Pan-Enterovirus Characterization Reveals Cryptic Circulation of Clinically Relevant Subtypes in Arizona Wastewater.

Background: Most seasonally circulating enteroviruses result in asymptomatic or mildly symptomatic infections. In rare cases, however, infection with some subtypes can result in paralysis or death. Of the 300 subtypes known, only poliovirus is reportable, limiting our understanding of the distribution of other enteroviruses that can cause clinical disease. Objective: The overarching objectives of this study were to: 1) describe the distribution of enteroviruses in Arizona during the late summer and fall of 2022, the time of year when they are thought to be most abundant, and 2) demonstrate the utility of viral pan-assay approaches for semi-agnostic discovery that can be followed up by more targeted assays and phylogenomics. Methods: This study utilizes pooled nasal samples collected from school-aged children and long-term care facility residents, and wastewater from multiple locations in Arizona during July-October of 2022. We used PCR to amplify and sequence a region common to all enteroviruses, followed by species-level bioinformatic characterization using the QIIME 2 platform. For Enterovirus-D68 (EV-D68), detection was carried out using RT-qPCR, followed by confirmation using near-complete whole EV-D68 genome sequencing using a newly designed tiled amplicon approach. Results: In the late summer and early fall of 2022, multiple enterovirus species were identified in Arizona wastewater, with Coxsackievirus A6, EV-D68, and Coxsackievirus A19 composing 86% of the characterized reads sequenced. While EV-D68 was not identified in pooled human nasal samples, and the only reported acute flaccid myelitis case in Arizona did not test positive for the virus, an in-depth analysis of EV-D68 in wastewater revealed that the virus was circulating from August through mid-October. A phylogenetic analysis on this relatively limited dataset revealed just a few importations into the state, with a single clade indicating local circulation. Significance: This study further supports the utility of wastewater-based epidemiology to identify potential public health threats. Our further investigations into EV-D68 shows how these data might help inform healthcare diagnoses for children presenting with concerning neurological symptoms.

Improved Subtyping of Staphylococcus aureus Clonal Complex 8 Strains Based on Whole-Genome Phylogenetic Analysis.

Strains of Staphylococcus aureus in clonal complex 8 (CC8), including USA300, USA500, and the Iberian clone, are prevalent pathogens in the United States, both inside and outside health care settings. Methods for typing CC8 strains are becoming obsolete as the strains evolve and diversify, and whole-genome sequencing has shown that some strain types fall into multiple sublineages within CC8. In this study, we attempt to clarify the strain nomenclature of CC8, classifying the major strain types based on whole-genome sequence phylogenetics using both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) genomes. We show that isolates of the Archaic and Iberian clones from decades ago make up the most basal clade of the main CC8 lineages and that at least one successful lineage of CC8, made up mostly of MSSA, diverged before the other well-known strain types USA500 and USA300. We also show that the USA500 type includes two clades separated by the previously described "Canadian epidemic MRSA" strain CMRSA9, that one clade containing USA500 also contains the USA300 clade, and that the USA300-0114 strain type is not a monophyletic group. Additionally, we present a rapid, simple CC8 strain-typing scheme using real-time PCR assays that target single nucleotide polymorphisms (SNPs) derived from our CC8 phylogeny and show the significant benefit of using more stable genomic markers based on evolutionary lineages over traditional S. aureus typing techniques. This more accurate and accessible S. aureus typing system may improve surveillance and better inform the epidemiology of this very important pathogen.IMPORTANCEStaphylococcus aureus is a major human pathogen worldwide in both community and health care settings. Surveillance for S. aureus strains is important to our understanding of their spread and to informing infection prevention and control. Confusion surrounding the strain nomenclature of one of the most prevalent lineages of S. aureus, clonal complex 8 (CC8), and the imprecision of current tools for typing S. aureus make surveillance and source tracing difficult and sometimes misleading. In this study, we clarify the CC8 strain designations and propose a new typing scheme for CC8 isolates that is rapid and easy to use. This typing scheme is based on relatively stable genomic markers, and we demonstrate its superiority over traditional typing techniques. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.

KlebSeq, a Diagnostic Tool for Surveillance, Detection, and Monitoring of Klebsiella pneumoniae.

Health care-acquired infections (HAIs) kill tens of thousands of people each year and add significantly to health care costs. Multidrug-resistant and epidemic strains are a large proportion of HAI agents, and multidrug-resistant strains of Klebsiella pneumoniae, a leading HAI agent, have caused an urgent public health crisis. In the health care environment, patient colonization by K. pneumoniae precedes infection, and transmission via colonization leads to outbreaks. Periodic patient screening for K. pneumoniae colonization has the potential to curb the number of HAIs. In this report, we describe the design and validation of KlebSeq, a highly informative screening tool that detects Klebsiella species and identifies clinically important strains and characteristics by using highly multiplexed amplicon sequencing without a live-culturing step. We demonstrate the utility of this tool on several complex specimen types, including urine, wound swabs and tissue, and several types of respiratory and fecal specimens, showing K. pneumoniae species and clonal group identification and antimicrobial resistance and virulence profiling, including capsule typing. Use of this amplicon sequencing tool to screen patients for Klebsiella carriage could inform health care staff of the risk of infection and outbreak potential. KlebSeq also serves as a model for next-generation molecular tools for public health and health care, as expansion of this tool can be used for several other HAI agents or applications.

Local Population Structure and Patterns of Western Hemisphere Dispersal for Coccidioides spp., the Fungal Cause of Valley Fever.

UNLABELLED: Coccidioidomycosis (or valley fever) is a fungal disease with high morbidity and mortality that affects tens of thousands of people each year. This infection is caused by two sibling species, Coccidioides immitis and C. posadasii, which are endemic to specific arid locales throughout the Western Hemisphere, particularly the desert southwest of the United States. Recent epidemiological and population genetic data suggest that the geographic range of coccidioidomycosis is expanding, as new endemic clusters have been identified in the state of Washington, well outside the established endemic range. The genetic mechanisms and epidemiological consequences of this expansion are unknown and require better understanding of the population structure and evolutionary history of these pathogens. Here we performed multiple phylogenetic inference and population genomics analyses of 68 new and 18 previously published genomes. The results provide evidence of substantial population structure in C. posadasii and demonstrate the presence of distinct geographic clades in central and southern Arizona as well as dispersed populations in Texas, Mexico, South America, and Central America. Although a smaller number of C. immitis strains were included in the analyses, some evidence of phylogeographic structure was also detected in this species, which has been historically limited to California and Baja, Mexico. Bayesian analyses indicated that C. posadasii is the more ancient of the two species and that Arizona contains the most diverse subpopulations. We propose a southern Arizona-northern Mexico origin for C. posadasii and describe a pathway for dispersal and distribution out of this region. IMPORTANCE: Coccidioidomycosis, or valley fever, is caused by the pathogenic fungi Coccidioides posadasii and C. immitis The fungal species and disease are primarily found in the American desert southwest, with spotted distribution throughout the Western Hemisphere. Initial molecular studies suggested a likely anthropogenic movement of C. posadasii from North America to South America. Here we comparatively analyze eighty-six genomes of the two Coccidioides species and establish local and species-wide population structures to not only clarify the earlier dispersal hypothesis but also provide evidence of likely ancestral populations and patterns of dispersal for the known subpopulations of C. posadasii.

Hypervirulent emm59 Clone in Invasive Group A Streptococcus Outbreak, Southwestern United States.

The hyper-virulent emm59 genotype of invasive group A Streptococcus was identified in northern Arizona in 2015. Eighteen isolates belonging to a genomic cluster grouped most closely with recently identified isolates in New Mexico. The continued transmission of emm59 in the southwestern United States poses a public health concern.

Whole-Genome Sequencing to Determine Origin of Multinational Outbreak of Sarocladium kiliense Bloodstream Infections.

We used whole-genome sequence typing (WGST) to investigate an outbreak of Sarocladium kiliense bloodstream infections (BSI) associated with receipt of contaminated antinausea medication among oncology patients in Colombia and Chile during 2013-2014. Twenty-five outbreak isolates (18 from patients and 7 from medication vials) and 11 control isolates unrelated to this outbreak were subjected to WGST to elucidate a source of infection. All outbreak isolates were nearly indistinguishable (<5 single-nucleotide polymorphisms), and >21,000 single-nucleotide polymorphisms were identified from unrelated control isolates, suggesting a point source for this outbreak. S. kiliense has been previously implicated in healthcare-related infections; however, the lack of available typing methods has precluded the ability to substantiate point sources. WGST for outbreak investigation caused by eukaryotic pathogens without reference genomes or existing genotyping methods enables accurate source identification to guide implementation of appropriate control and prevention measures.

Whole genome SNP typing to investigate methicillin-resistant Staphylococcus aureus carriage in a health-care provider as the source of multiple surgical site infections.

BACKGROUND: Prevention of nosocomial transmission of infections is a central responsibility in the healthcare environment, and accurate identification of transmission events presents the first challenge. Phylogenetic analysis based on whole genome sequencing provides a high-resolution approach for accurately relating isolates to one another, allowing precise identification or exclusion of transmission events and sources for nearly all cases. We sequenced 24 methicillin-resistant Staphylococcus aureus (MRSA) genomes to retrospectively investigate a suspected point source of three surgical site infections (SSIs) that occurred over a one-year period. The source of transmission was believed to be a surgical team member colonized with MRSA, involved in all surgeries preceding the SSI cases, who was subsequently decolonized. Genetic relatedness among isolates was determined using whole genome single nucleotide polymorphism (SNP) data. RESULTS: Whole genome SNP typing (WGST) revealed 283 informative SNPs between the surgical team member's isolate and the closest SSI isolate. The second isolate was 286 and the third was thousands of SNPs different, indicating the nasal carriage strain from the surgical team member was not the source of the SSIs. Given the mutation rates estimated for S. aureus, none of the SSI isolates share a common ancestor within the past 16 years, further discounting any common point source for these infections. The decolonization procedures and resources spent on the point source infection control could have been prevented if WGST was performed at the time of the suspected transmission, instead of retrospectively. CONCLUSIONS: Whole genome sequence analysis is an ideal method to exclude isolates involved in transmission events and nosocomial outbreaks, and coupling this method with epidemiological data can determine if a transmission event occurred. These methods promise to direct infection control resources more appropriately.

NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats.

Whole-genome sequencing (WGS) of bacterial isolates has become standard practice in many laboratories. Applications for WGS analysis include phylogeography and molecular epidemiology, using single nucleotide polymorphisms (SNPs) as the unit of evolution. NASP was developed as a reproducible method that scales well with the hundreds to thousands of WGS data typically used in comparative genomics applications. In this study, we demonstrate how NASP compares with other tools in the analysis of two real bacterial genomics datasets and one simulated dataset. Our results demonstrate that NASP produces similar, and often better, results in comparison with other pipelines, but is much more flexible in terms of data input types, job management systems, diversity of supported tools and output formats. We also demonstrate differences in results based on the choice of the reference genome and choice of inferring phylogenies from concatenated SNPs or alignments including monomorphic positions. NASP represents a source-available, version-controlled, unit-tested method and can be obtained from tgennorth.github.io/NASP.

Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic.

Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258.

Mapping the Evolution of Hypervirulent Klebsiella pneumoniae.

UNLABELLED: Highly invasive, community-acquired Klebsiella pneumoniae infections have recently emerged, resulting in pyogenic liver abscesses. These infections are caused by hypervirulent K. pneumoniae (hvKP) isolates primarily of capsule serotype K1 or K2. Hypervirulent K1 isolates belong to clonal complex 23 (CC23), indicating that this clonal lineage has a specific genetic background conferring hypervirulence. Here, we apply whole-genome sequencing to a collection of K. pneumoniae isolates to characterize the phylogenetic background of hvKP isolates with an emphasis on CC23. Most of the hvKP isolates belonged to CC23 and grouped into a distinct monophyletic clade, revealing that CC23 is a unique clonal lineage, clearly distinct from nonhypervirulent strains. Separate phylogenetic analyses of the CC23 isolates indicated that the CC23 lineage evolved recently by clonal expansion from a single common ancestor. Limited grouping according to geographical origin was observed, suggesting that CC23 has spread globally through multiple international transmissions. Conversely, hypervirulent K2 strains clustered in genetically unrelated groups. Strikingly, homologues of a large virulence plasmid were detected in all hvKP clonal lineages, indicating a key role in K. pneumoniae hypervirulence. The plasmid encodes two siderophores, aerobactin and salmochelin, and RmpA (regulator of the mucoid phenotype); all these factors were found to be restricted to hvKP isolates. Genomic comparisons revealed additional factors specifically associated with CC23. These included a distinct variant of a genomic island encoding yersiniabactin, colibactin, and microcin E492. Furthermore, additional novel genomic regions unique to CC23 were revealed which may also be involved in the increased virulence of this important clonal lineage. IMPORTANCE: During the last 3 decades, hypervirulent Klebsiella pneumoniae (hvKP) isolates have emerged, causing severe community-acquired infections primarily in the form of pyogenic liver abscesses. This syndrome has so far primarily been found in Southeast Asia, but increasing numbers of cases are being reported worldwide, indicating that the syndrome is turning into a globally emerging disease. We applied whole-genome sequencing to a collection of K. pneumoniae clinical isolates to reveal the phylogenetic background of hvKP and to identify genetic factors associated with the increased virulence. The hvKP isolates primarily belonged to clonal complex 23 (CC23), and this clonal lineage was revealed to be clearly distinct from nonhypervirulent strains. A specific virulence plasmid was found to be associated with hypervirulence, and novel genetic determinants uniquely associated with CC23 were identified. Our findings extend the understanding of the genetic background of the emergence of hvKP clones.

Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus.

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss.

Valley fever: finding new places for an old disease: Coccidioides immitis found in Washington State soil associated with recent human infection.

We used real-time polymerase chain reaction and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of valley fever, in Washington State linked to recent human infections and located outside the endemic range. Whole-genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients.

Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

MetaGeniE: characterizing human clinical samples using deep metagenomic sequencing.

With the decreasing cost of next-generation sequencing, deep sequencing of clinical samples provides unique opportunities to understand host-associated microbial communities. Among the primary challenges of clinical metagenomic sequencing is the rapid filtering of human reads to survey for pathogens with high specificity and sensitivity. Metagenomes are inherently variable due to different microbes in the samples and their relative abundance, the size and architecture of genomes, and factors such as target DNA amounts in tissue samples (i.e. human DNA versus pathogen DNA concentration). This variation in metagenomes typically manifests in sequencing datasets as low pathogen abundance, a high number of host reads, and the presence of close relatives and complex microbial communities. In addition to these challenges posed by the composition of metagenomes, high numbers of reads generated from high-throughput deep sequencing pose immense computational challenges. Accurate identification of pathogens is confounded by individual reads mapping to multiple different reference genomes due to gene similarity in different taxa present in the community or close relatives in the reference database. Available global and local sequence aligners also vary in sensitivity, specificity, and speed of detection. The efficiency of detection of pathogens in clinical samples is largely dependent on the desired taxonomic resolution of the organisms. We have developed an efficient strategy that identifies "all against all" relationships between sequencing reads and reference genomes. Our approach allows for scaling to large reference databases and then genome reconstruction by aggregating global and local alignments, thus allowing genetic characterization of pathogens at higher taxonomic resolution. These results were consistent with strain level SNP genotyping and bacterial identification from laboratory culture.

Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal.

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. Importance: Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic.

Real-time PCR assays for genotyping of Cryptococcus gattii in North America.

BACKGROUND: Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen. METHODS: We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human. RESULTS: Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA. CONCLUSIONS: The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.

Coccidioides immitis identified in soil outside of its known range - Washington, 2013.

Coccidioidomycosis ("valley fever") is caused by inhaling spores of the soil-dwelling fungi Coccidioides immitis or Coccidioides posadasii. Most infections are subclinical. When clinical manifestations do occur (typically 1-4 weeks after exposure), they are similar to those associated with influenza or community-acquired pneumonia. Disseminated disease is rare. Residual pulmonary nodules can lead to chronic lung disease. Fluconazole or other triazoles often are used for treatment, but mild cases often resolve without specific therapy. A total of 17,802 cases were reported in the United States in 2012.

Genome Sequence of Staphylococcus aureus Strain CA-347, a USA600 Methicillin-Resistant Isolate.

The Staphylococcus aureus clonal lineage CC45 is a predominant colonizer of healthy individuals in northern Europe and constitutes a highly basal cluster of the S. aureus population. Here, we report the complete genome sequence of S. aureus strain CA-347 (NRS648), a representative of the methicillin-resistant USA600 clone predominantly found in the United States.

Rapid and robust phylotyping of spa t003, a dominant MRSA clone in Luxembourg and other European countries.

BACKGROUND: spa typing is a common genotyping tool for methicillin-resistant Staphylococcus aureus (MRSA) in Europe. Given the high prevalence of dominant clones, spa-typing is proving to be limited in its ability to distinguish outbreak isolates from background isolates. New molecular tools need to be employed to improve subtyping of dominant local MRSA strains (e.g., spa type t003). METHODS: Phylogenetically critical, or canonical, SNPs (can-SNPs) were identified as subtyping targets through sequence analysis of 40 MRSA whole genomes from Luxembourg. Real-time PCR assays were designed around target SNPs and validated using a repository of 240 previously sub-typed and epidemiologically characterized Luxembourg MRSA isolates, including 153 community and hospital isolates, 69 isolates from long term care (LTC) facilities, and 21 prospectively analyzed MRSA isolates. Selected isolates were also analyzed by whole genome SNP typing (WGST) for comparison to the SNP assays and other subtyping techniques. RESULTS: Fourteen real-time PCR assays were developed and validated, including two assays to determine presence of spa t003 or t008. The other twelve assays successfully provided a high degree of resolution within the t003 subtype. WGST analysis of the LTC facility isolates provided greater resolution than other subtyping tools, identifying clusters indicative of ongoing transmission within LTC facilities. CONCLUSIONS: canSNP-based PCR assays are useful for local level MRSA phylotyping, especially in the presence of one or more dominant clones. The assays designed here can be easily adapted for investigating t003 MRSA strains in other regions in Western Europe. WGST provides substantially better resolution than other typing methods.

Evolution of a pathogen: a comparative genomics analysis identifies a genetic pathway to pathogenesis in Acinetobacter.

Acinetobacter baumannii is an emergent and global nosocomial pathogen. In addition to A. baumannii, other Acinetobacter species, especially those in the Acinetobacter calcoaceticus-baumannii (Acb) complex, have also been associated with serious human infection. Although mechanisms of attachment, persistence on abiotic surfaces, and pathogenesis in A. baumannii have been identified, the genetic mechanisms that explain the emergence of A. baumannii as the most widespread and virulent Acinetobacter species are not fully understood. Recent whole genome sequencing has provided insight into the phylogenetic structure of the genus Acinetobacter. However, a global comparison of genomic features between Acinetobacter spp. has not been described in the literature. In this study, 136 Acinetobacter genomes, including 67 sequenced in this study, were compared to identify the acquisition and loss of genes in the expansion of the Acinetobacter genus. A whole genome phylogeny confirmed that A. baumannii is a monophyletic clade and that the larger Acb complex is also a well-supported monophyletic group. The whole genome phylogeny provided the framework for a global genomic comparison based on a blast score ratio (BSR) analysis. The BSR analysis demonstrated that specific genes have been both lost and acquired in the evolution of A. baumannii. In addition, several genes associated with A. baumannii pathogenesis were found to be more conserved in the Acb complex, and especially in A. baumannii, than in other Acinetobacter genomes; until recently, a global analysis of the distribution and conservation of virulence factors across the genus was not possible. The results demonstrate that the acquisition of specific virulence factors has likely contributed to the widespread persistence and virulence of A. baumannii. The identification of novel features associated with transcriptional regulation and acquired by clades in the Acb complex presents targets for better understanding the evolution of pathogenesis and virulence in the expansion of the genus.