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Need for global water security has spurred growing interest in wastewater reuse to offset demand for municipal water. While reclaimed (i.e., nonpotable) microbial water quality regulations target fecal indicator bacteria, opportunistic pathogens (OPs), which are subject to regrowth in distribution systems and spread via aerosol inhalation and other noningestion routes, may be more relevant. This study compares the occurrences of five OP gene markers (Acanthamoebaspp., Legionellaspp., Mycobacteriumspp., Naegleria fowleri, Pseudomonas aeruginosa) in reclaimed versus potable water distribution systems and characterizes factors potentially contributing to their regrowth. Samples were collected over four sampling events at the point of compliance for water exiting treatment plants and at five points of use at four U.S. utilities bearing both reclaimed and potable water distribution systems. Reclaimed water systems harbored unique water chemistry (e.g., elevated nutrients), microbial community composition, and OP occurrence patterns compared to potable systems examined here and reported in the literature. Legionellaspp. genes, Mycobacteriumspp. genes, and total bacteria, represented by 16S rRNA genes, were more abundant in reclaimed than potable water distribution system samples (p≤ 0.0001). This work suggests that further consideration should be given to managing reclaimed water distribution systems with respect to nonpotable exposures to OPs.

Water reclamation provides a valuable resource for meeting nonpotable water demands. However, little is known about the potential for wastewater reuse to disseminate antibiotic resistance genes (ARGs). Here, samples were collected seasonally in 2014–2015 from four U.S. utilities’ reclaimed and potable water distribution systems before treatment, after treatment, and at five points of use (POU). Shotgun metagenomic sequencing was used to profile the resistome (i.e., full contingent of ARGs) of a subset (n= 38) of samples. Four ARGs (qnrA, blaTEM, vanA, sul1) were quantified by quantitative polymerase chain reaction. Bacterial community composition (via 16S rRNA gene amplicon sequencing), horizontal gene transfer (via quantification of intI1 integrase and plasmid genes), and selection pressure (via detection of metals and antibiotics) were investigated as potential factors governing the presence of ARGs. Certain ARGs were elevated in all (sul1; p≤ 0.0011) or some (blaTEM, qnrA; p≤ 0.0145) reclaimed POU samples compared to corresponding potable samples. Bacterial community composition was weakly correlated with ARGs (Adonis, R2= 0.1424–0.1734) and associations were noted between 193 ARGs and plasmid-associated genes. This study establishes that reclaimed water could convey greater abundances of certain ARGs than potable waters and provides observations regarding factors that likely control ARG occurrence in reclaimed water systems.

ABSTRACTHealth 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. pneumoniaeprecedes infection, and transmission via colonization leads to outbreaks. Periodic patient screening for K. pneumoniaecolonization 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 Klebsiellaspecies 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. pneumoniaespecies and clonal group identification and antimicrobial resistance and virulence profiling, including capsule typing. Use of this amplicon sequencing tool to screen patients for Klebsiellacarriage 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.

ABSTRACTIncreasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosisDNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of =1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.

RNA-based measurements have the potential for application across diverse areas of human health, including disease diagnosis, prognosis and therapeutic selection. Current clinical applications include infectious diseases, cancer, transplant medicine and fetal monitoring.RNA sequencing (RNA-seq) allows for the detection of a wide variety of RNA species, including mRNA, non-coding RNA, pathogen RNA, chimeric gene fusions, transcript isoforms and splice variants, and provides the capability to quantify known, pre-defined RNA species and rare RNA transcript variants within a sample. In addition to differential expression and detection of novel transcripts, RNA-seq also supports the detection of mutations and germline variation for hundreds to thousands of expressed genetic variants, facilitating assessment of allele-specific expression of these variants.Circulating RNAs and small regulatory RNAs, such as microRNAs, are very stable. These RNA species are vigorously being tested for their potential as biomarkers. However, there are currently few agreed upon methods for isolation or quantitative measurements and a current lack of quality controls that can be used to test platform accuracy and sample preparation quality.Analytical, bioinformatic and regulatory challenges exist, and ongoing efforts toward the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.

ABSTRACTA retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella entericaserotype Enteritidis and survey the population structure of commonly encountered S. entericaserotype Enteritidis outbreak isolates in the United States. A total of 52 S. entericaserotype 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. entericaserotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

ABSTRACTExserohilum rostratumwas the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohiluminfection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratumretrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ~136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

In a patient with positive serum serology for coccidioidomycosis, the differential diagnosis of concurrent pleural effusions can be challenging. We, therefore, sought to clarify the performance characteristics of biochemical, serologic, and nucleic-acid-based testing in an attempt to avoid invasive procedures. The utility of adenosine deaminase (ADA), coccidioidal serology, and polymerase chain reaction (PCR) in the evaluation of pleuropulmonary coccidioidomycosis has not been previously reported.

ABSTRACTMelioidosis 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-orf2assay to be superior in detecting B. pseudomalleidirectly 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.

ABSTRACTCyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.

One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

ABSTRACTOne of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

ABSTRACTThe recurring emergence of influenza virus strains that are resistant to available antiviral medications has become a global health concern, especially in light of the potential for a new influenza virus pandemic. Currently, virtually all circulating strains of influenza A virus in the United States are resistant to either of the two major classes of anti-influenza drugs (adamantanes and neuraminidase inhibitors). Thus, new therapeutic approaches that can be rapidly deployed and that will address the issue of recurring resistance should be developed. We have tested double and triple combinations of the approved anti-influenza drugs oseltamivir and amantadine together with ribavirin against three influenza virus strains using cytopathic effect inhibition assays in MDCK cells. We selected A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N2) as representatives of the wild-type versions of the predominant circulating seasonal influenza virus strains and A/Duck/MN/1525/81 (H5N1) as a representative of avian influenza virus strains. Dose-response curves were generated for all drug combinations, and the degree of drug interaction was quantified using a model that calculates the synergy (or antagonism) between the drugs in double and triple combinations. This report demonstrates that a triple combination of antivirals was highly synergistic against influenza A virus. Importantly, the synergy of the triple combination was 2- to 13-fold greater than the synergy of any double combination depending on the influenza virus subtype. These data support the investigation of a novel combination of oseltamivir, amantadine, and ribavirin as an effective treatment for both seasonal and pandemic influenza virus, allowing the efficient use of the existing drug supplies.

ABSTRACTThe “gold standard” for identifying Yersinia pestis-infected fleas has been inoculation of mice with pooled flea material. Inoculated mice are monitored for 21 days, and those that die are further analyzed for Y. pestisinfection by fluorescent-antibody assay and/or culture. PCR may provide a more rapid and sensitive alternative for identifying Y. pestisin fleas. To compare these assays, samples were prepared from 381 field-collected fleas. Each flea was analyzed individually by both PCR and mouse inoculation. Sixty of the 381 flea samples were positive forY. pestisby PCR; 48 of these PCR-positive samples caused death in mice (80.0% agreement). None of the 321 PCR-negative samples caused death. Among the 12 mice that survived inoculation with PCR-positive samples, 10 were later demonstrated by serology or culture to have been infected with Y. pestis. This suggests that death of inoculated mice is less reliable than PCR as an indicator of the presence of Y. pestisin flea samples. Mouse inoculation assays produce results that are comparable to PCR only when surviving as well as dead mice are analyzed for infection. The rapidity and sensitivity (10 to 100 CFU of Y. pestis) of PCR suggest that it could serve as a useful alternative to mouse inoculation for routine plague surveillance and outbreak investigations.

Fluoroquinolones (FQ) are crucial components of multidrug-resistant tuberculosis (MDR TB) treatment. Differing levels of resistance are associated with specific mutations within the quinolone-resistance-determining region (QRDR) of gyrA. We sequenced the QRDR from serial isolates of MDR TB patients in the Preserving Effective TB Treatment Study (PETTS) with baseline FQ resistance (FQR) or acquired FQ resistance (FQACQR) using an Ion Torrent Personal Genome Machine (PGM) to a depth of 10,000× and reported single nucleotide polymorphisms in ≥1% of reads.

As the COVID-19 pandemic swept across the United States, there was great differential impact on local and regional communities. One of the earliest and hardest hit regions was in New York, while at the same time Arizona (for example) had low incidence. That situation has changed dramatically, with Arizona now having the highest rate of disease increase in the country. Understanding the roots of the pandemic during the initial months is essential as the pandemic continues and reaches new heights. Genomic analysis and phylogenetic modeling of SARS-COV-2 in Arizona can help to reconstruct population composition and predict the earliest undetected introductions. This foundational work represents the basis for future analysis and understanding as the pandemic continues.

The appearance of Cryptococcus gattiiin the North American Pacific Northwest (PNW) in 1999 was an unexpected and is still an unexplained event. Recent phylogenomic analyses strongly suggest that this pathogenic fungus arrived in the PNW approximately 7 to 9 decades ago. In this paper, we theorize that the ancestors of the PNW C. gattiiclones arrived in the area by shipborne transport, possibly in contaminated ballast, and established themselves in coastal waters early in the 20th century.

Since 2002, there has been a gradual worldwide 1.3% annual decrease in the incidence of tuberculosis (TB). This is an encouraging statistic; however, it will not achieve the World Health Organization’s goal of eliminating TB by 2050, and it is being compounded by the persistent global incidence of drug-resistant tuberculosis (DR-TB) acquired by transmission and by treatment pressure. One key to effectively control tuberculosis and the spread of multiresistant strains is accurate information pertaining to drug resistance and susceptibility. Next-generation sequencing (NGS) has the potential to effectively change global health and the management of TB. Industry has focused primarily on using NGS for oncology diagnostics and human genomics, but the area in which NGS can rapidly impact health care is in the area of infectious disease diagnostics in low- and middle-income countries. To date, there has been a failure as a community to capitalize on the potential of NGS, especially at the reference laboratory level where it can provide actionable information pertaining to treatment options for patients. The rapid evolution of knowledge about the genetic foundations of tuberculosis drug resistance makes sequencing a versatile technology platform for providing rapid, accurate, and actionable results for treating this disease. No “plug-and-play” and “end-to-end” NGS solutions exist that provide clinically relevant sequence data from the Mycobacterium tuberculosiscomplex genome from primary clinical samples (e.g., sputum) in high-burden country reference laboratories, which is where they are most needed. However, such a system-based solution is underdeveloped by Foundation for Innovative Diagnostics (FIND), in collaboration with partners from academia, nongovernmental organizations, and industry. The solution is modular and is designed and developed to perform targeted amplicon sequencing directly from a patient’s primary sputum sample. This solution will initially allow reference laboratories to perform reflex NGS that provides a rapid and comprehensive analysis of a patient’s M. tuberculosiscomplex drug resistance profile, thereby facilitating optimization of a patient’s treatment, improving treatment outcomes, and reducing the spread of DR-TB. Such a system could also enable countries to implement culture-free drug resistance surveillance programs, which could bypass the need for expensive culture facilities, decrease a country’s dependence on external laboratories, and significantly expand the map of global surveillance capabilities. In addition, the introduction of such a system will provide a foundation for NGS to be used for genotypic testing for human immunodeficiency virus-infected patients, surveillance of other diseases, in-country capability for outbreak discovery and management, and a host of other diagnostic benefits that are currently limited to high-income countries.

Multidrug-resistant tuberculosis (TB) is an alarming threat, and targeted deep sequencing (DS) may be an effective method for rapid identification of drug-resistant profiles, including detection of heteroresistance. We evaluated the sensitivity and specificity of targeted DS versus phenotypic drug susceptibility testing (pDST) among patients starting first-line anti-TB therapy in Botswana. Overall, we found high concordance between DS and pDST.

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.

ABSTRACTMultidrug-resistant tuberculosis (TB) presents a major public health dilemma. Heteroresistance, the coexistence of drug-resistant and drug-susceptible strains or of multiple drug-resistant strains with discrete haplotypes, may affect accurate diagnosis and the institution of effective treatment. Subculture, or passage of cells onto fresh growth medium, is utilized to preserve Mycobacterium tuberculosiscell lines and is universally employed in TB diagnostics. The impact of such passages, typically performed in the absence of drug, on drug-resistant subpopulations is hypothesized to vary according to the competitive costs of genotypic resistance-associated variants. We applied ultradeep next-generation sequencing to 61 phenotypically rifampin-monoresistant (n= 17) and preextensively (n= 41) and extensively (n= 3) drug-resistant isolates with presumptive heteroresistance at two time points in serial subculture. We found significant dynamic loss of minor-variant resistant subpopulations across all analyzed resistance-determining regions, including eight isolates (13%) whose antibiogram data would have transitioned from resistant to susceptible for at least one drug through subculture. Surprisingly, some resistance-associated variants appeared to be selected for in subculture.

ABSTRACTCoccidioidomycosis (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 immitisand 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. posadasiiand 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. immitisstrains 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. posadasiiis 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. posadasiiand describe a pathway for dispersal and distribution out of this region.IMPORTANCECoccidioidomycosis, or valley fever, is caused by the pathogenic fungi Coccidioides posadasiiand 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. posadasiifrom North America to South America. Here we comparatively analyze eighty-six genomes of the two Coccidioidesspecies 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.

ABSTRACTHighly invasive, community-acquired Klebsiella pneumoniaeinfections 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. pneumoniaeisolates 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. pneumoniaehypervirulence. 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.IMPORTANCEDuring 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. pneumoniaeclinical 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.

ABSTRACTThe emergence of distinct populations of Cryptococcus gattiiin 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. gattiiisolates, 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 neoformansvar. grubiithat is rarely seen in global C. gattiibut that was present in all PNW populations. These findings provide greater understanding of C. gattiievolution in North America and support extensive evolution in, and dispersal from, South America.IMPORTANCECryptococcus gattiiemerged 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. gattiipopulations. 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. gattiiinto the PNW. Public health vigilance is warranted for emergence in regions where C. gattiiis not thought to be endemic.

ABSTRACTCholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio choleraeisolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. choleraeisolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. choleraeisolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks.IMPORTANCECholera is one of the ancient classical diseases and particularly prone to cause major outbreaks following major natural disasters, such as earthquakes and hurricanes, where the normal separation between sewage and drinking water is destroyed. This was the case following the 2010 earthquake in Haiti. Rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. Sequencing the genomes of bacteria can give detailed information on whether isolates from different sites share a common origin. We used this technology to sequence isolates of Vibrio choleraefrom Nepal, identify single-nucleotide polymorphisms (SNPs), and compare these high-resolution genotypes to the complete genome sequences of isolates from the Haiti outbreak. We provide support for the hypothesis that the isolates were brought to Haiti from Nepal.

Plague is a rare but highly virulent flea-borne zoonotic disease caused by the Gram-negative bacterium Yersinia pestis Yersin. Identifying areas at high risk of human exposure to the etiological agent of plague could provide a useful tool for targeting limited public health resources and reduce the likelihood of misdiagnosis by raising awareness of the disease. We created logistic regression models to identify landscape features associated with areas where humans have acquired plague from 1957 to 2004 in the four-corners region of the United States (Arizona, Colorado, New Mexico, and Utah), and we extrapolated those models within a geographical information system to predict where plague cases are likely to occur within the southwestern United States disease focus. The probability of an area being classified as high-risk plague habitat increased with elevation up to ≈2,300 m and declined as elevation increased thereafter, and declined with distance from key habitat types (e.g., southern Rocky Mountain piñon—juniper [Pinus edulis Engelm. and Juniperus spp.], Colorado plateau piñon-juniper woodland, Rocky Mountain ponderosa pine (Pinus ponderosa P.& C. Lawson var. scopulorum), and southern Rocky Mountain juniper woodland and savanna). The overall accuracy of the model was >82%. Our most conservative model predicted that 14.4% of the four-corners region represented a high risk of peridomestic exposure to Y. pestis.

We used a quantitative competitive polymerase chain reaction (PCR) (QC-PCR) to determine bacterial loads in 669 fleas collected in areas of confirmed and suspected plague epizootics. Fleas were collected out of rodent burrows (67.9%) and off of captured animals (24.1%) and rodent carcasses (8.1%). An initial PCR screening assay indicated that 12.1% (81/669) of all fleas were positive for Yersinia pestis. Fleas collected from burrows had significantly lower (?2 = 264.9, P < 0.0001) infection rates (6.8%) but significantly higher (Student t-test, P < 0.0001) bacterial loads (mean = 105.6 Y. pestis per flea) than fleas collected off of rodent carcasses (infection rate = 92.6%; mean bacterial load = 104.8 Y. pestis per flea). None of the fleas collected off of captured animals were positive for Y. pestis by PCR, although seven of the 176 captured animals were serologically positive for Y. pestis.