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Rhizopus spp. fungi are ubiquitous in the environment and a rare but substantial cause of infection in immunosuppressed persons and surgery patients. During 2005–2017, an abnormally high number of Rhizopus infections in surgery patients, with no apparent epidemiologic links, were reported in Argentina. To determine the likelihood of a common source of the cluster, we performed whole-genome sequencing on samples collected during 2006–2014. Most isolates were separated by >60 single-nucleotide polymorphisms, and we found no evidence for recombination or nonneutral mutation accumulation; these findings do not support common source or patient-to-patient transmission. Assembled genomes of most isolates were ≈25 Mbp, and multiple isolates had substantially larger assembled genomes (43–51 Mbp), indicative of infections with strain types that underwent genome expansion. Whole-genome sequencing has become an essential tool for studying epidemiology of fungal infections. Less discriminatory techniques may miss true relationships, possibly resulting in inappropriate attribution of point source.

Background Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. Results In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. Conclusions This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics.

In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China, causing severe morbidity and mortality. Since then, the virus has swept across the globe, causing millions of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, the Arizona Department of Health Services, and those collected as part of community surveillance projects at Arizona State University and the University of Arizona. Phylogenetic analysis of 84 genomes from across Arizona revealed a minimum of 11 distinct introductions inferred to have occurred during February and March. We show that >80% of our sequences descend from strains that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related case in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020., mBio, 11 (5), ISSN:2150-7511, ISSN:2161-2129

There has been a considerable upsurge of extensive, treatment recalcitrant, dermatophytosis presenting as tinea corporis and tinea cruris in India since the past few years. Genome analysis of Trichophyton species causing severe superficial dermatophytosis in North India confirmed a unique clade related to the T. mentagrophytes/interdigitale complex, seeming to belong to an early diverging clade of the complex. The Indian Trichophyton species genomes were highly related showing only up to 42 SNPs between any two isolates confirming their clonal origin. Other genetic approaches such as ITS sequencing and multigene phylogeny used in this study were contradictory or inconclusive to show the differentiation of these isolates from T. mentagrophytes/T. interdigitale. Remarkably, high rates of resistance to all three commonly used oral antifungals, i.e., 36% for terbinafine (MICs 4 to ≥32 mg/L), 39.5% for fluconazole (MIC range 32 to ≥64 mg/L) and griseofulvin (Geometric mean MIC ≥ 4 mg/L) were observed. Two important amino acid substitutions (Leu393Phe or Phe397Leu) leading to a terbinafine resistant phenotype were found in the squalene epoxidase protein of all tested terbinafine resistant isolates. All 20 examined genomes presented a high mobility group (HMG) domain transcription factor gene corresponding to mating type (+). Of these, three isolates also showed positivity for both alpha-box and HMG in the genome which might indicate hybridization or an incomplete sexual cycle. Therefore, we highlight the potential of this organism to rapidly spread alleles that might be driving antifungal resistance among its population. This new population of Trichophyton with high rates of in vitro antifungal resistance seems to be driving an ongoing outbreak of dermatophytosis in India. Our study highlights difficulties in identifying isolates from the Trichophyton mentagrophytes/interdigitale clade of the genus using currently available molecular tools. High resistance rates of terbinafine warrant further clinical studies to assess its utility in the treatment of dermatophytosis caused by this clonal strain.

Cryptococcus gattii molecular type VGII is one of the causes of cryptococcosis, a severe fungal disease that is acquired from the environment and affects many host species (including humans and koalas). In Australia, disease caused by C. gattii VGII is largely confined to western and central northern parts of the country, with sporadic cases reported in eastern Australia. We investigated an unusual case cluster of cryptococcosis, caused predominantly by C. gattii VGII, in a group of captive koalas in eastern Australia. This research identified that the movements of koalas between wildlife parks, including an initial transfer of a koala from Western Australia, introduced and subsequently spread C. gattii VGII in this captive environment. The spread of this pathogen by koalas could also impact other species, and these findings are significant in the implications they have for the management of koala transfers and captive environments., Cryptococcus gattii molecular type VGII is one of the etiologic agents of cryptococcosis, a systemic mycosis affecting a wide range of host species. Koalas (Phascolarctos cinereus) exhibit a comparatively high prevalence of cryptococcosis (clinical and subclinical) and nasal colonization, particularly in captivity. In Australia, disease associated with C. gattii VGII is typically confined to Western Australia and the Northern Territory (with sporadic cases reported in eastern Australia), occupying an enigmatic ecologic niche. A cluster of cryptococcosis in captive koalas in eastern Australia (five confirmed cases, a further two suspected), caused predominantly by C. gattii VGII, was investigated by surveying for subclinical disease, culturing koala nasal swabs and environmental samples, and genotyping cryptococcal isolates. URA5 restriction fragment length polymorphism analysis, multilocus sequence typing (MLST), and whole-genome sequencing (WGS) provided supportive evidence that the transfer of koalas from Western Australia and subsequently between several facilities in Queensland spread VGII into uncontaminated environments and environments in which C. gattii VGI was endemic. MLST identified VGII isolates as predominantly sequence type 7, while WGS further confirmed a limited genomic diversity and revealed a basal relationship with isolates from Western Australia. We hypothesize that this represents a founder effect following the introduction of a koala from Western Australia. Our findings suggest a possible competitive advantage for C. gattii VGII over VGI in the context of this captive koala environment. The ability of koalas to seed C. gattii VGII into new environments has implications for the management of captive populations and movements of koalas between zoos. IMPORTANCE Cryptococcus gattii molecular type VGII is one of the causes of cryptococcosis, a severe fungal disease that is acquired from the environment and affects many host species (including humans and koalas). In Australia, disease caused by C. gattii VGII is largely confined to western and central northern parts of the country, with sporadic cases reported in eastern Australia. We investigated an unusual case cluster of cryptococcosis, caused predominantly by C. gattii VGII, in a group of captive koalas in eastern Australia. This research identified that the movements of koalas between wildlife parks, including an initial transfer of a koala from Western Australia, introduced and subsequently spread C. gattii VGII in this captive environment. The spread of this pathogen by koalas could also impact other species, and these findings are significant in the implications they have for the management of koala transfers and captive environments.

Enteroviruses frequently result in respiratory and gastrointestinal illness; however, multiple subtypes, including poliovirus, can cause severe neurologic disease. Recent biennial increases (i.e., 2014, 2016, and 2018) in cases of non-polio acute flaccid paralysis have led to speculations that other enteroviruses, specifically enterovirus D68 (EV-D68), are emerging to fill the niche that was left from poliovirus eradication. A cluster of 11 suspect cases of pediatric acute flaccid myelitis (AFM) was identified in 2016 in Phoenix, AZ. Multiple genomic analyses identified the presence of EV-D68 in the majority of clinical AFM cases. Beyond limited detection of herpesvirus, no other likely etiologies were found in the cluster. These findings strengthen the likelihood that EV-D68 is a cause of AFM and show that the rapid molecular assays developed for this study are useful for investigations of AFM and EV-D68., Enteroviruses are a common cause of respiratory and gastrointestinal illness, and multiple subtypes, including poliovirus, can cause neurologic disease. In recent years, enterovirus D68 (EV-D68) has been associated with serious neurologic illnesses, including acute flaccid myelitis (AFM), frequently preceded by respiratory disease. A cluster of 11 suspect cases of pediatric AFM was identified in September 2016 in Phoenix, AZ. To determine if these cases were associated with EV-D68, we performed multiple genomic analyses of nasopharyngeal (NP) swabs and cerebrospinal fluid (CSF) material from the patients, including real-time PCR and amplicon sequencing targeting the EV-D68 VP1 gene and unbiased microbiome and metagenomic sequencing. Four of the 11 patients were classified as confirmed cases of AFM, and an additional case was classified as probable AFM. Real-time PCR and amplicon sequencing detected EV-D68 virus RNA in the three AFM patients from which NP swabs were collected, as well as in a fourth patient diagnosed with acute disseminated encephalomyelitis, a disease that commonly follows bacterial or viral infections, including enterovirus. No other obvious etiological causes for AFM were identified by 16S or RNA and DNA metagenomic sequencing in these cases, strengthening the likelihood that EV-D68 is an etiological factor. Herpes simplex viral DNA was detected in the CSF of the fourth case of AFM and in one additional suspect case from the cluster. Multiple genomic techniques, such as those described here, can be used to diagnose patients with suspected EV-D68 respiratory illness, to aid in AFM diagnosis, and for future EV-D68 surveillance and epidemiology.

In South Africa, Cryptococcus neoformans is the most common cause of adult meningitis. We performed multi locus sequence typing and fluconazole susceptibility testing of clinical C. neoformans isolates collected from 251 South African patients with cryptococcosis through national surveillance from 2005 to 2009. We examined the association between clinical characteristics of patients and genotype, and the effect of genotype on in-hospital mortality. We performed whole genome phylogenetic analysis of fifteen C. neoformans isolates with the molecular type VNB and tested their virulence in a Galleria mellonella model. Most isolates had the molecular type VNI (206/251, 82%), followed by VNII (25/251, 10%), VNB (15/251, 6%), and VNIV (5/251, 2%), 67 sequence types were identified. There were no differences in fluconazole minimum inhibitory concentration (MIC) values among molecular types and the majority of strains had low MIC values (MIC50 of 1 µg/mL and MIC90 of 4 µg/mL). Males were almost twice as likely of being infected with a non-VNI genotype (adjusted odds ratio [OR]: 1.65, 95% confidence interval [CI]: 0.25–10.99, p = 0.61). Compared to patients infected with a VNI genotype, those with a non-VNI genotype had a 50% reduced adjusted odds of dying in hospital (95% CI: 0.03–7.57, p = 0.62). However, for both these analyses, our estimates had wide confidence intervals spanning 1 with large p-values. Fifteen VNB strains were not as virulent in a G. mellonella larval model as the H99 reference strain. A majority of these VNB strains belonged to the VNBII clade and were very closely related by phylogenetic analysis.

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.

Staphylococcus 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., 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. IMPORTANCE Staphylococcus 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.

Coccidioides immitis and Coccidioides posadasii are soil fungi endemic to desert regions of the southwestern United States, and the causative agents of valley fever, or coccidioidomycosis. Studies have shown that the distribution of Coccidioides in soils is sporadic and cannot be explained by soil characteristics alone, suggesting that biotic and other abiotic factors should be examined. However, tools to reliably and robustly screen the large number of soils needed to investigate these potential associations have not been available. Thus, we developed a real-time polymerase chain reaction (PCR) assay for testing environmental samples by modifying CocciDx, an assay validated for testing clinical specimens to facilitate coccidioidomycosis diagnosis. For this study, we collected soil samples from previously established locations of C. posadasii in Arizona and new locations in fall 2013 and spring 2014, and screened the extracted DNA with the new assay known as CocciEnv. To verify the presence of Coccidioides in soil using an alternate method, we employed next generation amplicon sequencing targeting the ITS2 region. Results show our modified assay, CocciEnv, is a rapid and robust method for detecting Coccidioides DNA in complex environmental samples. The ability to test a large number of soils for the presence of Coccidioides is a much-needed tool in the understanding of the ecology of the organism and epidemiology of the disease and will greatly improve our understanding of this human pathogen.

Healthcare-acquired infections (HAIs) kill tens of thousands of people each year and add significantly to healthcare costs. Multidrug resistant and epidemic strains are a large proportion of HAI agents, and multidrug resistant strains ofKlebsiella pneumoniae, a leading HAI agent, have become an urgent public health crisis. In the healthcare environment, patient colonization ofK. pneumoniaeprecedes infection, and transmission via colonization leads to outbreaks. Periodic patient screening forK. pneumoniaecolonization has cost-effective and life-saving potential. In this study, we describe the design and validation of KlebSeq, a highly informative screening tool that detectsKlebsiellaspecies and identifies clinically important strains and characteristics 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, several types of respiratory, and fecal, showingK. pneumoniaespecies and clonal group identification and antimicrobial resistance and virulence profiling, including capsule typing. Use of this amplicon sequencing tool can be used to screen patients forK. pneumoniaecarriage to assess risk of infection and outbreak potential, and the expansion of this tool can be used for several other HAI agents or applications.

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei . In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1- orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia -like fimbrial/ Burkholderia thailandensis -like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica , whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica , including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica .

Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat.

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.

Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification., Author Summary Melioidosis is a bacterial disease caused by percutaneous inoculation, aspiration or ingestion of the soil bacteria Burkholderia pseudomallei. Melioidosis is primarily found in Southeast Asia and Northern Australia, and, to a lesser degree, nearby regions. A recent case of melioidosis in Southwestern United States (Southern Arizona) prompted a detailed epidemiological and molecular investigation to discover the source of infection. The authors describe the use of multiple genomic analysis tools to aid in this investigation. The results of these analyses uniformly identified Southeast Asia as the source of the strain that infected the patient, however the epidemiological investigation had determined the patient had no international travel or known exposures to Southeast Asian products. New cutting edge technologies, such as next generation sequencing, are quickly being adapted into epidemiologic investigations, particularly for cases and outbreaks of unknown origin, although older, mature technologies with larger existing databases will still be needed for appropriate comparative analyses.

We characterized the prevalence, antibiotic susceptibility profiles, and genotypes of Staphylococcus aureus among US meat and poultry samples (n = 136). S. aureus contaminated 47% of samples, and multidrug resistance was common among isolates (52%). S. aureus genotypes and resistance profiles differed significantly among sample types, suggesting food animal–specific contamination.

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei.

Background Circumcision is associated with significant reductions in HIV, HSV-2 and HPV infections among men and significant reductions in bacterial vaginosis among their female partners. Methodology/Principal Findings We assessed the penile (coronal sulci) microbiota in 12 HIV-negative Ugandan men before and after circumcision. Microbiota were characterized using sequence-tagged 16S rRNA gene pyrosequencing targeting the V3–V4 hypervariable regions. Taxonomic classification was performed using the RDP Naive Bayesian Classifier. Among the 42 unique bacterial families identified, Pseudomonadaceae and Oxalobactericeae were the most abundant irrespective of circumcision status. Circumcision was associated with a significant change in the overall microbiota (PerMANOVA p = 0.007) and with a significant decrease in putative anaerobic bacterial families (Wilcoxon Signed-Rank test p = 0.014). Specifically, two families—Clostridiales Family XI (p = 0.006) and Prevotellaceae (p = 0.006)—were uniquely abundant before circumcision. Within these families we identified a number of anaerobic genera previously associated with bacterial vaginosis including: Anaerococcus spp., Finegoldia spp., Peptoniphilus spp., and Prevotella spp. Conclusions/Significance The anoxic microenvironment of the subpreputial space may support pro-inflammatory anaerobes that can activate Langerhans cells to present HIV to CD4 cells in draining lymph nodes. Thus, the reduction in putative anaerobic bacteria after circumcision may play a role in protection from HIV and other sexually transmitted diseases.

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.

Background Rates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them. Results Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains. Conclusions The dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.

BackgroundBacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota.Methodology/principal findingsWe prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds--approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics.Conclusions/significanceThe 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure--reducing some bacteria while selecting for others.