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It is not possible to systematically screen the environment for rabies virus (RABV) using current approaches. We sought to determine under what conditions RABV is detectable from feces and other accessible samples from infected wildlife to broaden the number of biological samples that could be used to test for RABV. We employed a recently-developed quantitative RT-PCR assay called the "LN34 panlyssavirus real-time RT-PCR assay", which is highly sensitive and specific for all variants of RABV. We harvested and tested brain tissue, fecal, and/or mouth swab samples from 25 confirmed RABV positive bats of six species. To determine if rabies RNA lasts in feces sufficiently long post-defecation to use it as a surveillance tool, we tested fecal samples from 10 bats at the time of sample collection and after 24 hours of exposure to ambient conditions, with an additional test on six bats out to 72 hours. To assess whether we could pool fecal pellets and still detect a positive, we generated dilutions of known positives at 1:1, 1:10, 1:50, and 1:200. For six individuals for which matched brain, mouth swab, and fecal samples were tested, results were positive for 100%, 67%, and 67%, respectively. For the first time test to 24 hours, 63% of feces that were positive at time 0 were still positive after 24 hours, and 50% of samples at 72 hours were positive across all three replicates. Pooling tests revealed that fecal positives were detected at 1:10 dilution, but not at 1:50 or 1:200. Our preliminary results suggest that fecal samples hold promise for a rapid and non-invasive environmental screening system.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

As is the case globally, Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV (101/146, 70%), followed by VGI (40/146, 27%), VGII (3/146, 2%) and VGIII (2/146, 1%). Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294 (14/146, 10%) and ST155 (10/146, 7%) being most commonly observed. The fluconazole MIC50 and MIC90 values of 105 (of 146) randomly selected C. gattii isolates were 4 μg/ml and 16 μg/ml, respectively. VGIV isolates had a lower MIC50 value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers (99/136; 73% vs. 2/10; 20%), the confidence interval (CI) was wide (95% CI: 1.93-55.31, p = 0.006). Whole genome phylogeny of 98 isolates of South Africa's most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen.

BackgroundThe incidence of multidrug-resistant tuberculosis (MDR-TB) remains critically high in countries of the former Soviet Union, where >20% of new cases and >50% of previously treated cases have resistance to rifampin and isoniazid. Transmission of resistant strains, as opposed to resistance selected through inadequate treatment of drug-susceptible tuberculosis (TB), is the main driver of incident MDR-TB in these countries.Methods and findingsWe conducted a prospective, genomic analysis of all culture-positive TB cases diagnosed in 2018 and 2019 in the Republic of Moldova. We used phylogenetic methods to identify putative transmission clusters; spatial and demographic data were analyzed to further describe local transmission of Mycobacterium tuberculosis. Of 2,236 participants, 779 (36%) had MDR-TB, of whom 386 (50%) had never been treated previously for TB. Moreover, 92% of multidrug-resistant M. tuberculosis strains belonged to putative transmission clusters. Phylogenetic reconstruction identified 3 large clades that were comprised nearly uniformly of MDR-TB: 2 of these clades were of Beijing lineage, and 1 of Ural lineage, and each had additional distinct clade-specific second-line drug resistance mutations and geographic distributions. Spatial and temporal proximity between pairs of cases within a cluster was associated with greater genomic similarity. Our study lasted for only 2 years, a relatively short duration compared with the natural history of TB, and, thus, the ability to infer the full extent of transmission is limited.ConclusionsThe MDR-TB epidemic in Moldova is associated with the local transmission of multiple M. tuberculosis strains, including distinct clades of highly drug-resistant M. tuberculosis with varying geographic distributions and drug resistance profiles. This study demonstrates the role of comprehensive genomic surveillance for understanding the transmission of M. tuberculosis and highlights the urgency of interventions to interrupt transmission of highly drug-resistant M. tuberculosis.

BackgroundAccurate, comprehensive, and timely detection of drug-resistant tuberculosis (TB) is essential to inform patient treatment and enable public health surveillance. This is crucial for effective control of TB globally. Whole-genome sequencing (WGS) and targeted next-generation sequencing (NGS) approaches have potential as rapid in vitro diagnostics (IVDs), but the complexity of workflows, interpretation of results, high costs, and vulnerability of instrumentation have been barriers to broad uptake outside of reference laboratories, especially in low- and middle-income countries. A new, solid-state, tabletop sequencing instrument, Illumina iSeq100, has the potential to decentralize NGS for individual patient care.Methods and findingsIn this study, we evaluated WGS and targeted NGS for TB on both the new iSeq100 and the widely used MiSeq (both manufactured by Illumina) and compared sequencing performance, costs, and usability. We utilized DNA libraries produced from Mycobacterium tuberculosis clinical isolates for the evaluation. We conducted WGS on three strains and observed equivalent uniform genome coverage with both platforms and found the depth of coverage obtained was consistent with the expected data output. Utilizing the standardized, cloud-based ReSeqTB bioinformatics pipeline for variant analysis, we found the two platforms to have 94.0% (CI 93.1%-94.8%) agreement, in comparison to 97.6% (CI 97%-98.1%) agreement for the same libraries on two MiSeq instruments. For the targeted NGS approach, 46 M. tuberculosis-specific amplicon libraries had 99.6% (CI 98.0%-99.9%) agreement between the iSeq100 and MiSeq data sets in drug resistance-associated SNPs. The upfront capital costs are almost 5-fold lower for the iSeq100 ($19,900 USD) platform in comparison to the MiSeq ($99,000 USD); however, because of difference in the batching capabilities, the price per sample for WGS was higher on the iSeq100. For WGS of M. tuberculosis at the minimum depth of coverage of 30x, the cost per sample on the iSeq100 was $69.44 USD versus $28.21 USD on the MiSeq, assuming a 2 × 150 bp run on a v3 kit. In terms of ease of use, the sequencing workflow of iSeq100 has been optimized to only require 27 minutes total of hands-on time pre- and post-run, and the maintenance is simplified by a single-use cartridge-based fluidic system. As these are the first sequencing attempts on the iSeq100 for M. tuberculosis, the sequencing pool loading concentration still needs optimization, which will affect sequencing error and depth of coverage. Additionally, the costs are based on current equipment and reagent costs, which are subject to change.ConclusionsThe iSeq100 instrument is capable of running existing TB WGS and targeted NGS library preparations with comparable accuracy to the MiSeq. The iSeq100 has reduced sequencing workflow hands-on time and is able to deliver sequencing results in

West Nile Virus (WNV) has been detected annually in Maricopa County, Arizona, since 2003. With this in mind, we sought to determine if contemporary strains are endemic to the county or are annually imported. As part of this effort, we developed a new protocol for tiled amplicon sequencing of WNV to efficiently attain greater than 99% coverage of 14 WNV genomes collected directly from positive mosquito pools distributed throughout Maricopa County between 2014 and 2017. Bayesian phylogenetic analyses revealed that contemporary genomes fall within two major lineages; NA/WN02 and SW/WN03. We found that all of the Arizona strains possessed an amino acid substitution known to be under positive selection, which has arisen independently at least four times in Arizona. The SW/WN03 strains exhibited transient behavior, with at least 10 separate introductions into Arizona when considering both historical and contemporary strains. However, NA/WN02 strains are geographically differentiated and appear to be endemic in Arizona, with two clades that have been circulating for four and seven years. This establishment in Maricopa County provides the first evidence of local overwintering by a WNV strain over the course of several years in Arizona. Within a national context, the placement of eleven contemporary Arizona strains in the NA/WN02 lineage indicates while WNV first entered the northeastern United States in 1999, the most ancestral extant strains of WNV are now circulating in the American southwest.

Cryptococcosis is an important fungal infection in immunocompromised individuals, especially those infected with HIV. In Brazil, despite the free availability of antiretroviral therapy (ART) in the public health system, the mortality rate due to Cryptococcus neoformans meningitis is still high. To obtain a more detailed picture of the population genetic structure of this species in southeast Brazil, we studied 108 clinical isolates from 101 patients and 35 environmental isolates. Among the patients, 59% had a fatal outcome mainly in HIV-positive male patients. All the isolates were found to be C. neoformans var. grubii major molecular type VNI and mating type locus alpha. Twelve were identified as diploid by flow cytometry, being homozygous (AαAα) for the mating type and by PCR screening of the STE20, GPA1, and PAK1 genes. Using the ISHAM consensus multilocus sequence typing (MLST) scheme, 13 sequence types (ST) were identified, with one being newly described. ST93 was identified from 81 (75%) of the clinical isolates, while ST77 and ST93 were identified from 19 (54%) and 10 (29%) environmental isolates, respectively. The southeastern Brazilian isolates had an overwhelming clonal population structure. When compared with populations from different continents based on data extracted from the ISHAM-MLST database (mlst.mycologylab.org) they showed less genetic variability. Two main clusters within C. neoformans var. grubii VNI were identified that diverged from VNB around 0.58 to 4.8 million years ago.

The emerging pathogen Cryptococcus gattii causes life-threatening disease in immunocompetent and immunocompromised hosts. Of the four major molecular types (VGI-VGIV), the molecular type VGIII has recently emerged as cause of disease in otherwise healthy individuals, prompting a need to investigate its population genetic structure to understand if there are potential genotype-dependent characteristics in its epidemiology, environmental niche(s), host range and clinical features of disease. Multilocus sequence typing (MLST) of 122 clinical, environmental and veterinary C. gattii VGIII isolates from Australia, Colombia, Guatemala, Mexico, New Zealand, Paraguay, USA and Venezuela, and whole genome sequencing (WGS) of 60 isolates representing all established MLST types identified four divergent sub-populations. The majority of the isolates belong to two main clades, corresponding either to serotype B or C, indicating an ongoing species evolution. Both major clades included clinical, environmental and veterinary isolates. The C. gattii VGIII population was genetically highly diverse, with minor differences between countries, isolation source, serotype and mating type. Little to no recombination was found between the two major groups, serotype B and C, at the whole and mitochondrial genome level. C. gattii VGIII is widespread in the Americas, with sporadic cases occurring elsewhere, WGS revealed Mexico and USA as a likely origin of the serotype B VGIII population and Colombia as a possible origin of the serotype C VGIII population. Serotype B isolates are more virulent than serotype C isolates in a murine model of infection, causing predominantly pulmonary cryptococcosis. No specific link between genotype and virulence was observed. Antifungal susceptibility testing against six antifungal drugs revealed that serotype B isolates are more susceptible to azoles than serotype C isolates, highlighting the importance of strain typing to guide effective treatment to improve the disease outcome.

BACKGROUND:Histoplasma capsulatum comprises a worldwide complex of saprobiotic fungi mainly found in nitrogen/phosphate (often bird guano) enriched soils. The microconidia of Histoplasma species may be inhaled by mammalian hosts, and is followed by a rapid conversion to yeast that can persist in host tissues causing histoplasmosis, a deep pulmonary/systemic mycosis. Histoplasma capsulatum sensu lato is a complex of at least eight clades geographically distributed as follows: Australia, Netherlands, Eurasia, North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B) and Africa. With the exception of the Eurasian cluster, those clades are considered phylogenetic species. METHODOLOGY/PRINCIPAL FINDINGS:Increased Histoplasma sampling (n = 234) resulted in the revision of the phylogenetic distribution and population structure using 1,563 aligned nucleotides from four protein-coding regions. The LAm B clade appears to be divided into at least two highly supported clades, which are geographically restricted to either Colombia/Argentina or Brazil respectively. Moreover, a complex population genetic structure was identified within LAm A clade supporting multiple monophylogenetic species, which could be driven by rapid host or environmental adaptation (~0.5 MYA). We found two divergent clades, which include Latin American isolates (newly named as LAm A1 and LAm A2), harboring a cryptic cluster in association with bats. CONCLUSIONS/SIGNIFICANCE:At least six new phylogenetic species are proposed in the Histoplasma species complex supported by different phylogenetic and population genetics methods, comprising LAm A1, LAm A2, LAm B1, LAm B2, RJ and BAC-1 phylogenetic species. The genetic isolation of Histoplasma could be a result of differential dispersion potential of naturally infected bats and other mammals. In addition, the present study guides isolate selection for future population genomics and genome wide association studies in this important pathogen complex.

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.

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

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.

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 tuberculosis complex 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. tuberculosis complex 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.

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.

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.

Case reports of Apophysomyces spp. in immunocompetent hosts have been a result of traumatic deep implantation of Apophysomyces spp. spore-contaminated soil or debris. On May 22, 2011 a tornado occurred in Joplin, MO, leaving 13 tornado victims with Apophysomyces trapeziformis infections as a result of lacerations from airborne material. We used whole genome sequence typing (WGST) for high-resolution phylogenetic SNP analysis of 17 outbreak Apophysomyces isolates and five additional temporally and spatially diverse Apophysomyces control isolates (three A. trapeziformis and two A. variabilis isolates). Whole genome SNP phylogenetic analysis revealed three clusters of genotypically related or identical A. trapeziformis isolates and multiple distinct isolates among the Joplin group; this indicated multiple genotypes from a single or multiple sources. Though no linkage between genotype and location of exposure was observed, WGST analysis determined that the Joplin isolates were more closely related to each other than to the control isolates, suggesting local population structure. Additionally, species delineation based on WGST demonstrated the need to reassess currently accepted taxonomic classifications of phylogenetic species within the genus Apophysomyces.

Burkholderia pseudomallei is a gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.

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.

Widespread resistance among circulating influenza A strains to at least one of the anti-influenza drugs is a major public health concern. A triple combination antiviral drug (TCAD) regimen comprised of amantadine, oseltamivir, and ribavirin has been shown to have synergistic and broad spectrum activity against influenza A strains, including drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the TCAD regimen on resistance in influenza in vitro, including: 1) serial passage at constant drug concentrations, 2) serial passage at escalating drug concentrations, and 3) evaluation of the contribution of each component of the TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in influenza A, treatment with the TCAD regimen resulted in the sustained suppression of drug resistant viruses, whereas treatment with amantadine alone or the amantadine-oseltamivir double combination led to the rapid selection of resistant variants which comprised ∼100% of the population. Furthermore, the TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each drug in the TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize combination drug regimens for the suppression of resistance may be broadly applicable to other infectious diseases.

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen.

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.

Whole-genome sequencing has advanced our understanding of the population structure of the pathogenic species complex Cryptococcus gattii, which has allowed for the phylogenomic specification of previously described major molecular type groupings and novel lineages. Recently, isolates collected in Mexico in the 1960s were determined to be genetically distant from other known molecular types and were classified as VGVI. We sequenced four clinical isolates and one veterinary isolate collected in the southwestern United States and Argentina from 2012 to 2021. Phylogenomic analysis groups these genomes with those of the Mexican VGVI isolates, expanding VGVI into a clade and establishing this molecular type as a clinically important population. These findings also potentially expand the known Cryptococcus ecological range with a previously unrecognized endemic area.

Antimicrobial resistance (AMR) in the nosocomial pathogen, Acinetobacter baumannii, is becoming a serious public health threat. While some mechanisms of AMR have been reported, understanding novel mechanisms of resistance is critical for identifying emerging resistance. One of the first steps in identifying novel AMR mechanisms is performing genotype/phenotype association studies; however, performing these studies is complicated by the plastic nature of the A. baumannii pan-genome. In this study, we compared the antibiograms of 12 antimicrobials associated with multiple drug families for 84 A. baumannii isolates, many isolated in Arizona, USA. in silico screening of these genomes for known AMR mechanisms failed to identify clear correlations for most drugs. We then performed a bacterial genome wide association study (bGWAS) looking for associations between all possible 21-mers; this approach generally failed to identify mechanisms that explained the resistance phenotype. In order to decrease the genomic noise associated with population stratification, we compared four phylogenetically-related pairs of isolates with differing susceptibility profiles. RNA-Sequencing (RNA-Seq) was performed on paired isolates and differentially-expressed genes were identified. In these isolate pairs, five different potential mechanisms were identified, highlighting the difficulty of broad AMR surveillance in this species. To verify and validate differential expression, amplicon sequencing was performed. These results suggest that a diagnostic platform based on gene expression rather than genomics alone may be beneficial in certain surveillance efforts. The implementation of such advanced diagnostics coupled with increased AMR surveillance will potentially improve A. baumannii infection treatment and patient outcomes.

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 (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.

Burkholderia pseudomallei is a bacterium endemic to Southeast Asia and northern Australia, but it has not been found to occur endemically in the United States. We report an ostensibly autochthonous case of melioidosis in the United States. Despite an extensive investigation, the source of exposure was not identified.