Clinical relevance of topical antibiotic use in co-selecting for multidrug-resistant Staphylococcus aureus: Insights from in vitro and ex vivo models.

Topical antibiotic preparations, such as fusidic acid (FA) or mupirocin, are used in the prevention and treatment of superficial skin infections caused by staphylococci. Previous genomic epidemiology work has suggested an association between the widespread use of topical antibiotics and the emergence of methicillin resistant Staphylococcus aureus in some settings. In this study, we provide experimental proof of co-selection for multidrug resistance in S. aureus following exposure to FA or mupirocin. Through targeted mutagenesis and phenotypic analyses, we confirmed that fusC carriage confers resistance to FA, and mupA carriage confers high-level resistance to mupirocin in multiple S. aureus genetic backgrounds. In vitro experiments demonstrated that carriage of fusC and mupA confer a competitive advantage in the presence of sub-inhibitory concentrations of FA and mupirocin, respectively. Further, we used a porcine skin colonisation model to show that clinically relevant concentrations of topical antibiotics can co-select for presence of unrelated antimicrobial resistance determinants, such as mecA, blaZ, and qacA, in fusC or mupA harbouring S. aureus These findings provide valuable insights on the role of acquired FA or mupirocin resistance in co-selecting for broader antibiotic resistance in S. aureus, prompting greater need for judicious use of topical antibiotics.

Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections.

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

Biphasic Outbreak of Invasive Group A Streptococcus Disease in Eldercare Facility, New Zealand.

A 3-month outbreak of invasive group A Streptococcus disease at an eldercare facility, in which 5 persons died, was biphasic. Although targeted chemoprophylaxis contained the initial outbreak, a second phase of the outbreak occurred after infection control processes ended. To retrospectively investigate the genomic epidemiology of the biphasic outbreak, we used whole-genome sequencing and multiple bioinformatics approaches. Analysis of isolates from the outbreak and isolates prospectively collected during the outbreak response indicated a single S. pyogenes emm81 clone among residents and staff members. Outbreak isolates differed from nonoutbreak emm81 isolates by harboring an integrative conjugative genomic element that contained the macrolide resistance determinant erm(TR). This study shows how retrospective high-resolution genomic investigations identified rapid spread of a closed-facilty clonal outbreak that was controlled, but not readily cleared, by infection control management procedures.

Co-circulation of Multidrug-resistant Shigella Among Men Who Have Sex With Men in Australia.

BACKGROUND: In urban Australia, the burden of shigellosis is either in returning travelers from shigellosis-endemic regions or in men who have sex with men (MSM). Here, we combine genomic data with comprehensive epidemiological data on sexual exposure and travel to describe the spread of multidrug-resistant Shigella lineages. METHODS: A population-level study of all cultured Shigella isolates in the state of Victoria, Australia, was undertaken from 1 January 2016 through 31 March 2018. Antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatic analyses of 545 Shigella isolates were performed at the Microbiological Diagnostic Unit Public Health Laboratory. Risk factor data on travel and sexual exposure were collected through enhanced surveillance forms or by interviews. RESULTS: Rates of antimicrobial resistance were high, with 17.6% (95/541) and 50.6% (274/541) resistance to ciprofloxacin and azithromycin, respectively. There were strong associations between antimicrobial resistance, phylogeny, and epidemiology. Specifically, 2 major MSM-associated lineages were identified: a Shigellasonnei lineage (n = 159) and a Shigella flexneri 2a lineage (n = 105). Of concern, 147/159 (92.4%) of isolates within the S. sonnei MSM-associated lineage harbored mutations associated with reduced susceptibility to recommended oral antimicrobials: namely, azithromycin, trimethoprim-sulfamethoxazole, and ciprofloxacin. Long-read sequencing demonstrated global dissemination of multidrug-resistant plasmids across Shigella species and lineages, but predominantly associated with MSM isolates. CONCLUSIONS: Our contemporary data highlight the ongoing public health threat posed by resistant Shigella, both in Australia and globally. Urgent multidisciplinary public health measures are required to interrupt transmission and prevent infection.

Genomic Analysis of Fluoroquinolone- and Tetracycline-Resistant Campylobacter jejuni Sequence Type 6964 in Humans and Poultry, New Zealand, 2014-2016.

In 2014, antimicrobial drug-resistant Campylobacter jejuni sequence type 6964 emerged contemporaneously in poultry from 3 supply companies in the North Island of New Zealand and as a major cause of campylobacteriosis in humans in New Zealand. This lineage, not previously identified in New Zealand, was resistant to tetracycline and fluoroquinolones. Genomic analysis revealed divergence into 2 major clades; both clades were associated with human infection, 1 with poultry companies A and B and the other with company C. Accessory genome evolution was associated with a plasmid, phage insertions, and natural transformation. We hypothesize that the tetO gene and a phage were inserted into the chromosome after conjugation, leaving a remnant plasmid that was lost from isolates from company C. The emergence and rapid spread of a resistant clone of C. jejuni in New Zealand, coupled with evolutionary change in the accessory genome, demonstrate the need for ongoing Campylobacter surveillance among poultry and humans.

Remodeling of pSK1 Family Plasmids and Enhanced Chlorhexidine Tolerance in a Dominant Hospital Lineage of Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation have been shaped in part by mobile genetic elements (MGEs), facilitating the global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs, in particular, how changes in the structure of multidrug resistance (MDR) plasmids may influence important staphylococcal phenotypes, is incomplete. Here, we undertook a population and functional genomics study of 212 methicillin-resistant S. aureus (MRSA) sequence type 239 (ST239) isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have coevolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics, we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s and found that multiple structural variants have arisen. Plasmid maintenance and stability were linked to IS256- and IS257-mediated chromosomal integration and disruption of the plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin, trimethoprim, and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA isolates through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing the rates of gentamicin resistance and reduced chlorhexidine susceptibility and (ii) changes in the plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a potential evolutionary response of ST239 MRSA to biocides, one of which may contribute to the ongoing persistence and adaptation of this lineage within health care institutions.

Genome-by-genome approach for fast bacterial genealogical relationship evaluation.

Motivation: Large-scale whole-genome sequencing dataset-based studies are becoming increasingly common in pathogen surveillance and outbreak investigations. A highly discriminative and time-efficient bioinformatics tool is needed to transform large amounts of sequencing data into usable biological information. To replace the intuitive, yet inefficient, way of gene-by-gene allele calling algorithm, a new algorithm using genome-by-genome approach was developed. Results: Tests showed that the program equipped with the new algorithm achieved significant improvements in allele calling efficiency compared to a conventional gene-by-gene approach. The new program, Fast-GeP, rendered a fast and easy way to infer high-resolution genealogical relationships between bacterial isolates using whole-genome sequencing data. Availability and implementation: FAST-GeP is freely available from: https://github.com/jizhang-nz/fast-GeP. Supplementary information: Supplementary data are available at Bioinformatics online.

Agricultural Origins of a Highly Persistent Lineage of Vancomycin-Resistant Enterococcus faecalis in New Zealand.

Enterococcus faecalis and Enterococcus faecium are human and animal gut commensals. Vancomycin-resistant enterococci (VRE) are important opportunistic pathogens with limited treatment options. Historically, the glycopeptide antibiotics vancomycin and avoparcin selected for the emergence of vancomycin resistance in human and animal isolates, respectively, resulting in global cessation of avoparcin use between 1997 and 2000. To better understand human- and animal-associated VRE strains in the postavoparcin era, we sequenced the genomes of 231 VRE isolates from New Zealand (NZ; 75 human clinical, 156 poultry) cultured between 1998 and 2009. E. faecium lineages and their antibiotic resistance carriage patterns strictly delineated between agricultural and human reservoirs, with bacitracin resistance ubiquitous in poultry but absent in clinical E. faecium strains. In contrast, one E. faecalis lineage (ST108) predominated in both poultry and human isolates in the 3 years following avoparcin discontinuation. Both phylogenetic and antimicrobial susceptibility (i.e., ubiquitous bacitracin resistance in both poultry and clinical ST108 isolates) analyses suggest an agricultural origin for the ST108 lineage. VRE isolate resistomes were carried on multiple, heterogeneous plasmids. In some isolate genomes, bacitracin, erythromycin, and vancomycin resistance elements were colocalized, indicating multiple potentially linked selection mechanisms.IMPORTANCE Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence.

Current and Emerging Topical Antibacterials and Antiseptics: Agents, Action, and Resistance Patterns.

Bacterial skin infections represent some of the most common infectious diseases globally. Prevention and treatment of skin infections can involve application of a topical antimicrobial, which may be an antibiotic (such as mupirocin or fusidic acid) or an antiseptic (such as chlorhexidine or alcohol). However, there is limited evidence to support the widespread prophylactic or therapeutic use of topical agents. Challenges involved in the use of topical antimicrobials include increasing rates of bacterial resistance, local hypersensitivity reactions (particularly to older agents, such as bacitracin), and concerns about the indiscriminate use of antiseptics potentially coselecting for antibiotic resistance. We review the evidence for the major clinical uses of topical antibiotics and antiseptics. In addition, we review the mechanisms of action of common topical agents and define the clinical and molecular epidemiology of antimicrobial resistance in these agents. Moreover, we review the potential use of newer and emerging agents, such as retapamulin and ebselen, and discuss the role of antiseptic agents in preventing bacterial skin infections. A comprehensive understanding of the clinical efficacy and drivers of resistance to topical agents will inform the optimal use of these agents to preserve their activity in the future.

Burkholderia lata Infections from Intrinsically Contaminated Chlorhexidine Mouthwash, Australia, 2016.

Burkholderia lata was isolated from 8 intensive care patients at 2 tertiary hospitals in Australia. Whole-genome sequencing demonstrated that clinical and environmental isolates originated from a batch of contaminated commercial chlorhexidine mouthwash. Genomic analysis identified efflux pump-encoding genes as potential facilitators of bacterial persistence within this biocide.

Topical Antibiotic Use Coselects for the Carriage of Mobile Genetic Elements Conferring Resistance to Unrelated Antimicrobials in Staphylococcus aureus.

Topical antibiotics, such as mupirocin and fusidic acid, are commonly used in the prevention and treatment of skin infections, particularly those caused by staphylococci. However, the widespread use of these agents is associated with increased resistance to these agents, potentially limiting their efficacy. Of particular concern is the observation that resistance to topical antibiotics is often associated with multidrug resistance, suggesting that topical antibiotics may play a role in the emergence of multidrug-resistant (MDR) strains. New Zealand (NZ) has some of the highest globally recorded rates of topical antibiotic usage and resistance. Using a combination of Pacific Biosciences single-molecule real-time (SMRT) whole-genome sequencing, Illumina short-read sequencing, and Bayesian phylogenomic modeling on 118 new multilocus sequence type 1 (ST1) community Staphylococcus aureus isolates from New Zealand and 61 publically available international ST1 genome sequences, we demonstrate a strong correlation between the clinical introduction of topical antibiotics and the emergence of MDR ST1 S. aureus We also provide in vitro experimental evidence showing that exposure to topical antibiotics can lead to the rapid selection of MDR S. aureus isolates carrying plasmids that confer resistance to multiple unrelated antibiotics, from within a mixed population of competitor strains. These findings have important implications regarding the impact of the indiscriminate use of topical antibiotics.

Genomic Analysis of Multiresistant Staphylococcus capitis Associated with Neonatal Sepsis.

Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen.

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium.

Background: Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. Objectives: To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. Methods: The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. Results: The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. Conclusions: The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates.

Genomic epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in New Zealand.

Background: Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health. No studies to date have examined the genomic epidemiology of gonorrhoea in the Western Pacific Region, where the incidence of gonorrhoea is particularly high. Methods: A population-level study of N. gonorrhoeae in New Zealand (October 2014 to May 2015). Comprehensive susceptibility testing and WGS data were obtained for 398 isolates. Relatedness was inferred using phylogenetic trees, and pairwise core SNPs. Mutations and genes known to be associated with resistance were identified, and correlated with phenotype. Results: Eleven clusters were identified. In six of these clusters, >25% of isolates were from females, while in eight of them, >15% of isolates were from females. Drug resistance was common; 98%, 32% and 68% of isolates were non-susceptible to penicillin, ciprofloxacin and tetracycline, respectively. Elevated MICs to extended-spectrum cephalosporins (ESCs) were observed in 3.5% of isolates (cefixime MICs ≥ 0.12 mg/L, ceftriaxone MICs ≥ 0.06 mg/L). Only nine isolates had penA XXXIV genotypes, three of which had decreased susceptibility to ESCs (MIC = 0.12 mg/L). Azithromycin non-susceptibility was identified in 43 isolates (10.8%); two of these isolates had 23S mutations (C2611T, 4/4 alleles), while all had mutations in mtrR or its promoter. Conclusions: The high proportion of females in clusters suggests transmission is not exclusively among MSM in New Zealand; re-assessment of risk factors for transmission may be warranted in this context. As elevated MICs of ESCs and/or azithromycin were found in closely related strains, targeted public health interventions to halt transmission are urgently needed.

The changing landscape of vancomycin-resistant Enterococcus faecium in Australia: a population-level genomic study.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm. Methods: A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid. Results: vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission. Conclusions: Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.

Incorporating Whole-Genome Sequencing into Public Health Surveillance: Lessons from Prospective Sequencing of Salmonella Typhimurium in Australia.

In Australia, the incidence of Salmonella Typhimurium has increased dramatically over the past decade. Whole-genome sequencing (WGS) is transforming public health microbiology, but poses challenges for surveillance. To compare WGS-based approaches with conventional typing for Salmonella surveillance, we performed concurrent WGS and multilocus variable-number tandem-repeat analysis (MLVA) of Salmonella Typhimurium isolates from the Australian Capital Territory (ACT) for a period of 5 months. We exchanged data via a central shared virtual machine and performed comparative genomic analyses. Epidemiological evidence was integrated with WGS-derived data to identify related isolates and sources of infection, and we compared WGS data for surveillance with findings from MLVA typing. We found that WGS data combined with epidemiological data linked an additional 9% of isolates to at least one other isolate in the study in contrast to MLVA and epidemiological data, and 19% more isolates than epidemiological data alone. Analysis of risk factors showed that in one WGS-defined cluster, human cases had higher odds of purchasing a single egg brand. While WGS was more sensitive and specific than conventional typing methods, we identified barriers to uptake of genomic surveillance around complexity of reporting of WGS results, timeliness, acceptability, and stability. In conclusion, WGS offers higher resolution of Salmonella Typhimurium laboratory surveillance than existing methods and can provide further evidence on sources of infection in case and outbreak investigations for public health action. However, there are several challenges that need to be addressed for effective implementation of genomic surveillance in Australia.

Seven Salmonella Typhimurium Outbreaks in Australia Linked by Trace-Back and Whole Genome Sequencing.

Salmonella Typhimurium is a common cause of foodborne illness in Australia. We report on seven outbreaks of Salmonella Typhimurium multilocus variable-number tandem-repeat analysis (MLVA) 03-26-13-08-523 (European convention 2-24-12-7-0212) in three Australian states and territories investigated between November 2015 and March 2016. We identified a common egg grading facility in five of the outbreaks. While no Salmonella Typhimurium was detected at the grading facility and eggs could not be traced back to a particular farm, whole genome sequencing (WGS) of isolates from cases from all seven outbreaks indicated a common source. WGS was able to provide higher discriminatory power than MLVA and will likely link more Salmonella Typhimurium cases between states and territories in the future. National harmonization of Salmonella surveillance is important for effective implementation of WGS for Salmonella outbreak investigations.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

A Supervised Statistical Learning Approach for Accurate Legionella pneumophila Source Attribution during Outbreaks.

Public health agencies are increasingly relying on genomics during Legionnaires' disease investigations. However, the causative bacterium (Legionella pneumophila) has an unusual population structure, with extreme temporal and spatial genome sequence conservation. Furthermore, Legionnaires' disease outbreaks can be caused by multiple L. pneumophila genotypes in a single source. These factors can confound cluster identification using standard phylogenomic methods. Here, we show that a statistical learning approach based on L. pneumophila core genome single nucleotide polymorphism (SNP) comparisons eliminates ambiguity for defining outbreak clusters and accurately predicts exposure sources for clinical cases. We illustrate the performance of our method by genome comparisons of 234 L. pneumophila isolates obtained from patients and cooling towers in Melbourne, Australia, between 1994 and 2014. This collection included one of the largest reported Legionnaires' disease outbreaks, which involved 125 cases at an aquarium. Using only sequence data from L. pneumophila cooling tower isolates and including all core genome variation, we built a multivariate model using discriminant analysis of principal components (DAPC) to find cooling tower-specific genomic signatures and then used it to predict the origin of clinical isolates. Model assignments were 93% congruent with epidemiological data, including the aquarium Legionnaires' disease outbreak and three other unrelated outbreak investigations. We applied the same approach to a recently described investigation of Legionnaires' disease within a UK hospital and observed a model predictive ability of 86%. We have developed a promising means to breach L. pneumophila genetic diversity extremes and provide objective source attribution data for outbreak investigations.IMPORTANCE Microbial outbreak investigations are moving to a paradigm where whole-genome sequencing and phylogenetic trees are used to support epidemiological investigations. It is critical that outbreak source predictions are accurate, particularly for pathogens, like Legionella pneumophila, which can spread widely and rapidly via cooling system aerosols, causing Legionnaires' disease. Here, by studying hundreds of Legionella pneumophila genomes collected over 21 years around a major Australian city, we uncovered limitations with the phylogenetic approach that could lead to a misidentification of outbreak sources. We implement instead a statistical learning technique that eliminates the ambiguity of inferring disease transmission from phylogenies. Our approach takes geolocation information and core genome variation from environmental L. pneumophila isolates to build statistical models that predict with high confidence the environmental source of clinical L. pneumophila during disease outbreaks. We show the versatility of the technique by applying it to unrelated Legionnaires' disease outbreaks in Australia and the UK.

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

The prevalence of fusidic acid (FA) resistance amongStaphylococcus aureusstrains in New Zealand (NZ) is among the highest reported globally, with a recent study describing a resistance rate of approximately 28%. Three FA-resistantS. aureusclones (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 thefusCgene. 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 offusCin FA-resistant NZ isolates is unknown. To better understand the rapid emergence of FA-resistantS. 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,fusCwas invariably located within staphylococcal cassette chromosome (SCC) elements, suggesting that SCC-mediated horizontal transfer is the primary mechanism offusCdissemination. The genotypic association offusCwithmecAhas important implications for the emergence of MRSA clones in populations with high usage of fusidic acid. In addition, we found thatfusCwas colocated with a recently described virulence factor (tirS) in dominant NZS. aureusclones, suggesting a fitness advantage. This study points to the likely molecular mechanisms responsible for the successful emergence and spread of FA-resistantS. aureus.