Characterisation of invasive clinical Haemophilus influenzae isolates in Queensland, Australia using whole-genome sequencing.

Haemophilus influenzae is an important aetiological organism of both adult and child respiratory disease. The number of non-typeable (NTHi) invasive H. influenzae isolates referred to the Queensland (QLD) Public Health Microbiology laboratory has increased notably year-by-year. In this study we used whole-genome sequencing to molecularly characterise 100 referred invasive H. influenzae, including 74 NTHi isolates over a 15-year period, observing the carriage of capsular and putative virulence genes, including the major adhesins, antimicrobial resistance genes and population diversity. Encapsulated isolates were largely clonal, however NTHi isolates displayed high genetic variability by MLST and single nucleotide polymorphism typing with no dominant clone observed. The only mechanism for ß-lactam resistance identified in the QLD isolates was ß-lactamase production. No single set of virulence determinants was conclusively associated with invasive QLD NTHi isolates.

Discovery of Streptococcus pneumoniae serogroup 35 variants in Australian patients.

OBJECTIVES: Streptococcus pneumoniae isolates from Australian invasive pneumococcal disease cases displaying an atypical 35B phenotype. Whole genome sequencing was used to analyse these strains and identify changes to the capsule gene regions. METHODS: Four atypical serogroup 35 isolates from Australian reference laboratories were unable to be assigned to one of the four known group 35 serotypes by the Quellung serotyping method. Genetic characterization of the capsule locus was performed by bioinformatic analysis of whole genome sequencing data for all isolates. RESULTS: Genetic analysis identified four independent disruptions to the wciG gene, which encodes an O-acetyltransferase responsible for the O-acetylation of the 6Galß1 residue in the capsular polysaccharide repeat unit of serotype 35B. CONCLUSIONS: This is the first published report on the incidence and capsular gene characteristics of a S. pneumoniae 35B variant.

Epidemiological typing of Neisseria gonorrhoeae and detection of markers associated with antimicrobial resistance directly from urine samples using next generation sequencing.

OBJECTIVES: To investigate the potential for next generation sequencing (NGS) to be used directly on clinical specimens that have tested positive for Neisseria gonorrhoeae by nucleic acid amplification testing (NAAT), to generate information on epidemiological genotyping and antimicrobial resistance (AMR) markers. METHODS: DNA was extracted from 13 N. gonorrhoeae NAAT-positive urine specimens, enriched for microbial DNA and sequenced using the Ion Torrent PGM workflow. Sequences that aligned to the human genome were filtered out and the remaining sequences were de novo assembled. The resulting contigs were searched for regions of interest using Ridom SeqSphere. MLST and NG-MAST alleles were assigned according to the schemes at PubMLST.org and NG-MAST.net, respectively. RESULTS: In total, 11 of the 13 samples tested generated a sufficient number of N. gonorrhoeae sequence reads to provide full coverage of the genome at a depth of 6-130×. Complete MLST and NG-MAST sequence types could be generated for each of these samples. The presence of 10 different AMR markers was investigated, and both previously reported and novel mutations were identified in genes associated with reduced susceptibility to several antimicrobials. CONCLUSIONS: We found that sequencing the entire genome of N. gonorrhoeae directly from clinical samples is possible using NGS, and that multiple levels of N. gonorrhoeae typing information can be generated. As NAAT only testing becomes more common, this method could be used to detect both known and novel mutations associated with AMR and to generate genotyping information, supporting AMR and epidemiological surveillance in the absence of culturing.

Molecular characterization of an Australian serotype 1 Streptococcus pneumoniae outbreak.

Serotype 1 Streptococcus pneumoniae is a cause of invasive pneumococcal disease (IPD) worldwide and has been associated with IPD outbreaks, while carriage is rarely detected in healthy adults or children. This study details an Australian multi-state and territory outbreak of serotype 1 S. pneumoniae IPD between 2010 and 2012. Molecular characterization demonstrated the outbreak was largely due to the clonal expansion of sequence type 306, MLVA type 261 S. pneumoniae serotype 1.

Real-time investigation of a Legionella pneumophila outbreak using whole genome sequencing.

Legionella pneumophila is the main pathogen responsible for outbreaks of Legionnaires' disease, which can be related to contaminated water supplies such as cooling towers or water pipes. We combined conventional molecular methods and whole genome sequence (WGS) analysis to investigate an outbreak of L. pneumophila in a large Australian hospital. Typing of these isolates using sequence-based typing and virulence gene profiling, was unable to discriminate between outbreak and non-outbreak isolates. WGS analysis was performed on isolates during the outbreak, as well as on unlinked isolates from the Public Health Microbiology reference collection. The more powerful resolution provided by analysis of whole genome sequences allowed outbreak isolates to be distinguished from isolates that were temporally and spatially unassociated with the outbreak, demonstrating that this technology can be used in real-time to investigate L. pneumophila outbreaks.

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household.

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters.