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Understanding soil microbiome responses to different land‐management strategies provides insights into tallgrass prairie restoration. However, few studies have examined these interactions. We addressed this issue by surveying samples from the University of Nebraska at Omaha's Glacier Creek Preserve, where a long‐term burn experiment was established in 1982, with the mow treatments incorporated in 2002. In 2015, we collected 89 soil samples and obtained ~2.3 million bacterial and archaeal 16S rRNA sequence reads and ~2.4 million fungal ITS reads. Analysis of the sequences showed that dominant phyla were Proteobacteria (~30.0%) and Acidobacteria (~22.8%) in bacteria and Ascomycota (~66.2%) in fungi. We found that burning and mowing had a distinct effect on soil bacterial and fungal diversity: soils exposed to the burn treatments possessed a relatively high bacterial diversity but a relatively low fungal diversity. We also observed that burning and mowing had different effects on different microbial groups within bacteria and fungi. The burn treatments led to a relatively high abundance of bacterial groups such as Gemmatimonadetes and Armatimonadetes that prefer dry or warmer soils and arbuscular mycorrhizal (AM) fungi, whereas the mow treatments resulted in increased abundances of Nitrospirae bacteria. Moreover, functional prediction revealed that soil samples from the burn treatments were enriched with AM and plant saprotrophic fungi, and fungal plant pathogens were enriched in the mow samples. Considering soil functional differences between burn and mow treatments and an increased plant production after burning treatments, we speculate that burning could be a better strategy in the tallgrass land restoration, with certain fungal groups as an imperative indicator. HIGHLIGHTS: Understanding soil microbiome responses to different land‐management strategies provides insights into tallgrass prairie restoration We showed differences in the soil bacterial and fungal communities between burn and mow treatments. Soil fungi were enriched in AM fungi and plant saprotroph functions after burning, but fungi with putative plant pathogens after mowing. The burn treatment is a better strategy for tallgrass prairie land restoration in this study region.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Background: Enterococcus faecium is a major cause of clinical infections, often due to multidrug-resistant (MDR) strains. Whole genome sequencing (WGS) is a powerful tool to study MDR bacteria and their antimicrobial resistance (AMR) mechanisms. Here we use WGS to characterize E. faecium clinical isolates and test the feasibility of rules-based genotypic prediction of AMR. Methods: Clinical isolates were divided into derivation and validation sets. Phenotypic susceptibility testing for ampicillin, vancomycin, high-level gentamicin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, and linezolid was performed using the VITEK 2 automated system, with confirmation and discrepancy resolution by broth microdilution, disk diffusion, or gradient diffusion when needed. WGS was performed to identify isolate lineage and AMR genotype. AMR prediction rules were derived by analyzing the genotypic-phenotypic relationship in the derivation set. Results: Phylogenetic analysis demonstrated that 88% of isolates in the collection belonged to hospital-associated clonal complex 17. Additionally, 12% of isolates had novel sequence types. When applied to the validation set, the derived prediction rules demonstrated an overall positive predictive value of 98% and negative predictive value of 99% compared to standard phenotypic methods. Most errors were falsely resistant predictions for tetracycline and doxycycline. Further analysis of genotypic-phenotypic discrepancies revealed potentially novel pbp5 and tet(M) alleles that provide insight into ampicillin and tetracycline class resistance mechanisms. The prediction rules demonstrated generalizability when tested on an external dataset. Conclusions: Known AMR genes and mutations can predict E. faecium phenotypic susceptibility with high accuracy for most routinely tested antibiotics, providing opportunities for advancing molecular diagnostics.

Background Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations. Results We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities. Conclusions The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa.

Vaginal microbiota composition affects several important reproductive health outcomes. Lactobacillus crispatus-dominant bacterial communities have favorable associations whereas anaerobe-dominant communities deficient of lactobacilli are linked to poor outcomes, including bacterial vaginosis (BV). Lactobacillus iners, the most abundant vaginal species worldwide, has adverse associations compared to L. crispatus, but standard metronidazole treatment for BV promotes L. iners-dominance, likely contributing to post-treatment relapse. L. iners is under-studied because it fails to grow in standard Lactobacillus media in vitro. Here we trace this in vitro phenotype to a species-specific cysteine requirement associated with limitations in cysteine-related transport mechanisms and show that vaginal cysteine concentrations correlate with Lactobacillus abundance in vivo. We demonstrate that cystine uptake inhibitors selectively impede L. iners growth and that combining an inhibitor with metronidazole thus promotes L. crispatus dominance of defined BV-like communities. These findings identify a novel target for therapeutic vaginal microbiota modulation to improve reproductive health.

Background Cervicovaginal microbiota domination by Lactobacillus crispatus is associated with beneficial health outcomes, whereas L. iners dominance has more adverse associations. However bacterial vaginosis (BV) treatment with metronidazole (MTZ) typically leads to domination by L. iners rather than L. crispatus. L. iners differs from other lactobacilli by its inability to grow in MRS media. We hypothesized that exploring this growth difference would identify targets for selective L. iners inhibition. Methods Bacteria were grown anaerobically. Nutrient uptake and metabolism were assessed using UPLC-MS/MS and isotopically labeled substrates. Bacterial genome annotation employed Prodigal, Roary, and EggNOG. Competition experiments with mock mixed communities were analyzed by 16S rRNA gene sequencing. We confirmed result generalizability using a diverse collection of South African and North American strains and genomes. Results Supplementing MRS broth with L-cysteine (Cys) or L-cystine permitted robust L. iners growth, while L. crispatus grew without Cys supplementation. Despite their different growth requirements, neither species could synthesize Cys via canonical pathways. Adding the cystine uptake inhibitors S-methyl-L-cysteine (SMC, Fig 1) or seleno-DL-cystine (SDLC) blocked growth of L. iners but not other lactobacilli, suggesting L. iners lacks mechanisms other lactobacilli use to exploit complex exogenous Cys sources. Notably, cydABCD, an operon with Cys/glutathione transport and redox homeostasis activities, is absent from L. iners but present in non-iners Lactobacillus species. Consistent with possible roles for cydABCD in explaining the observed phenotypes, (1) L. iners failed to take up exogenous glutathione and (2) supplementing MRS with reducing agents permitted L. iners growth, which could be blocked by SMC or SDLC. In growth competitions testing L. iners and L. crispatus within mock BV-like communities, SMC plus MTZ outperformed MTZ alone in promoting L. crispatus dominance (Figs 2&3). Figure 1: S-methyl-L-cysteine (SMC) selectively blocks growth of L. iners but not other cervicovaginal Lactobacillus species in cysteine-supplemented MRS broth. Growth was measured by optical density and inhibition calculated relative to Cys-supplemented no-inhibitor control during exponential growth. Values displayed are median (+/- maximum/minimum) for 3 replicates from a single experiment. In all panels, representative data are shown from 1 of >=2 independent experiments for each bacterial strain and media condition. Results are representative of multiple strains for L. iners (n = 16), L. crispatus (n = 7), and L. jensenii (n = 2). Figure 2: Relative abundance of L. crispatus, L. iners, or various BV-associated bacteria in mock bacterial communities grown in rich, non-selective media with or without metronidazole (MTZ) and/or SMC. Relative abundance was determined by bacterial 16S rRNA gene sequencing. Data are shown for three representative mock communities with 5 replicates per media condition. Figure 3: Ratio of L. crispatus to other species in the mock bacterial communities depicted in Figure 2. Statistical significance determined via 1-way ANOVA of log10-transformed ratios with post-hoc Tukey test; selected pairwise comparisons are shown (***, p < 0.001). Conclusion L. iners has unique requirements for exogenous cysteine/cystine or a reduced environment for growth. Targeting cystine uptake to inhibit L. iners is a potential strategy for shifting cervicovaginal microbiota towards L. crispatus-dominant communities. Disclosures Douglas S. Kwon, MD, PhD, Day Zero Diagnostics (Consultant, Shareholder, Other Financial or Material Support, co-founder)

Background Whole genome sequencing (WGS) is a powerful tool to uncover transmission patterns and antimicrobial resistance (AMR) mechanisms of Enterococcus faecium, a major cause of hospital-acquired infections. Most E. faecium genomic studies include isolates from outbreak investigations rather than routine sampling. Additionally, the use of WGS to predict E. faecium AMR has not been tested systematically. Here we use WGS to characterize over 400 E. faecium clinical isolates to assess their strain diversity and AMR mechanisms. Methods Clinical E. faecium isolates from the MGH Microbiology Laboratory were collected at random from 1/2016-12/2017 (derivation set; 193 isolates) and with enrichment for more resistant isolates from 1/2018-9/2019 (validation set; 226 isolates). Species identification was performed using the bioMérieux VITEK MS instrument. Susceptibility testing was performed using the AST-GP75 card (bioMérieux VITEK 2), with confirmation by disk diffusion or ETEST when needed. Bacterial DNA from isolates was extracted, purified, sequenced (Illumina NextSeq), and quality filtered. Samples with >20x genome coverage were analyzed with SRST2 and AliView. Results MLST analysis of the derivation set demonstrated strikingly high diversity compared to previously published studies, with the three most frequent types (ST412, ST18, ST736) comprising fewer than half of samples. We identified and confirmed four novel MLST types comprising 12% of samples. We next analyzed the derivation isolate set to determine which genes and SNPs, if applicable, predicted resistance to seven antibiotics routinely tested at our institution: ampicillin, ciprofloxacin, doxycycline, high-level gentamicin, levofloxacin, tetracycline, and vancomycin. These rules were uniformly applied to the validation isolate set and demonstrated that genotypic AMR prediction has an overall positive predictive value of 97.0% and negative predictive value of 97.1% compared to standard susceptibility methods. Table 1. Summary of validation set predictions of antimicrobial susceptibility based on defined genotypic features. * The intermediate category is considered with the susceptible category. Conclusion In a diverse and challenging set of clinical E. faecium isolates, known AMR genes and SNPs can be simply applied to predict phenotypic susceptibility with high accuracy for seven routinely tested antibiotics. Further testing will be performed to resolve phenotype-genotype discrepancies. Summary of validation set predictions of antimicrobial susceptibility based on defined genotypic features. * The intermediate category is considered with the susceptible category. Disclosures Melis N. Anahtar, MD, PhD, Day Zero Diagnostics (Other Financial or Material Support, Co-founder, consultant, equity holder) Virginia M. Pierce, MD, Selux Diagnostics, Inc. (Grant/Research Support) Douglas S. Kwon, MD, PhD, Day Zero Diagnostics (Consultant, Shareholder, Other Financial or Material Support, co-founder)

Vaginal microbiota has been postulated as a key contributor to the disproportionately higher HIV acquisition risk in women. The commensal bacterial communities in the vaginal tract have been implicated in HIV pathogenesis, with strict anaerobes such as Gardnerella and Prevotella causing inflammation and increased frequency of HIV target cells. Young African women are up to six times more likely to be infected with HIV than their male counterparts. The underlying biological mechanisms for increased susceptibility to HIV infection are not fully known, particularly among pregnant women who are also at risk of transmitting the infection to their unborn babies. We characterized the vaginal microbiome of pregnant women receiving antenatal care. Using 16S rRNA sequencing, we analyzed the richness and abundances of the commensal bacterial communities within the female genital tract. Data was analyzed using qiime2 version 2018, Dada2 plugin, and Naive-Bayes classifier for Taxonomic assignment. We report that 19% (35/179) of pregnant women had a Lactobacillus-dominant vaginal microbiota profile. Our findings show that the main cervicotypes (“CTs”) were CT1 which was predominantly non-iners Lactobacillus (6%, 11/179), CT2 which was dominated by L. iners (13%, 24/179), CT3 that was Gardnerella dominant (49%, 87/179) and CT4, a mixed CT co-dominated by L. iners, Gardnerella and Atopobium. Cervical lavage of women with non-Lactobacillus CT had significantly higher levels of inflammatory cytokines IL-1beta, TNF-alpha, and chemokines IL-6 and IL-8. Highly diverse cervicotype (CT4) was associated with inflammation, a known catalyst of HIV acquisition and transmission, within pregnant women regardless of HIV sero-status.

Existing studies characterizing gut microbiome variation in the United States suffer from population ascertainment biases, with individuals of American Indian ancestry being among the most underrepresented. Here, we describe the first gut microbiome diversity study of an American Indian community. We partnered with the Cheyenne and Arapaho (C&A), federally recognized American Indian tribes in Oklahoma, and compared gut microbiome diversity and metabolic function of C&A participants to individuals of non-native ancestry in Oklahoma (NNIs). While the C&A and NNI participants share microbiome features common to industrialized populations, the C&A participants had taxonomic profiles characterized by a reduced abundance of the anti-inflammatory bacterial genus Faecalibacterium, along with a fecal metabolite profile similar to dysbiotic states described for metabolic disorders. American Indians are known to be at elevated risk for metabolic disorders. While many aspects of this health disparity remain poorly understood, our results support the need to further study the microbiome as a contributing factor. As the field of microbiome research transitions to therapeutic interventions, it raises concerns that the continued exclusion and lack of participation of American Indian communities in these studies will further exacerbate health disparities. To increase momentum in fostering these much needed partnerships, it is essential that the scientific community actively engage in and recruit these vulnerable populations in basic research through a strategy that promotes mutual trust and understanding, as outlined in this study.

Identifying the origin of a biological invasion has important applications to the effective control of the invaders. This is more critical for invasive agricultural pests that cause severe economic losses. The brown marmorated stink bug, Halyomorpha halys, originally from East Asia, has become a principal agricultural pest in the US since its first detection in Pennsylvania in 1996. This species is responsible for crop failures on many mid-Atlantic farms and current control efforts rely on heavy insecticide applications because no other options are available. To examine the genetic diversity and identify the source region of the US introductions, we sequenced portions of the mitochondrial cytochrome c oxidase subunit II gene, 12S ribosomal RNA gene and control region in populations from the US, China, South Korea and Japan. We detected high genetic divergence among native populations and traced the origin of US H. halys to the Beijing area in China. We observed much lower genetic diversity in exotic compared to native populations—two mitochondrial haplotypes in 55 US specimens versus 43 haplotypes in 77 native specimens. A single introduction of small propagule size matches the invasion history in the US. For the effective control of the US population, we suggest that surveys on egg parasitoids and insecticide resistance in natives should focus on the Beijing area in China.

We examine the genetic structure and evolutionary history of the mitten crab Eriocheir sensu stricto in East Asia by employing a genome scan – amplified fragment length polymorphism. Population analysis reveals three divergent clades in Eriocheir s. s., which dominate the East China Sea–Yellow Sea, the Sea of Japan (plus Okinawa) and the South China Sea, respectively, mostly in agreement with our previous mtDNA analysis. With the tropical South China Sea inferred as the origin, the East China Sea–Yellow Sea and the Sea of Japan clades in the north diverged successively from the ancestral clade during the mid-Pleistocene. The divergence of the three clades likely resulted from isolation of the three marginal seas caused by sea level change in the Pleistocene. Two sympatric zones, one of the East China Sea–Yellow Sea and the South China Sea clades in southeast China and the other of the East China Sea–Yellow Sea and the Sea of Japan clades in Vladivostok, are demonstrated to be hybrid zones, with hybridization occurring currently in the former but historically in the latter. Adaptive speciation is observed in the divergence process of the three clades, possibly because of selection from accumulated temperature. Our study indicates that the genetic structure and evolutionary history of Eriocheir s. s. have been primarily affected by Pleistocene glacial cycles, secondarily by divergent selection and drainage isolation, but only minimally by human activities.

Repetitive DNA sequences not only exist abundantly in eukaryotic nuclear genomes, but also occur as tandem repeats in many animal mitochondrial DNA (mtDNA) control regions. Due to concerted evolution, these repetitive sequences are highly similar or even identical within a genome. When long repetitive regions are the targets of amplification for the purpose of sequencing, multiple amplicons may result if one primer has to be located inside the repeats. Here, we show that, without separating these amplicons by gel purification or cloning, directly sequencing the mitochondrial repeats with the primer outside repetitive region is feasible and efficient. We exemplify it by sequencing the mtDNA control region of the mosquito Aedes albopictus, which harbors typical large tandem DNA repeats. This one-way sequencing strategy is optimal for population surveys.

We examined the impact of Pleistocene glacial cycles on geographical distribution and genetic structure of the mitten crab Eriocheir sensu stricto in East Asia using sequence variation of mitochondrial cytochrome c oxidase I and cytochrome b gene segments. Phylogenies revealed four distinct but shallow structured lineages in Eriocheir s. s. Three lineages dominated the East China Sea-Yellow Sea, the Sea of Japan and the South China Sea on the margins of the region, and one lineage occurred on Okinawa Island. This geographical distribution represents a general phylogeographic pattern in East Asia, which is closely associated with the fluctuations of marginal seas and islands during the Pleistocene. The four lineages are estimated to have diverged during the mid-Pleistocene. Demographic expansions were observed in each lineage, starting within the second-to-latest interglacial period in the marginal sea lineages ( approximately 70-130ka) and within the last glacial period in the Okinawa lineage ( approximately 25-80ka). Expansions have probably taken place northward along the coast of the East China Sea-Yellow Sea, following the rise of sea levels. Centered on the southern Korean Peninsula, expansions have likely occurred northward along the west coast and eastward along the south coast of the Sea of Japan. Each marginal sea has served as a single refugium during glacial periods. Two secondary contact regions were identified, one of the East China Sea-Yellow Sea and South China Sea lineages, and another of the East China Sea-Yellow Sea and Sea of Japan lineages. Phylogeography of Eriocheir s. s. provides insights into the evolutionary history and mechanism for generating biodiversity in East Asia.

Quaternary glacial cycles have played an important role in shaping the biodiversity in temperate regions. This is well documented in Northern Hemisphere, but much less understood for Southern Hemisphere. We used mitochondrial DNA and nuclear elongation factor 1α intron sequences to examine the Pleistocene glacial impacts on the phylogeographical pattern of the freshwater crab Aegla alacalufi in Chilean Patagonia. Phylogenetic analyses, which separated the glaciated populations on eastern continent into a north group (seven populations) and a south group (one population), revealed a shallow phylogenetic structure in the north group but a deep one in the non-glaciated populations on western islands, indicating the significant influence of glaciation on these populations. Phylogenies also identified the Yaldad population on Chiloé Island as a potentially unrecognized new species. The non-glaciated populations showed higher among population genetic divergence than the glaciated ones, but lower population genetic diversity was not detected in the latter. The two glaciated groups, which diverged from the non-glaciated populations at ~96,800-29,500 years ago and ~104,200-73,800 years ago, respectively, seem to have different glacial refugia. Unexpectedly, the non-glaciated islands did not serve as refugia for them. Demographic expansion was detected in the glaciated north group, with a constant population increase after the last glacial maximum. Nested clade analyses suggest a possible colonization from western islands to eastern continent. After arriving on the continent and surviving the last glacial period there, populations likely have expanded from high to low altitude, following the flood of melting ice. Aegla alacalufi genetic diversity has been primarily affected by Pleistocene glaciation and minimally by drainage isolation.

Background: Multiple loci and population genetic methods were employed to study the phylogeographic history of the Patagonian freshwater crab Aegla neuquensis (Aeglidae: Decopoda). This taxon occurs in two large river systems in the Patagonian Steppe, from the foothills of the Andes Mountains east to the Atlantic Ocean. Methodology/Principal Findings: A nuclear phylogeny and multilocus nested clade phylogeographic analysis detected a fragmentation event between the Negro and Chico-Chubut river systems. This event occurred approximately 137 thousand years ago. An isolation-with-migration analysis and maximum-likelihood estimates of gene flow showed asymmetrical exchange of genetic material between these two river systems exclusively in their headwaters. We used information theory to determine the best-fit demographic history between these two river systems under an isolation-with-migration model. The best-fit model suggests that the Negro and the ancestral populations have the same effective population sizes; whereas the Chico-Chubut population is smaller and shows that gene flow from the Chico-Chubut into the Negro is four times higher than in the reverse direction. Much of the Chico-Chubut system appears to have only been recently colonized while the Negro populations appear to have been in place for most of the evolutionary history of this taxon. Conclusions/Significance: Due to mitochondrial introgression, three nuclear loci provided different phylogeographic resolution than the three mitochondrial genes for an ancient fragmentation event observed in the nuclear phylogeny. However, the mitochondrial locus provided greater resolution on more recent evolutionary events. Our study, therefore, demonstrates the need to include both nuclear and mitochondrial loci for a more complete understanding of evolutionary histories and associated phylogeographic events. Our results suggest that gene flow between these systems, before and after fragmentation was through periodic paleolakes that formed in the headwaters region. Fragmentation between the Negro and Chico-Chubut systems was driven by the disappearance of these paleolakes during the Patagonian Glaciation.