Abundance and diversity of resistomes differ between healthy human oral cavities and gut.

The global threat of antimicrobial resistance has driven the use of high-throughput sequencing techniques to monitor the profile of resistance genes, known as the resistome, in microbial populations. The human oral cavity contains a poorly explored reservoir of these genes. Here we analyse and compare the resistome profiles of 788 oral cavities worldwide with paired stool metagenomes. We find country and body site-specific differences in the prevalence of antimicrobial resistance genes, classes and mechanisms in oral and stool samples. Within individuals, the highest abundances of antimicrobial resistance genes are found in the oral cavity, but the oral cavity contains a lower diversity of resistance genes compared to the gut. Additionally, co-occurrence analysis shows contrasting ARG-species associations between saliva and stool samples. Maintenance and persistence of antimicrobial resistance is likely to vary across different body sites. Thus, we highlight the importance of characterising the resistome across body sites to uncover the antimicrobial resistance potential in the human body.

The Human Mucosal Mycobiome and Fungal Community Interactions.

With the advent of high-throughput sequencing techniques, the astonishing extent and complexity of the microbial communities that reside within and upon us has begun to become clear. Moreover, with advances in computing and modelling methods, we are now beginning to grasp just how dynamic our interactions with these communities are. The diversity of both these communities and their interactions-both within the community and with us-are dependent on a multitude of factors, both microbial- and host-mediated. Importantly, it is becoming clear that shifts in the makeup of these communities, or their responses, are linked to different disease states. Although much of the work to define these interactions and links has been investigating bacterial communities, recently there has been significant growth in the body of knowledge, indicating that shifts in the host fungal communities (mycobiome) are also intimately linked to disease status. In this review, we will explore these associations, along with the interactions between fungal communities and their human and microbial habitat, and discuss the future applications of systems biology in determining their role in disease status.

Interspecies transfer of the penicillin-binding protein 3-encoding gene ftsI between Haemophilus influenzae and Haemophilus haemolyticus can confer reduced susceptibility to ß-lactam antimicrobial agents.

Mutations in ftsI, encoding penicillin-binding protein 3, can cause decreased ß-lactam susceptibility in Haemophilus influenzae. Sequencing of ftsI from clinical strains has indicated interspecies recombination of ftsI between H. influenzae and Haemophilus haemolyticus. This study documented apparently unrestricted homologous recombination of ftsI between H. influenzae and H. haemolyticus in vitro. Transfer of ftsI from resistant isolates conferred similar but not identical increases in the MICs of susceptible strains of H. influenzae and H. haemolyticus.

Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus.

OBJECTIVES: To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. METHODS: The ftsI genes of 100 Haemophilus isolates comprising genetically defined ß-lactamase-negative ampicillin-susceptible (gBLNAS), ß-lactamase-positive ampicillin-resistant (gBLPAR), ß-lactamase-negative ampicillin-resistant (gBLNAR) and ß-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. RESULTS: Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. CONCLUSIONS: We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance.

PCR screening for the N526K substitution in isolates of Haemophilus influenzae and Haemophilus haemolyticus.

OBJECTIVES: Firstly, to evaluate the current PBP3-S primers of Hasegawa et al. (Microb Drug Resist 2003; 9: 39-46) and develop new primers for the amplification of N526 in isolates of Haemophilus haemolyticus. Secondly, to develop a new PCR assay for the detection (by amplification) of the N526K substitution, encoded by either the AAA or AAG single nucleotide polymorphism (SNP) at position 1576-1578 of the ftsI gene, in isolates of both Haemophilus influenzae and H. haemolyticus. METHODS: A total of 50 H. influenzae and 50 H. haemolyticus isolates, comprising N526 and N526K genotypes, were used to evaluate the performance of SNP-based PCR primers for the detection of the ß-lactamase-negative ampicillin resistance (BLNAR)-defining N526K substitution in H. influenzae and H. haemolyticus, using a real-time PCR platform. RESULTS: The PBP3-S primers of Hasegawa et al. failed to amplify H. haemolyticus isolates, irrespective of their N526/N526K status, owing to an inability of the forward primer to bind the H. haemolyticus ftsI sequence, giving an overall sensitivity of 100% and a specificity of 40% when using all of the isolates. However, the PBP3-N526 and PBP3-N526K PCR primers designed in this study were 100% sensitive and specific, and 84% sensitive and 100% specific, respectively, for the detection of N526K-positive isolates. CONCLUSIONS: Although antibiotic resistance surveillance studies on H. influenzae should include a definitive test for H. influenzae/H. haemolyticus identification, the new primers from this study will not only allow for PCR characterization of both H. influenzae and H. haemolyticus with respect to the N526K BLNAR substitutions, they will also stop incorrect characterization of susceptible H. haemolyticus isolates as low-BLNAR H. influenzae.

Prevalence and mechanisms of ß-lactam resistance in Haemophilus haemolyticus.

OBJECTIVES: To compare the phenotypic and genotypic ß-lactam resistance profiles of non-typeable Haemophilus influenzae (NTHi) and the closely phylogenetically related Haemophilus haemolyticus. METHODS: XV-dependent Haemophilus species isolated as normal flora from nasopharyngeal and throat swabs (n = 312) were screened by PCR for markers to determine NTHi and H. haemolyticus identity. All NTHi and H. haemolyticus isolates were subsequently tested for susceptibilities to ampicillin and amoxicillin/clavulanate, and characterized with respect to the presence of blaTEM, blaROB and ftsI gene mutations. RESULTS: Of the 312 isolates, 236 (75%) were identified as NTHi, 61 (20%) as H. haemolyticus and 15 (5%) as equivocal. PCR for resistance genes showed 15.7% (37/236) of NTHi and 13.1% (8/61) of H. haemolyticus isolates were blaTEM positive and none was positive for blaROB. The blaTEM genes of both species were encoded on similar replicons and associated with the same promoter types. Altered penicillin-binding protein 3 due to the N526K substitution accounted for 31% of both NTHi (73/236) and H. haemolyticus (19/61) isolates, respectively. The presence of N526K in both NTHi and H. haemolyticus was associated with slightly raised ampicillin MICs compared with the H. influenzae Rd and H. haemolyticus ATCC 33390 control strains. In addition, some NTHi gBLNAR-associated substitutions were seen in H. haemolyticus with and without N526K, and appear to represent part of the baseline genotype of that species. CONCLUSIONS: The phenotypic and genotypic ß-lactam resistance in NTHi and H. haemolyticus is very similar, such that H. haemolyticus may represent a reservoir for ß-lactam resistance determinants for NTHi.

An evaluation of SNP-based PCR methods for the detection of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae.

Forty-four previously characterized strains of Haemophilus influenzae were used to evaluate the specificity of previously published SNP PCR primers for the detection of the N526K substitution in PBP3 of BLNAR isolates using real-time PCR. Hasegawa et al. primers that amplify strains without a substitution at 526 and fail to amplify strains with N526K were 100% sensitive and specific for detecting N526K. However, primer sets of Hasegawa et al. and Nakamura et al. designed to amplify strains with N526K, but not strains without a substitution, were unable to do this reliably because the primers were specific for N526K encoded by AAG and failed to amplify strains with N526K encoded by AAA. A review of N526K strains deposited on GenBank revealed an even distribution of AAG and AAA codons for N526K in European and Australian BLNAR strains, whereas only the AAG codon was seen in Japanese strains. The exclusive presence of the AAG codon in Japanese strains appears to be independent of the use of the SNP PCR primers evaluated here and remains unexplained.

Prevalence and genotypic characteristics of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae in Australia.

OBJECTIVES: To determine the prevalence of ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae in Australia and characterize the associated amino acid substitutions in penicillin-binding protein 3. METHODS: Two hundred consecutive non-repeat clinical isolates of H. influenzae were collected and ß-lactamase-negative isolates were screened for reduced ampicillin susceptibility using an ampicillin 2 µg disc (breakpoint <17 mm) and Etest (breakpoint ≥0.25 mg/L). All screen-positive isolates had their ampicillin MICs determined by reference broth microdilution and their ftsI genes were sequenced. RESULTS: No BLNAR strains (MIC ≥4 mg/L) were found, but 5 (2.5%) low BLNAR (L-BLNAR) strains (MIC ≥2 mg/L) and 36 (18%) genetic BLNAR (gBLNAR) strains (R517H or N526K) were found. Of the gBLNAR strains, four had the R517H substitution and the remainder had N526K, while no strains had combined N526K and M377I/S385T/L389F substitutions. A number of strains with neither R517H nor N526K substitutions that did not meet the gBLNAR definition had other BLNAR-associated substitutions. CONCLUSIONS: BLNAR and L-BLNAR strains are uncommon in Australia, while gBLNAR strains are more common than previously recognized.