Jason C. Kwong

Whole genome sequencing in clinical and public health microbiology

SummaryGenomics and whole genome sequencing (WGS) have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology. The growth and availability of bench-top WGS analysers has facilitated the feasibility of genomics in clinical and public health microbiology. Given current resource and infrastructure limitations, WGS is most applicable to use in public health laboratories, reference laboratories, and hospital infection control-affiliated laboratories. As WGS represents the pinnacle for strain characterisation and epidemiological analyses, it is likely to replace traditional typing methods, resistance gene detection and other sequence-based investigations (e.g., 16S rDNA PCR) in the near future. Although genomic technologies are rapidly evolving, widespread implementation in clinical and public health microbiology laboratories is limited by the need for effective semi-automated pipelines, standardised quality control and data interpretation, bioinformatics expertise, and infrastructure.

Prospective whole genome sequencing enhances national surveillance of Listeria monocytogenes

ABSTRACT Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance of Listeria monocytogenes has the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423 L. monocytogenes isolates underwent WGS, and comparisons uncovered a diverse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferred in silico from the WGS data were highly concordant (>99%) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97 L. monocytogenes isolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospective L. monocytogenes surveillance and investigated for other pathogens relevant to public health.

Faecal microbiota transplantation for severe Clostridium difficile infection in the intensive care unit.

We describe a case of faecal microbiota transplantation (FMT) used for severe binary toxin-positive Clostridium difficile infection in an intensive care setting. The patient was admitted to the ICU of a tertiary hospital and failed traditional maximal pharmacological management. Adjunctive therapy with FMT given through gastroscopy resulted in resolution of the C. difficile-related symptoms. Although there is a growing experience with FMT for recurrent C. difficile infection, published evidence in severe disease is very limited. In a landscape of increasingly severe C. difficile infection, adjunctive FMT may be considered a useful early treatment option.

Managing a nosocomial outbreak of carbapenem-resistant Klebsiella pneumoniae: an early Australian hospital experience.

Carbapenems are traditionally reserved as the last line of defence for treatment of serious infections with multiresistant Gram‐negative bacilli. Reports of Klebsiella pneumoniae carbapenemase (KPC)‐producing organisms have been emerging globally, but rare in Australasia to date. We describe an outbreak of KPC‐2 producing K. pneumoniae at an Australian hospital.

Rapid Emergence and Evolution of Staphylococcus aureus Clones Harboring fusC-Containing Staphylococcal Cassette Chromosome Elements

ABSTRACT The prevalence of fusidic acid (FA) resistance among Staphylococcus aureus strains in New Zealand (NZ) is among the highest reported globally, with a recent study describing a resistance rate of approximately 28%. Three FA-resistant S. aureus clones (ST5 MRSA, ST1 MSSA, and ST1 MRSA) have emerged over the past decade and now predominate in NZ, and in all three clones FA resistance is mediated by the fusC gene. In particular, ST5 MRSA has rapidly become the dominant MRSA clone in NZ, although the origin of FA-resistant ST5 MRSA has not been explored, and the genetic context of fusC in FA-resistant NZ isolates is unknown. To better understand the rapid emergence of FA-resistant S. aureus, we used population-based comparative genomics to characterize a collection of FA-resistant and FA-susceptible isolates from NZ. FA-resistant NZ ST5 MRSA displayed minimal genetic diversity and represented a phylogenetically distinct clade within a global population model of clonal complex 5 (CC5) S. aureus. In all lineages, fusC was invariably located within staphylococcal cassette chromosome (SCC) elements, suggesting that SCC-mediated horizontal transfer is the primary mechanism of fusC dissemination. The genotypic association of fusC with mecA has important implications for the emergence of MRSA clones in populations with high usage of fusidic acid. In addition, we found that fusC was colocated with a recently described virulence factor (tirS) in dominant NZ S. aureus clones, suggesting a fitness advantage. This study points to the likely molecular mechanisms responsible for the successful emergence and spread of FA-resistant S. aureus.

Managing Severe Community-Acquired Pneumonia Due to Community Methicillin-Resistant Staphylococcus aureus (MRSA)

Community-associated methicillin-resistant Staphylococcus aureus (MRSA) is a rare, but significant cause of community-acquired pneumonia (CAP). A number of virulence determinants have been implicated in the development of severe community MRSA pneumonia, characterized by multilobar cavitating necrosis in patients without usual risk-factors for pneumonia. Optimal management is uncertain, and is extrapolated from anecdotal experiences with small case series, randomized studies of hospital-acquired pneumonia, and laboratory investigations using in vitro experiments and animal models of MRSA pneumonia. Adequate clinical suspicion, early diagnosis and administration of appropriate antibiotics are necessary for best patient outcomes, although some patients will still do badly even with early anti-MRSA therapy. Vancomycin or linezolid have been recommended as first-line therapy, possibly in combination with other antibiotics. Newer antibiotics such as ceftaroline are still being evaluated.

New aspirations: the debate on aspiration pneumonia treatment guidelines.

Aspiration pneumonia occurs most commonly in patients with a predisposition to aspiration (eg, those with neurological bulbar dysfunction). There is limited evidence regarding the involvement of anaerobes in most cases of aspiration pneumonia. Most patients respond to treatment for aspiration pneumonia without specific anti‐anaerobic therapy such as metronidazole. Metronidazole has adverse side effects, and widespread use where not indicated can promote carriage of multiresistant intestinal flora such as vancomycin‐resistant enterococci. Use of metronidazole may be appropriate in patients with aspiration pneumonia and evidence of a lung abscess, necrotising pneumonia, putrid sputum or severe periodontal disease.

A phylogenomic framework for assessing the global emergence and evolution of clonal complex 398 methicillin-resistant Staphylococcus aureus

Distinct clones of methicillin-resistant Staphylococcus aureus (MRSA) have emerged as important causes of infection in individuals who have exposure to livestock (livestock-associated MRSA; LA-MRSA). Clonal complex 398 (CC398) is the most prevalent LA-MRSA clone, and has been reported from several geographical settings, including Europe, the Americas and Asia. To understand the factors contributing to the global dissemination of this clone, we analysed CC398 MRSA isolates from New Zealand (NZ), a geographically isolated country with an economy strongly dependent on livestock farming. We supplemented the NZ CC398 MRSA collection with global datasets of CC398 MRSA and CC398 methicillin-susceptible S. aureus. Here, we demonstrate multiple sporadic incursions of CC398 MRSA into NZ, as well as recent importation and spread of a swine-associated clade related to the European LA-MRSA lineage. Within a larger global phylogenomic framework, Bayesian modelling suggested that this NZ clade emerged in the late 2000s, with a probable origin in swine from Western Europe. Elucidating the factors responsible for the incursion and spread of LA-MRSA in geographically distant regions, such as NZ, provides important insights into global pathways of S. aureus transmission, and will inform strategies to control importation and spread.

Sharing Is Caring: International Sharing of Data Enhances Genomic Surveillance of Listeria monocytogenes

TO THE EDITOR—We read with interest the 2 recent reports by Jackson et al and Jensen et al on the use of whole-genome sequencing (WGS) for surveillance and outbreak investigation of Listeria monocytogenes [1, 2]. We have also used WGS in Australia for this purpose [3] and, on the basis of our experience, we wish to highlight the importance of international collaborations and real-time data exchange. An elderly man presented with confusion and fever for investigation and was subsequently admitted to hospital. Blood cultures were positive for L. monocytogenes, with the case notified to the state department of health, and the isolate was referred to state and national Listeria reference laboratories. The isolate underwent molecular typing and WGS (European Nucleotide Archive accession number ERS946910) in accordance with a national Listeria surveillance program [3], and epidemiologic information was obtained using a standardized case questionnaire. As the isolate was not epidemiologically or genomically linked to any other Australian isolate, the case was deemed to be sporadic. WGS data were also submitted to the US Food and Drug Administration GenomeTrakr network, an enhanced real-time surveillance program, for comparison with international isolates [1]. Upon analysis, the isolate was found to be almost identical to a US cluster of isolates from stone fruit,

Emergence of multidrug resistance in locally-acquired human infections with Salmonella Typhimurium in Australia owing to a new clade harbouring blaCTX-M-9

Antimicrobial resistance in non-typhoidal Salmonella is a critical problem globally, with the emergence of resistance to third-generation cephalosporins (3GCs) a particular concern. The aim of this study was to use whole-genome sequencing (WGS) to characterise recently identified human and non-human isolates of 3GC-resistant Salmonella enterica subsp. enterica serovar Typhimurium from Australia. The Illumina NextSeq sequencing platform was used to determine the genome sequences of 78 S. Typhimurium definitive type 44 isolated in Australia between 1992 and 2016, including 31 3GC-resistant isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. We report the emergence of 3GC resistance in locally-acquired Australian S. Typhimurium for the first time. Phenotypically resistant isolates of human and animal origin were geographically restricted and were found by WGS all to be closely related and to carry blaCTX-M-9. Dairy cattle were the suspected source based on geographical clustering of animal isolates, which were predominantly bovine in origin. In conclusion, locally-acquired human cases of S. Typhimurium carrying blaCTX-M-9 were identified that appear to be of bovine origin, raising concerns regarding the human impact of off-label use of ceftiofur in cattle.