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Madagascar reports more human plague (causative agent: Yersinia pestis) cases annually than any other country (1), often several hundred each season (typically September-March) (2). Y. pestis persists in multiple rural [...]

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

Burkholderia thailandensis, a gram-negative bacterium found in the environment, poses a public health threat both because of its ability to cause infections as an opportunistic pathogen and potential misidentification as [...]

ABSTRACT Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and C. posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation was made available. In the current study, the genome of the Silveira strain was sequenced using Single Molecule Real Time Sequencing (SMRT) PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

Coccidioides posadasii is a pathogenic fungus that causes coccidioidomycosis in many arid regions of the Americas. One of these regions is bordered by the Caribbean Sea, and the surrounding landscape may play an important role in the dispersion of C. posadasii across South America through southeastern Mexico, Honduras, Guatemala, and Venezuela. Comparative phylogenomic analyses of C. posadasii reveal that clinical strains from Venezuela are genetically distinct from the North American populations found in (i) Arizona and (ii) Texas, Mexico, and the rest of South America (TX/MX/SA). We find evidence for admixture between the Venezuela and the North American populations of C. posadasii in Central America. Additionally, the proportion of Venezuelan alleles in the admixed population decreases as latitude (and distance from Venezuela) increases. Our results indicate that the population in Venezuela may have been subjected to a recent bottleneck and shows a strong population structure. This analysis provides insight into potential for Coccidioides spp. to invade new regions.IMPORTANCE Valley Fever is a fungal disease caused by two species of fungi: Coccidioides immitis and C. posadasii These fungi are found throughout the arid regions of North and South America; however, our understanding of genetic diversity and disease in South America is limited. In this report, we analyze 10 new genomes of Coccidioides posadasii from regions bordering the Caribbean Sea. We show that these populations are distinct and that isolates from Venezuela are likely a result of a recent bottleneck. These data point to patterns that might be observed when investigating recently established populations.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCRpositive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culturenegative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a gram-negative soil-dwelling bacterium and the causative agent of melioidosis (1). B. pseudomallei is endemic to tropical regions around the world (1), but its environmental distribution in [...]

Francisella tularensis, a Tier 1 select agent (7), has 3 subspecies: tularensis (type A), holarctica (type B), and mediasiatica (Appendix 1 Figure, https://wwwnc. cdc.gov/EID/article/25/5/18-0363-App1.pdf). In humans, disease is caused by [...]

Pathogen genome sequencing has become a routine part of our response to active outbreaks of infectious disease and should be an important part of our preparations for future epidemics. In this Essay, we discuss the innovations that have enabled routine pathogen genome sequencing, as well as how genome sequences can be used to understand and control the spread of infectious disease. We also explore the impact of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic on the field of pathogen genomics and outline the challenges we must address to further improve the utility of pathogen genome sequencing in the future. The SARS-CoV-2 pandemic had a major impact on the field of pathogen genomics. This Essay discusses innovations that have enabled routine pathogen genome sequencing, as well as how genome sequences can be used to understand and control the spread of infectious disease and discusses outstanding challenges to improve pathogen genome sequencing in the future. [ABSTRACT FROM AUTHOR]

Tularemia is a disease caused primarily by 2 subspecies of Francisella tularensis: F. tularensis subsp. tularensis, which is restricted to North America; and F. tularensis subsp. holarctica, which is found [...]

Because they are difficult to culture, obtaining genomic information from Leptospira spp. is challenging, hindering the overall understanding of leptospirosis. We designed and validated a culture-independent DNA capture and enrichment system for obtaining Leptospira genomic information from complex human and animal samples. It can be utilized with a variety of complex sample types and diverse species as it was designed using the pan-genome of all known pathogenic Leptospira spp. This system significantly increases the proportion of Leptospira DNA contained within DNA extracts obtained from complex samples, oftentimes reaching >95% even when some estimated starting proportions were <1%. Sequencing enriched extracts results in genomic coverage similar to sequenced isolates, thereby enabling enriched complex extracts to be analyzed together with whole genome sequences from isolates, which facilitates robust species identification and high-resolution genotyping. The system is flexible and can be readily updated when new genomic information becomes available. Implementation of this DNA capture and enrichment system will improve efforts to obtain genomic data from unculturable Leptospira-positive human and animal samples. This, in turn, will lead to a better understanding of the overall genomic diversity and gene content of Leptospira spp. that cause leptospirosis, aiding epidemiology and the development of improved diagnostics and vaccines. [ABSTRACT FROM AUTHOR]

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O. lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O. lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites. [ABSTRACT FROM AUTHOR]

Melioidosis is an underreported human disease of tropical and sub-tropical regions caused by the saprophyte Burkholderia pseudomallei. Although most global melioidosis cases are reported from tropical regions in Southeast Asia and northern Australia, there are multiple occurrences from sub-tropical regions, including the United States (U.S.). Most melioidosis cases reported from the continental U.S. are the result of acquiring the disease during travel to endemic regions or from contaminated imported materials. Only two human melioidosis cases from the continental U.S. have likely acquired B. pseudomallei directly from local environments and these cases lived only ~7 km from each other in rural Texas. In this study, we assessed the risk of acquiring melioidosis from the environment within the continental U.S. by surveying for B. pseudomallei in the environment in Texas where these two human melioidosis cases likely acquired their infections. We sampled the environment near the homes of the two cases and at additional sampling locations in surrounding counties in Texas that were selected based on ecological niche modeling. B. pseudomallei was not detected at the residences of these two cases or in the surrounding region. These negative data are important to demonstrate that B. pseudomallei is rare in the environment in the U.S. even at locations where locally acquired human cases likely have occurred, documenting the low risk of acquiring B. pseudomallei infection from the environment in the continental U.S. [ABSTRACT FROM AUTHOR]

The migratory behavior of wild birds contributes to the geographical spread of ticks and their microorganisms. In this study, we aimed to investigate the dispersal and co-occurrence of Francisella and spotted fever group Rickettsia (SFGR) in ticks infesting birds migrating northward in the African-Western Palaearctic region (AWPR). Birds were trapped with mist nests across the Mediterranean basin during the 2014 and 2015 spring migration. In total, 575 ticks were collected from 244 birds. We screened the ticks for the species Francisella tularensis, the genus Francisella, and SFGR by microfluidic real-time PCR. Confirmatory analyses and metagenomic sequencing were performed on tick samples that putatively tested positive for F. tularensis during initial screenings. Hyalomma rufipes was the most common tick species and had a high prevalence of Francisella, including co-occurrence of Francisella and SFGR. Metagenomic analysis of total DNA extracted from two H. rufipes confirmed the presence of Francisella, Rickettsia, and Midichloria. Average nucleotide identity and phylogenetic inference indicated the highest identity of the metagenome-assembled genomes to a Francisella-like endosymbiont (FLE), Rickettsia aeschlimannii, and Midichloria mitochondrii. The results of this study suggest that (i) FLE- and SFGR-containing ticks are dispersed by northbound migratory birds in the AWPR, (ii) H. rufipes likely is not involved in transmission of F. tularensis in the AWPR, and (iii) a dual endosymbiosis of FLEs and Midichloria may support some of the nutritional requirements of H. rufipes. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology. Methodology/Principal findings: We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup. Conclusions/Significance: Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics. Author summary: Leptospirosis is a common zoonotic disease worldwide, but more prevalent in the tropics. Cases are more common following severe weather events, possibly due to flooding, which may more readily distribute soil and/or water contaminated with Leptospira spp., the disease agents. Human cases increased following the 2017 hurricanes that ravaged Puerto Rico (Maria and Irma), prompting environmental sampling of soil and water to assess the presence, abundance, and possible persistence of pathogenic leptospires in these environments. The goal was to better understand these potential reservoirs of human and animal disease. Diverse and novel groups of pathogenic Leptospira were abundant and widespread in soil and water in Puerto Rico and sometimes were consistently detected in these environments for >1 year. However, most groups we identified have not previously been described from humans and/or other animals, so the disease potential of these novel organisms is unknown. The results of this study reveal a tremendous amount of previously uncharacterized Leptospira diversity in soil and water in Puerto Rico. The description and characterization of these novel types improves our understanding of the genus Leptospira, and will aid in the development of improved diagnostics and preventative tools to advance public health outcomes. [ABSTRACT FROM AUTHOR]

Melioidosis, an infection caused by Burkholderia pseudomallei , has a very high risk of mortality when treated, with an even higher risk of fatality if undiagnosed or not treated appropriately. It is endemic to Asia, Australia, South America, and the Caribbean; however, the number of melioidosis cases reported in the United States has been increasing. Therefore, physicians should be aware of this clinical entity and its possible presentations. Mycotic aneurysms due to B. pseudomallei are extremely rare, accounting for ~1%–2% of cases. Here we describe a rare case of melioidosis presenting as a mycotic aneurysm in the United States, highlight the potential for diagnostic challenges and epidemiologic concerns, and provide a review of mycotic aneurysm cases due to B. pseudomallei published to date. [ABSTRACT FROM AUTHOR]

Background Pneumonic plague (PP), caused by Yersinia pestis , is the most feared clinical form of plague due to its rapid lethality and potential to cause outbreaks. PP outbreaks are now rare due to antimicrobial therapy. Methods A PP outbreak in Madagascar involving transmission of a Y. pestis strain resistant to streptomycin, the current recommended first-line treatment in Madagascar, was retrospectively characterized using epidemiology, clinical diagnostics, molecular characterization, and animal studies. Results The outbreak occurred in February 2013 in the Faratsiho district of Madagascar and involved 22 cases, including 3 untreated fatalities. The 19 other cases participated in funeral practices for the fatal cases and fully recovered after combination antimicrobial therapy: intramuscular streptomycin followed by oral co-trimoxazole. The Y. pestis strain that circulated during this outbreak is resistant to streptomycin resulting from a spontaneous point mutation in the 30S ribosomal protein S12 (rpsL) gene. This same mutation causes streptomycin resistance in 2 unrelated Y. pestis strains, one isolated from a fatal PP case in a different region of Madagascar in 1987 and another isolated from a fatal PP case in China in 1996, documenting this mutation has occurred independently at least 3 times in Y. pestis. Laboratory experiments revealed this mutation has no detectable impact on fitness or virulence, and revertants to wild-type are rare in other species containing it, suggesting Y. pestis strains containing it could persist in the environment. Conclusions Unique antimicrobial resistant (AMR) strains of Y. pestis continue to arise in Madagascar and can be transmitted during PP outbreaks. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796–1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1–2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei. Author summary: Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. The evaluation of diversity within B. pseudomallei has largely been conducted utilizing clinical isolates despite almost all infections emerging from environmental exposure. In this study, we surveyed the genomic diversity of 169 isolates collected from a single soil sample in Ubon Ratchathani Province, Thailand. Seven different sequence types were identified, and substantial within-sequence-type gene diversity was observed. To test for differential virulence, 30 isolates were challenged in a mouse melioidosis model. A small number of isolates killed all mice, but most killed none, demonstrating the variable virulence potential of different B. pseudomallei isolates present in the single sample. A comparative genomics analysis identified multiple genes associated with virulence, including Hcp1, a component of the type VI secretion system and a known virulence factor. The results of this study have implications for the comprehensive environmental surveillance, environmental exposure, and differential virulence of B. pseudomallei. [ABSTRACT FROM AUTHOR]

Plague is a re‐emerging zoonotic disease and a major public health concern in several portions of the world, especially in Madagascar. We report on the presence of different subtypes of Yersinia pestis co‐occurring in the same locality. After confirmation of a human plague case in Ambohitromby Commune (Ankazobe District) via isolation of Y. pestis, we undertook small mammal trapping to identify the circulation of Y. pestis amongst rodents in this locality; blood samples were collected from rodents for seroprevalence analysis. Of the 60 individuals of Rattus rattus captured, one yielded an isolate of Y. pestis, 13 others were positive for F1 antigen of Y. pestis using a rapid diagnostic test, and 4 were PCR positive targeting the caf1 and pla genes; 28/60 (46.7%) of the captured R. rattus were seropositive for Y. pestis. Whole‐genome SNP analyses revealed that the two isolates obtained from the human case, and the R. rattus belonged to two different subtypes of Y. pestis (s05 and s13, respectively) that were circulating concurrently in Ambohitromby in 2016. Three Y. pestis subtypes (s03, s05 and s13) have now been isolated from Ambohitromby. Subtype s05 had been persisting there for >10 years but one or both of the other subtypes may have been introduced from the Central Highlands region as they were not observed in previous years (s13) or only observed once previously (s03). High seroprevalence against Y. pestis in R. rattus suggests that a portion of the local murine population may have acquired resistance to Y. pestis. Future research should focus on genomically characterizing Y. pestis strains circulating in Ankazobe District and other plague‐endemic regions of Madagascar to better understand the overall phylogeography of Y. pestis. [ABSTRACT FROM AUTHOR]

Yersinia pestis, causative agent of plague, occurs throughout the western United States in rodent populations and periodically causes epizootics in susceptible species, including black‐tailed prairie dogs (Cynomys ludovicianus). How Y. pestis persists long‐term in the environment between these epizootics is poorly understood but multiple mechanisms have been proposed, including, among others, a separate enzootic transmission cycle that maintains Y. pestis without involvement of epizootic hosts and persistence of Y. pestis within epizootic host populations without causing high mortality within those populations. We live‐trapped and collected fleas from black‐tailed prairie dogs and other mammal species from sites with and without black‐tailed prairie dogs in 2004 and 2005 and tested all fleas for presence of Y. pestis. Y. pestis was not detected in 2126 fleas collected in 2004 but was detected in 294 fleas collected from multiple sites in 2005, before and during a widespread epizootic that drastically reduced black‐tailed prairie dog populations in the affected colonies. Temporal and spatial patterns of Y. pestis occurrence in fleas and genotyping of Y. pestis present in some infected fleas suggest Y. pestis was introduced multiple times from sources outside the study area and once introduced, was dispersed between several sites. We conclude Y. pestis likely was not present in these black‐tailed prairie dog colonies prior to epizootic activity in these colonies. Although we did not identify likely enzootic hosts, we found evidence that deer mice (Peromyscus maniculatus) may serve as bridging hosts for Y. pestis between unknown enzootic hosts and black‐tailed prairie dogs. [ABSTRACT FROM AUTHOR]

Abstract Background Acinetobacter baumannii has recently emerged as a significant global pathogen, with a surprisingly rapid acquisition of antibiotic resistance and spread within hospitals and health care institutions. This study examines the genomic content of three A. baumannii strains isolated from distinct body sites. Isolates from blood, peri-anal, and wound sources were examined in an attempt to identify genetic features that could be correlated to each isolation source. Results Pulsed-field gel electrophoresis, multi-locus sequence typing and antibiotic resistance profiles demonstrated genotypic and phenotypic variation. Each isolate was sequenced to high-quality draft status, which allowed for comparative genomic analyses with existing A. baumannii genomes. A high resolution, whole genome alignment method detailed the phylogenetic relationships of sequenced A. baumannii and found no correlation between phylogeny and body site of isolation. This method identified genomic regions unique to both those isolates found on the surface of the skin or in wounds, termed colonization isolates, and those identified from body fluids, termed invasive isolates; these regions may play a role in the pathogenesis and spread of this important pathogen. A PCR-based screen of 74 A. baumanii isolates demonstrated that these unique genes are not exclusive to either phenotype or isolation source; however, a conserved genomic region exclusive to all sequenced A. baumannii was identified and verified. Conclusions The results of the comparative genome analysis and PCR assay show that A. baumannii is a diverse and genomically variable pathogen that appears to have the potential to cause a range of human disease regardless of the isolation source.

Of the seven currently known botulinum neurotoxin-producing species of Clostridium , C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes. [ABSTRACT FROM AUTHOR]

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host. Author summary: Pathogens frequently jump between different hosts, and associated adaptation may lead to the emergence of new infectious agents. Such host-jumping evolution is witnessed through endpoint analyses but these cannot capture genetic changes in lineages that have gone extinct. In this study, we have identified and monitored an example of the adaptive evolution of a bacterium often deadly to its mammalian host, in an unprecedented case whereby disease lessened through time and the pathogen adapted to become a part of the commensal human flora. We used genomic analyses to characterize more than 16 years of this evolutionary process and the stepwise mutations that control pathogen interactions with the patient. Soon after infection, mutational changes occurred that allowed the bacterium to remain in the airways without causing disease. This shift towards avirulence was determined based on clinical data and virulence testing in an animal model. In addition, mutations occurred that contributed to the persistence of the bacteria in the patient's lungs. Finally, we found evidence for the evolutionary emergence and persistence of two distinct lineages of the bacterium over the last 13 years, presenting interesting questions about niche utilization. Bacteria are ubiquitous in the human body and almost all are beneficial or benign. In this study, we document the evolutionary conversion of a normally deadly bacterium towards a commensal. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species. [ABSTRACT FROM AUTHOR]

Onchocerca lupi, Rodonaja 1967, is an emerging, zoonotic filarial nematode parasite that causes ocular disease in dogs, cats, wild canids, and humans. It is the causative agent of ocular onchocercosis in canines with increasing incidence in both North America and the Old World during the early twenty-first century. We report the complete mitochondrial genome of an O. lupi isolate from a dog from Arizona, southwestern USA, and its genetic differentiation from related Onchocerca species. The whole mitochondrial genome was obtained from whole genome sequencing of genomic DNA isolated from an adult worm. This mitogenome is 13,766 bp in size and contains 36 genes and a control region. This mitogenome provides a valuable resource for future studies involving epidemiological surveillance, population genetics, phylogeography, and comparative mitogenomics of this emerging pathogen and other parasitic nematodes. [ABSTRACT FROM AUTHOR]

Comparison between spectroscopy, size-restricted spectroscopy and quantitative binding approaches were performed for normalization of large, multiplex amplicon pools for performance and efficiency. The quantitative binding approach showed superiority compared to other approaches and represented an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included.

A single software platform, mothur, is used to trim, screen, and align sequences, calculate distances, assign sequences to operational taxonomic units and also to describe the [alpha] and [beta] diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. The analysis of the huge sequence is completed in less than 2 hours with a laptop computer.

16S rRNA gene sequence analysis is used for characterizing the microbial diversity of septic tank effluent (STE) and the biomat that is formed due to STE infiltration on soil. The results have shown that microbial communities are different within control soil, STE, and the biomat and that microbes found in STE are not found in the biomat.

A phylogenetic comparison of microbial communities in hypersaline evaporites is conducted on crusts by using culture-dependent rRNA gene sequence analysis. Many sequences are shared between evaporites, showing that similar environments select for specific microbial lineages from a global metacommunity.

A microbial community analysis using 16S rRNA gene sequencing is performed on borehole water and granite rock core from Henderson Mine near Empire, Colorado. The studies have shown that geochemical variance has a key impact on microbial community structure in deep, subsurface systems.

Background: Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated. Methodology/Principal findings: We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp. Conclusions/Significance: B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America. [ABSTRACT FROM AUTHOR]

The global dissemination of Klebsiella pneumoniae and Klebsiella pneumoniae carbapenemase (KPC) has been largely attributed to a few high-risk sequence types (STs) (ST258, ST11, ST512) associated with human disease. ST101 is an emerging clone that has been identified in different parts of the world with the potential to become a global, persistent public health threat. Recent research suggests the ST101 lineage is associated with an 11% increase in mortality rate in comparison to non-ST101 infections. In this study, we generated a high-quality, near-finished genome assembly of a multidrug-resistant (MDR) isolate from Italy (isolate 4743) that is a single locus variant of ST101 (ST1685). We demonstrate that the 4743 genome contains virulence features such as an integrative conjugative element carrying the yersiniabactin siderophore (ICEKp3), the mannose-resistant Klebsiella -like (type III) fimbriae cluster (mrkABCDFHIJ), the ferric uptake system (kfuABC), the yersiniabactin receptor gene fyuA , a capsular K type K17, and an O antigen type of O1. K. pneumoniae 4743 carries the bla KPC-2 carbapenemase gene along with genes conferring resistance to aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, macrolides, lincosamides, and streptogramin B. A comparative genomics analysis of 44 ST101 genomes as well as newly sequenced isolate 4743 identified variable antimicrobial resistance (AMR) resistance profiles and incompatibility plasmid types, but similar virulence factor profiles. Using Bayesian methodologies, we estimate the common ancestor for the ST101 lineage emerged in 1990 (95% HPD: 1965 to 2007) and isolates within the lineage acquired bla KPC after the divergence from its parental clonal group and dissemination. The identification of virulence factors and antibiotic resistance genes acquired by this newly emerging clone provides insight into the reported increased mortality rates and highlights its potential success as a persistent nosocomial pathogen. With a combination of both colistin resistance, carbapenem resistance, and several known virulence factors, the ST101 genetic repertoire may be a "perfect storm" allowing for a newly emerging, high-risk, extensively antibiotic resistant clone. This high-risk clone appears adept at acquiring resistance and may perpetuate the dissemination of extensive antimicrobial resistance. Greater focus on the acquisition of virulence factors and antibiotic resistance genes is crucial for understanding the spread of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Yersinia pestis was introduced to Brazil during the third plague pandemic and currently exists in several recognized foci. There is currently limited available phylogeographic data regarding Y. pestis in Brazil. We generated whole genome sequences for 411 Y. pestis strains from six Brazilian foci to investigate the phylogeography of Y. pestis in Brazil; these strains were isolated from 1966 to 1997. All 411 strains were assigned to a single monophyletic clade within the 1.ORI population, indicating a single Y. pestis introduction was responsible for the successful establishment of endemic foci in Brazil. There was a moderate level of genomic diversity but little population structure among the 411 Brazilian Y. pestis strains, consistent with a radial expansion wherein Y. pestis spread rapidly from the coast to the interior of Brazil and became ecologically established. Overall, there were no strong spatial or temporal patterns among the Brazilian strains. However, strains from the same focus tended to be more closely related and strains isolated from foci closer to the coast tended to fall in more basal positions in the whole genome phylogeny than strains from more interior foci. Overall, the patterns observed in Brazil are similar to other locations affected during the 3rd plague pandemic such as in North America and Madagascar. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the environmental bacillus that causes melioidosis; a disease clinically significant in Australia and Southeast Asia but emerging in tropical and sub-tropical regions around the globe. Previous studies have placed the ancestral population of the organism in Australia with a single lineage disseminated to Southeast Asia. We have previously characterized B. pseudomallei isolates from New Guinea and the Torres Strait archipelago; remote regions that share paleogeographic ties with Australia. These studies identified regional biogeographical boundaries. In this study, we utilize whole-genome sequencing to reconstruct ancient evolutionary relationships and ascertain correlations between paleogeography and present-day distribution of this bacterium in Australasia. Our results indicate that B. pseudomallei from New Guinea fall into a single clade within the Australian population. Furthermore, clades from New Guinea are region-specific; an observation possibly linked to limited recent anthropogenic influence in comparison to mainland Australia and Southeast Asia. Isolates from the Torres Strait archipelago were distinct yet scattered among those from mainland Australia. These results provide evidence that the New Guinean and Torres Strait lineages may be remnants of an ancient portion of the Australian population. Rising sea levels isolated New Guinea and the Torres Strait Islands from each other and the Australian mainland, and may have allowed long-term isolated evolution of these lineages, providing support for a theory of microbial biogeography congruent with that of macro flora and fauna. Moreover, these findings indicate that contemporary microbial biogeography theories should consider recent and ongoing impacts of globalisation and human activity. [ABSTRACT FROM AUTHOR]

Asymptomatic colonization with extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae has been described for humans, various mammal species, and birds. Here, antimicrobial resistant bacteria were recovered from dog feces originating in Germany, Kosovo, Afghanistan, Croatia, and Ukraine, with a subset of mostly E. coli isolates obtained from a longitudinal collection over twelve months. In vitro antimicrobial resistance testing revealed various patterns of resistance against single or all investigated beta-lactam antibiotics, with none of the 101 isolates resistant against two tested carbapenem antibiotics. Whole genome sequence analysis revealed bacteria species-specific patterns for 23 antimicrobial resistance coding DNA sequences (CDS) that were unapparent from the in vitro analysis alone. Phylogenetic analysis of single nucleotide polymorphisms (SNP) revealed clonal bacterial isolates originating from different dogs, suggesting transmission between dogs in the same community. However, individual resistant E. coli clones were not detected over a period longer than seven days. Multi locus sequence typing (MLST) of 85 E. coli isolates revealed 31 different sequence types (ST) with an accumulation of ST744 (n = 9), ST10 (n = 8), and ST648 (n = 6), although the world-wide hospital-associated CTX-M beta-lactamase producing ST131 was not detected. Neither the antimicrobial resistance CDSs patterns nor the phylogenetic analysis revealed an epidemiological correlation among the longitudinal isolates collected from a period longer than seven days. No genetic linkage could be associated with the geographic origin of isolates. In conclusion, healthy dogs frequently carry ESBL-producing bacteria, independent to prior treatment, which may be transmitted between individual dogs of the same community. Otherwise, these antimicrobial resistant bacteria share few commonalities, making their presence eerily unpredictable. [ABSTRACT FROM AUTHOR]

Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism’s biological potential, the proteins expressed provide a functional readout of the organism’s response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples. [ABSTRACT FROM AUTHOR]