entire ftsI gene among the clinical isolates in vivo. Chapter 7 extended on the work of Chapter 6 using an in vitro approach. Transformation experiments, using reference recipients and fully characterised N526K-positive isolates of H. influenzae and H. haemolyticus as donors, were performed to investigate potential inter- and intra-species ftsI recombination events. Both inter- and intra-species recombination of the ftsI gene frequently occurred in H. influenzae and H. haemolyticus isolates, and resulted in the formation of mosaic ftsI genes that carry the N526K-positive resistance genotype. In summary, the major findings of this thesis are that a widely used SNP-based PCR algorithm is unreliable for N526K-positive BLNAR detection because of a previously unrecognized SNP encoding the N526K substitution, and because of ftsI sequence divergence with H. haemolyticus that might mis-identify as H. influenzae. As a result, a new SNP-based PCR algorithm was developed and shown to be 100% sensitive and specific for detection of the N526K substitution. Additionally, this thesis presents for the first time the phenotypic and genotypic ˜í‚â§-lactam susceptibility profiles of H. haemolyticus isolates, and highlights the potential role H. haemolyticus plays in the emergence and dissemination of ˜í‚â§-lactam resistance determinants in H. influenzae. Finally, this thesis has characterised homologous (inter- and intra-species) recombination events involving the ftsI gene in both in vivo and in vitro models. Such ftsI recombination events were shown to occur frequently between H. influenzae and H. haemolyticus isolates, and frequently resulted in the formation of mosaic ftsI genes that contribute to the dissemination and diversification of the N526K-positive resistance mechanism in Haemophilus species.