Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird.

St. Louis Encephalitis Virus (SLEV) has been seasonally detected within the Culex spp . populations within Maricopa County, Arizona and Coachella Valley, California since an outbreak in Maricopa County in 2015. Previous work revealed that the outbreak was caused by an importation of SLEV genotype III, which had only been detected within Argentina in prior years. However, little is known about when the importation occurred or the population dynamics since its arrival into the southwestern United States. In this study, we wanted to determine if the annual detection of SLEV in Maricopa and Riverside counties is due to enzootic cycling or new importations. To address this question, we analyzed 143 SLEV genomes (138 sequenced as part of this study) using the Bayesian phylogenetic analysis software, BEAST, to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Arizona and California form two distinct populations with little evidence of transmission among the two populations since the onset of the outbreak. Interestingly, the SLEV variants in Coachella Valley appear to be annually imported from a nearby source, whereas the Arizona population is locally sourced each year. Finally, the earliest representatives of SLEV genotype III in the southwestern US formed a polytomy that includes both California and Arizona samples. We propose that the initial outbreak could have resulted from an introductory population of SLEV, perhaps in one or more bird flocks migrating north in 2013, rather than a single variant introduced by one bird.

Due to the large number of negative tests, individually screening large populations for rare pathogens can be wasteful and expensive. Sample pooling methods improve the efficiency of large-scale pathogen screening campaigns by reducing the number of tests and reagents required to accurately categorize positive and negative individuals. Such methods rely on group testing theory which mainly focuses on minimizing the total number of tests; however, many other practical concerns and tradeoffs must be considered when choosing an appropriate method for a given set of circumstances. Here we use computational simulations to determine how several theoretical approaches compare in terms of (a) the number of tests, to minimize costs and save reagents, (b) the number of sequential steps, to reduce the time it takes to complete the assay, (c) the number of samples per pool, to avoid the limits of detection, (d) simplicity, to reduce the risk of human error, and (e) robustness, to poor estimates of the number of positive samples. We found that established methods often perform very well in one area but very poorly in others. Therefore, we introduce and validate a new method which performs fairly well across each of the above criteria making it a good general use approach.

Dental caries, also known as cavities or dental decay, effects children at a rate five times higher than asthma in the United States. This disease is highly preventable but causes extreme pain and costs millions of dollars to treat every year. The presence of Streptococcus mutansand Streptococcus sobrinusplays a major role in the development and progression of tooth decay; therefore, it is important to establish colonization of these bacteria to assess the risk of developing the disease. Streptococcus bacteria are difficult to grow, extract, and sequence due to strict necessary growth conditions. In this study, we evaluated the performance of different storage and culturing protocols and developed strategies for reducing interference by unwanted bacterial and fungal genomes when sequencing extracted samples. We compared storage conditions of samples at various temperatures, and with and without glycerol. We decided the best storage method was at −80 °C in a specialized solution known as Aimes. When sequencing cultures, we encountered various unwanted bacterial and fungal genomes. To reduce this, we modified our culturing methods by including growth in anaerobic conditions and using serial isolation streaking. These modifications have limited the growth of aerobic specimens and increased culture purity before extraction. With this study, we will be able to better understand the oral microbiome and aim to identify virulence factors in S. mutans and S. sobrinus that contribute to the high rates of dental caries in children.

Staphylococcus aureus is the leading cause of soft tissue and skin infection, but also causes pneumonia, endocarditis, and bone infection. The same infections can be caused by methicillin resistant S. aureus (MRSA), but are much more difficult to treat because MRSA is highly resistant to antibiotics. In ethnic and socioeconomic minority groups, methicillin resistance in S. aureus is 30 % higher than the general population. As a result, methicillin resistance is likely to have higher prevalence in jails and prisons where the demographic is disproportionately represented by ethnic minorities, individuals with lower average socioeconomic status, and less access to healthcare. Jail inmates are an understudied but very important population because diseases acquired in jails can be easily introduced into the general population and vice versa because cycling into and out of jail is very common. The presence of S. aureus is an important determinant of the development of soft tissue and other infections, making the detection of this bacteria and the identification of antibiotic resistance essential in assessing risk of disease. In this study, we identified S. aureus in samples from a United States county jail through bacterial plating and DNA extraction. Methicillin resistance was identified in S. aureus positive samples through PCR and sequencing. Our analysis has found elevated rates of S. aureus in the jail population being studied. This project will determine if rates of S. aureus and methicillin resistance in S. aureus isolated from this demographic are disproportionate to the general population.

Bats and their associated guano microbiota provide important terrestrial and subterranean ecosystem services and serve as a reservoir for a wide range of epizootic and zoonotic diseases. Unfortunately, large-scale studies of bats and their guano microbiotas are limited by the time and cost of sample collection, which requires specially trained individuals to work at night to capture bats when they are most active. Indirectly surveying bat gut microbiota through guano deposits could be a more cost-effective alternative, but it must first be established whether the postdefecation exposure to an aerobic environment has a large impact on the guano microbial community. A number of recent studies on mammalian feces have shown that the impact of aerobic exposure is highly species specific; therefore, it is difficult to predict how exposure will affect the bat guano microbiota without empirical data. In our study, we collected fresh guano samples from 24 individuals of 10 bat species that are common throughout the arid environments of the American southwest and subjected the samples to 0, 1, and 12hr of exposure. The biodiversity decreased rapidly after the shift from an anaerobic to an aerobic environmentmuch faster than previously reported in mammalian species. However, the relative composition of the core guano microbiota remained stable and, using highly sensitive targeted PCR methods, we found that pathogens present in the original, non-exposed samples could still be recovered after 12hr of exposure. These results suggest that with careful sample analysis protocols, a more efficient passive collection strategy is feasible; for example, guano could be collected on tarps placed near the roost entrance. Such passive collection methods would greatly reduce the cost of sample collection by allowing more sites or roosts to be surveyed with a fraction of trained personnel, time, and effort investments needed.

BACKGROUND:Targeted PCR amplicon sequencing (TAS) techniques provide a sensitive, scalable, and cost-effective way to query and identify closely related bacterial species and strains. Typically, this is accomplished by targeting housekeeping genes that provide resolution down to the family, genera, and sometimes species level. Unfortunately, this level of resolution is not sufficient in many applications where strain-level identification of bacteria is required (biodefense, forensics, clinical diagnostics, and outbreak investigations). Adding more genomic targets will increase the resolution, but the challenge is identifying the appropriate targets. VaST was developed to address this challenge by finding the minimum number of targets that, in combination, achieve maximum strain-level resolution for any strain complex. The final combination of target regions identified by the algorithm produce a unique haplotype for each strain which can be used as a fingerprint for identifying unknown samples in a TAS assay. VaST ensures that the targets have conserved primer regions so that the targets can be amplified in all of the known strains and it also favors the inclusion of targets with basal variants which makes the set more robust when identifying previously unseen strains.RESULTS:We analyzed VaST's performance using a number of different pathogenic species that are relevant to human disease outbreaks and biodefense. The number of targets required to achieve full resolution ranged from 20 to 88% fewer sites than what would be required in the worst case and most of the resolution is achieved within the first 20 targets. We computationally and experimentally validated one of the VaST panels and found that the targets led to accurate phylogenetic placement of strains, even when the strains were not a part of the original panel design.CONCLUSIONS:VaST is an open source software that, when provided a set of variant sites, can find the minimum number of sites that will provide maximum resolution of a strain complex, and it has many different run-time options that can accommodate a wide range of applications. VaST can be an effective tool in the design of strain identification panels that, when combined with TAS technologies, offer an efficient and inexpensive strain typing protocol.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

Recent studies have demonstrated diversity in the lung microbiomes of chronic obstructive pulmonary disease and healthy individuals. Lung microbial communities may not just serve as a predictor of cancer development, but also as a target of pharmacological cancer prevention strategies. We sought to characterize the lung microbiome diversity within patients with lung cancer for comparison to those with other cancers and those without cancer. Signed informed consent was obtained from patients ages ≥18 years undergoing a clinically indicated bronchoscopy. A bronchial lavage (BAL) was collected for research purposes after completing routine bronchoscopic procedures. Samples were prepped and DNA was extracted and 515F/806R 16S rRNA primers used to amplify Variable Region 4. Amplicons were sequenced and grouped into 100% operational taxonomic units (OTUs) using vsearch. Taxonomy was assigned, a phylogenetic tree was constructed, and sequences aligned for phylogenetic diversity calculations, including Faith's Phylogenetic Diversity and weighted and unweighted UniFrac. OTUs that were significantly different across sample categories were identified using DESeq2. There were 137 unique BAL samples collected. One patient had an adverse research procedure event that resolved after temporary supplemental oxygen and overnight observation. BALs were from 68 non-small cell lung cancer (NSCLC), 12 small cell lung cancer (SCLC), 52 other cancers, and 5 non-cancer patients. 58 NSCLC were current/former smokers (average 43 pack-years), while all the SCLC were current/former smokers (average 56 pack-years). 22 other cancers were current/former smokers (average of 27 pack-years). Overall, 51 samples (37.2%) had sufficient sequencing reads (>20,000) for subsequent analyses. There were multiple bacterial taxonomic groups in each sample, however, phylogenetic diversity was low compared to other body sites. There were no statistical differences in alpha/beta diversity between ever-smokers and never-smokers, NSCLC vs SCLC, lung cancer vs non-cancer, and location of BAL collection (upper vs lower airways and right vs left lung). There were a number of statistically significant differences by taxonomy (False Discovery Rate adjusted p<0.01 and listing genus only). Adenocarcinoma vs non-adenocarcinoma had more Streptococcus and Veillonella; less Haemophilus. For NSCLC vs SCLC, Rothia was more prevalent in SCLC. BALs from the upper airways had more prevalent Streptococcus. BALs from the right lung had more prevalent Capnocytophaga and Parvimonas; less Moraxella and Selenomonas. We report significant taxonomic differences by tumor type and location of BAL sampling and overall low phylogenetic diversity. Future validation of this work can be used to modify bacterial colonization in a lung cancer prevention strategy or for early diagnostics/therapeutics.

The insertion sequence IS1111 is used for molecular detection of Coxiella burnetii but also found in Coxiella -like endosymbionts of ticks, presenting a risk of false positive detection of C. burnetii . Limited IS1111 sequences from Coxiella -like bacteria restrict in silico assessment of IS1111 assays. However, Coxiella -like bacteria detectable by IS1111 assays appear to be rare in tick populations, limiting the impact of false positives on C. burnetii prevalence estimations. C. burnetii can be distinguished from Coxiella -like bacteria using C. burnetii SNP genotyping assays. Such assays can be used for detection, but are best used as post hoc tests given the extreme sensitivity of assays that target the multiple copy IS1111. Recently, Duron (2015) demonstrated the presence of IS1111 in Coxiella -like endosymbionts of ticks. This work adds to our knowledge of sequence diversity within IS1111 from Coxiella -like endosymbionts. Duron et al . (2015) also show that Coxiella -like endosymbionts are widespread among tick species. Detection assays for the Q fever pathogen, C. burnetii , often target portions of IS1111 (Kim et al . 2005; Klee et al . 2006; Loftis et al . 2006; Panning et al . 2008; de Bruin et al . 2011); the repetitive nature of IS1111 provides multiple targets for primer annealing, resulting in unparalleled detection sensitivity. Given the presence of IS1111 in Coxiella -like bacteria, Duron and colleagues (Duron 2015 …