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Abstract Background The obligate intracellular bacterial family Chlamydiaceae comprises a number of different species that cause disease in various vertebrate hosts including humans. Chlamydia suis, primarily found in the gastrointestinal tract of pigs, is the only species of the Chlamydiaceae family to have naturally gained tetracycline resistance (TetR), through a genomic island (Tet-island), integrated into the middle of chromosomal invasin-like gene inv. Previous studies have hypothesised that the uptake of the Tet-island from a host outside the Chlamydiaceae family was a unique event, followed by spread among C. suis through homologous recombination. In vitro recombination studies have shown that Tet-island exchange between C. suis strains is possible. Our aim in this study was to gain a deeper understanding of the interclade recombination of the Tet-island, among currently circulating C. suis field strains compared to in vitro-generated recombinants, using published whole genome sequences of C. suis field strains (n = 35) and in vitro-generated recombinants (n = 63). Results We found that the phylogeny of inv better reflected the phylogeny of the Tet-island than that of the whole genome, supporting recombination rather than site-specific insertion as the means of transfer. There were considerable differences between the distribution of recombinations within in vitro-generated strains compared to that within the field strains. These differences are likely because in vitro-generated recombinants were selected for a tetracycline and rifamycin/rifampicin resistant background, leading to the largest peak of recombination across the Tet-island. Finally, we found that interclade recombinations across the Tet-island were more variable in length downstream of the Tet-island than upstream. Conclusions Our study supports the hypothesis that the occurrence of TetR strains in both clades of C. suis came about through interclade recombination after a single ancestral horizontal gene transfer event.

Background Tracking the spread of antibiotic resistant bacteria is critical to reduce global morbidity and mortality associated with human and animal infections. There is a need to understand the role that wild animals in maintenance and transfer of antibiotic resistance genes (ARGs). Methods This study used metagenomics to identify and compare the abundance of bacterial species and ARGs detected in the gut microbiomes from sympatric humans and wild mouse lemurs in a forest-dominated, roadless region of Madagascar near Ranomafana National Park. We examined the contribution of human geographic location toward differences in ARG abundance and compared the genomic similarity of ARGs between host source microbiomes. Results Alpha and beta diversity of species and ARGs between host sources were distinct but maintained a similar number of detectable ARG alleles. Humans were differentially more abundant for four distinct tetracycline resistance-associated genes compared to lemurs. There was no significant difference in human ARG diversity from different locations. Human and lemur microbiomes shared 14 distinct ARGs with highly conserved in nucleotide identity. Synteny of ARG-associated assemblies revealed a distinct multidrug-resistant gene cassette carrying dfrA1 and aadA1 present in human and lemur microbiomes without evidence of geographic overlap, suggesting that these resistance genes could be widespread in this ecosystem. Further investigation into intermediary processes that maintain drug-resistant bacteria in wildlife settings is needed.

ABSTRACT Staphylococcus aureus causes both hospital- and community-acquired infections in humans worldwide. Due to the high incidence of infection, S. aureus is also one of the most sampled and sequenced pathogens today, providing an outstanding resource to understand variation at the bacterial subspecies level. We processed and downsampled 83,383 public S. aureus Illumina whole-genome shotgun sequences and 1,263 complete genomes to produce 7,954 representative substrains. Pairwise comparison of average nucleotide identity revealed a natural boundary of 99.5% that could be used to define 145 distinct strains within the species. We found that intermediate frequency genes in the pangenome (present in 10%–95% of genomes) could be divided into those closely linked to strain background (“strain-concentrated”) and those highly variable within strains (“strain-diffuse”). Non-core genes had different patterns of chromosome location. Notably, strain-diffuse genes were associated with prophages; strain-concentrated genes were associated with the vSaβ genome island and rare genes (84,000 genomes and subsampled to remove redundancy. We found that individual samples sharing >99.5% of their genome could be grouped into strains. We also showed that a portion of genes that are present in intermediate frequency in the species are strongly associated with some strains but completely absent from others, suggesting a role in strain specificity. This work lays the foundation for understanding individual gene histories of the S. aureus species and also outlines strategies for processing large bacterial genomic data sets.

ABSTRACT The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, symbionts in the gardens of fungus-farming ants. We ask how the gain and loss of various biosynthetic pathways correspond to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes composed of seven to eight chromosomes. The genomes are highly syntenic with macrosynteny decreasing with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. An ancestral state reconstruction analysis of biosynthetic pathways revealed that, while many secondary metabolites are shared with non-ant-associated Sordariomycetes, 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their fungal hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.IMPORTANCEMicrobial symbionts interact with their hosts and competitors through a remarkable array of secondary metabolites and natural products. Here, we highlight the highly streamlined genomic features of attine-associated fungal symbionts. The genomes of Escovopsis species, as well as species from other symbiont genera, many of which are common with the gardens of fungus-growing ants, are defined by seven chromosomes. Despite a high degree of metabolic conservation, we observe some variation in the symbionts’ potential to produce secondary metabolites. As the phylogenetic distribution of the encoding biosynthetic gene clusters coincides with attine transitions in agricultural systems, we highlight the likely role of these metabolites in mediating adaptation by a group of highly specialized symbionts.

ABSTRACT The continued emergence of Neisseria gonorrhoeae strains that express resistance to multiple antibiotics, including the last drug for empiric monotherapy (ceftriaxone), necessitates the development of new treatment options to cure gonorrheal infections. Toward this goal, we recently reported that corallopyronin A (CorA), which targets the switch region of the β′ subunit (RpoC) of bacterial DNA-dependent RNA polymerase (RNAP), has potent anti-gonococcal activity against a panel of multidrug-resistant clinical strains. Moreover, in that study, CorA could eliminate gonococcal infection of primary human epithelial cells and gonococci in a biofilm state. To determine if N. gonorrhoeae could develop high-level resistance to CorA in a single step, we sought to isolate spontaneous mutants expressing any CorA resistance phenotypes. However, no single-step mutants with high-level CorA resistance were isolated. High-level CorA resistance could only be achieved in this study through a multi-step pathway involving over-expression of the MtrCDE drug efflux pump and single amino acid changes in the β and β′ subunits (RpoB and RpoC, respectively) of RNAP. Molecular modeling of RpoB and RpoC interacting with CorA was used to deduce how the amino acid changes in RpoB and RpoC could influence gonococcal resistance to CorA. Bioinformatic analyses of whole genome sequences of clinical gonococcal isolates indicated that the CorA resistance determining mutations in RpoB/C, identified herein, are very rare (≤ 0.0029%), suggesting that the proposed pathway for resistance is predictive of how this phenotype could potentially evolve if CorA is used therapeutically to treat gonorrhea in the future.IMPORTANCEThe continued emergence of multi-antibiotic-resistant strains of Neisseria gonorrhoeae necessitates the development of new antibiotics that are effective against this human pathogen. We previously described that the RNA polymerase-targeting antibiotic corallopyronin A (CorA) has potent activity against a large collection of clinical strains that express different antibiotic resistance phenotypes including when such gonococci are in a biofilm state. Herein, we tested whether a CorA-sensitive gonococcal strain could develop spontaneous resistance. Our finding that CorA resistance could only be achieved by a multi-step process involving over-expression of the MtrCDE efflux pump and single amino acid changes in RpoB and RpoC suggests that such resistance may be difficult for gonococci to evolve if this antibiotic is used in the future to treat gonorrheal infections that are refractory to cure by other antibiotics.

BackgroundWe aimed to compare patient characteristics, MRSA sequence types, and biofilm production of MRSA strains that did and did not cause a foreign body infection in patients with MRSA bloodstream infections (BSI)MethodsAll adult patients with MRSA BSI hospitalized in two hospitals were identified by clinical microbiology laboratory surveillance. Only patients who had at least one implanted foreign body during the episode of BSI were included.ResultsIn July 2018 - March 2022, of 423 patients identified with MRSA BSI, 118 (28%) had ≥1 foreign body. Among them, 51 (43%) had one or more foreign body infections. In multivariable analysis, factors associated with foreign body infection were history of MRSA infection in the last year (OR=4.7 [1.4-15.5], p=0.012) community-associated BSI (OR=68.1 [4.2-1114.3], p=0.003); surgical site infection as source of infection (OR=11.8 [2-70.4], p=0.007); presence of more than one foreign body (OR=3.4 [1.1-10.7], p=0.033); interval between foreign body implantation and infection

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient) and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity, (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure, (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection.IMPORTANCEUpon infection in the cystic fibrosis (CF) lung, Pseudomonas aeruginosa rapidly acquires genetic mutations, especially in genes involved in antimicrobial resistance (AMR), often resulting in diverse, treatment-resistant populations. However, the role of bacterial population diversity within the context of chronic infection is still poorly understood. In this study, we found that hypermutator strains of P. aeruginosa in the CF lung undergoing treatment with tobramycin evolved increased sensitivity to tobramycin relative to non-hypermutators within the same population. This finding suggests that antimicrobial treatment may only exert weak selection pressure on P. aeruginosa populations in the CF lung. We further found no evidence for collateral sensitivity in these clinical populations, suggesting that collateral sensitivity may not be a robust, naturally occurring phenomenon for this microbe.

ABSTRACTPacific Islanders (PI) are a vulnerable population with a high prevalence of Chlamydia trachomatis (Ct) sexually transmitted infections and remain underrepresented in research. Here, 258 vaginal and 92 paired endocervical samples from women of diverse ethnicities in Fiji were analyzed using metagenomic shotgun sequencing. Using VALENCIA, a sub-community state type (subCST) classifier, 93 (36.04%) vaginal and 38 (41.30%) endocervical microbiomes did not match reference subCSTs. We, therefore, designed four unique subCSTs, IV-D0, IV-D1, IV-D2, and IV-E—differentially dominated by Gardnerella vaginalis, Prevotella spp., and Lactobacillus iners—to improve microbiome classifications. Ct-infected paired endocervical and vaginal microbiomes were significantly more likely to have differential species relative abundance (81.58%) than uninfected pairs (35.84%; adjusted odds ratio, 7.93; 95% confidence interval, 2.93–21.93; P < 0.0001). Among paired microbiomes, the endocervix had a significantly higher alpha diversity and independently a higher alpha diversity for high-risk human papilloma virus (HPV) types compared to low-risk and no HPV. The Ct-infected endocervical network compared to all others had smaller clusters—indicating lower species abundance and decreased weight of interactions among bacterial groups within clusters—and no interactions with potentially beneficial Lactobacillus spp. These data suggest a distinct influence on this network from Ct-infected endocervical cells. Furthermore, the high relative abundance of G. vaginalis and Prevotella spp. may generate polymicrobial biofilms that predispose to and/or promote Ct and possibly HPV persistence and pathogenicity. While these data expand on the existing repertoire of endocervical and vaginal microbiomes and fill in knowledge gaps regarding PIs, prospective studies are needed to define causal associations to develop successful interventions.IMPORTANCEChlamydia trachomatis (Ct) is the most common sexually transmitted bacterium globally. Endocervical and vaginal microbiome interactions are rarely examined within the context of Ct or among vulnerable populations. We evaluated 258 vaginal and 92 paired endocervical samples from Fijian women using metagenomic shotgun sequencing. Over 37% of the microbiomes could not be classified into sub-community state types (subCSTs). We, therefore, developed subCSTs IV-D0, IV-D1, IV-D2, and IV-E—dominated primarily by Gardnerella vaginalis—to improve classification. Among paired microbiomes, the endocervix had a significantly higher alpha diversity and, independently, higher diversity for high-risk human papilloma virus (HPV) genotypes compared to low-risk and no HPV. Ct-infected endocervical networks had smaller clusters without interactions with potentially beneficial Lactobacillus spp. Overall, these data suggest that G. vaginalis may generate polymicrobial biofilms that predispose to and/or promote Ct and possibly HPV persistence and pathogenicity. Our findings expand on the existing repertoire of endocervical and vaginal microbiomes and fill in knowledge gaps regarding Pacific Islanders.

ABSTRACT Increasing antibiotic resistance of Neisseria gonorrhoeae, the causative agent of gonorrhea, is a growing global concern that has renewed vaccine development efforts. The gonococcal OmpA protein was previously identified as a vaccine candidate due to its surface exposure, conservation, stable expression, and involvement in host–cell interactions. We previously demonstrated that the transcription of ompA can be activated by the MisR/MisS two-component system. Interestingly, earlier work suggested that the availability of free iron also influences ompA expression, which we confirmed in this study. In the present study, we found that iron regulation of ompA was independent of MisR and searched for additional regulators. A DNA pull-down assay with the ompA promoter from gonococcal lysates obtained from bacteria grown in the presence or absence of iron identified an XRE (Xenobiotic Response Element) family member protein encoded by NGO1982. We found that an NGO1982 null mutant of N. gonorrhoeae strain FA19 displayed a reduced level of ompA expression compared to the wild-type (WT) parent strain. Given this regulation, and the capacity of this XRE-like protein to regulate a gene involved in peptidoglycan biosynthesis (ltgA), along with its presence in other Neisseria sp., we termed the NGO1982-encoded protein as NceR (Neisseria cell envelope regulator). Critically, results from DNA-binding studies indicated that NceR regulates ompA through a direct mechanism. Thus, ompA expression is subject to both iron-dependent (NceR) and -independent (MisR/MisS) pathways. Hence, levels of the vaccine antigen candidate OmpA in circulating gonococcal strains could be influenced by transcriptional regulatory systems and the availability of iron. IMPORTANCE Herein, we report that the gene encoding a conserved gonococcal surface-exposed vaccine candidate (OmpA) is activated by a heretofore undescribed XRE family transcription factor, which we term NceR. We report that NceR regulation of ompA expression in N. gonorrhoeae is mediated by an iron-dependent mechanism, while the previously described MisR regulatory system is iron-independent. Our study highlights the importance of defining the complexity of coordinated genetic and physiologic systems that regulate genes encoding vaccine candidates to better understand their availability during infection.

IntroductionChlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression.MethodsWe used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site.ResultsThe 78 C. trachomatis genomes from participants resolved into two major clades of the C. trachomatis phylogeny (the “prevalent urogenital and anorectal” clade and “non-prevalent urogenital and anorectal” clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains.DiscussionThe absence of large numbers of fixed SNPs between C. trachomatis genomes within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant genetic variation in different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage.

ABSTRACT Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections. Vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here, we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. Primers were designed to generate 10 amplicons covering 16 genes associated with the VISA phenotype. We sequenced amplicon pools as long reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. Each amplicon in the pool was sequenced to high (>1,000×) coverage, and no relationship was found between amplicon length and coverage. We also were able to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of the average minor allele frequency (6.5%) at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic resistance genotype inference using rapid nanopore sequencing from clinical samples. IMPORTANCE Bacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia that is also commonly antibiotic resistant. In this work, we develop a method to accelerate detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA), through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both rapidly identifies VISA genotypes and incorporates the most comprehensive database of VISA genetic determinants known to date.

IntroductionUSA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage.MethodsHere, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome, to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage.ResultsPhylogenetic analysis with these genomes confirms that the NAE and SAE USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

Abstract Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.

ABSTRACT Gonorrhea remains a major global public health problem because of the high incidence of infection (estimated 82 million cases in 2020) and the emergence and spread of Neisseria gonorrhoeae strains resistant to previous and current antibiotics used to treat infections. Given the dearth of new antibiotics that are likely to enter clinical practice in the near future, there is concern that cases of untreatable gonorrhea might emerge. In response to this crisis, the World Health Organization (WHO), in partnership with the Global Antibiotic Research and Development Partnership (GARDP), has made the search for and development of new antibiotics against N. gonorrhoeae a priority. Ideally, these antibiotics should also be active against other sexually transmitted organisms, such as Chlamydia trachomatis and/or Mycoplasma genitalium, which are often found with N. gonorrhoeae as co-infections. Corallopyronin A is a potent antimicrobial that exhibits activity against Chlamydia spp. and inhibits transcription by binding to the RpoB switch region. Accordingly, we tested the effectiveness of corallopyronin A against N. gonorrhoeae. We also examined the mutation frequency and modes of potential resistance against corallopyronin A. We report that corallopyronin A has potent antimicrobial action against antibiotic-susceptible and antibiotic-resistant N. gonorrhoeae strains and could eradicate gonococcal infection of cultured, primary human cervical epithelial cells. Critically, we found that spontaneous corallopyronin A-resistant mutants of N. gonorrhoeae are exceedingly rare (≤10−10) when selected at 4× the MIC. Our results support pre-clinical studies aimed at developing corallopyronin A for gonorrheal treatment regimens. IMPORTANCE The high global incidence of gonorrhea, the lack of a protective vaccine, and the emergence of N. gonorrhoeae strains expressing resistance to currently used antibiotics demand that new treatment options be developed. Accordingly, we investigated whether corallopyronin A, an antibiotic which is effective against other pathogens, including C. trachomatis, which together with gonococci frequently cause co-infections in humans, could exert anti-gonococcal action in vitro and ex vivo, and potential resistance emergence. We propose that corallopyronin A be considered a potential future treatment option for gonorrhea because of its potent activity, low resistance development, and recent advances in scalable production.

ABSTRACT Chlamydia trachomatis is a sexually transmitted pathogen and a global public health concern. Little is known about the microbial composition and function across endocervical, vaginal, and rectal microbiomes in the context of C. trachomatis infection. We evaluated the microbiomes of 10 age-matched high-risk Fijian women with and without C. trachomatis using metagenomic shotgun sequencing (MSS). Lactobacillus iners and Lactobacillus crispatus dominated the vagina and endocervix of uninfected women. Species often found in higher relative abundance in bacterial vaginosis (BV)—Mageeibacillus indolicus, Prevotella spp., Sneathia spp., Gardnerella vaginalis, and Veillonellaceae spp.—were dominant in C. trachomatis-infected women. This combination of BV pathogens was unique to Pacific Islanders compared to previously studied groups. The C. trachomatis-infected endocervix had a higher diversity of microbiota and microbial profiles that were somewhat different from those of the vagina. However, community state type III (CST-III) and CST-IV predominated, reflecting pathogenic microbiota regardless of C. trachomatis infection status. Rectal microbiomes were dominated by Prevotella and Bacteroides, although four women had unique microbiomes with Gardnerella, Akkermansia, Bifidobacterium, and Brachyspira. A high level of microbial similarity across microbiomes in two C. trachomatis-infected women suggested intragenitorectal transmission. A number of metabolic pathways in the endocervix, driven by BV pathogens and C. trachomatis to meet nutritional requirements for survival/growth, 5-fold higher than that in the vagina indicated that endocervical microbial functions are likely more diverse and complex than those in the vagina. Our novel findings provide the impetus for larger prospective studies to interrogate microbial/microbiome interactions that promote C. trachomatis infection and better define the unique genitorectal microbiomes of Pacific Islanders. IMPORTANCE Chlamydia trachomatis is the primary cause of bacterial sexually transmitted infections worldwide, with a disturbing increase in annual rates. While there is a plethora of data on healthy and pathogenic vaginal microbiomes—defining microbial profiles and associations with sexually transmitted infections (STIs)—far fewer studies have similarly examined the endocervix or rectum. Further, vulnerable populations, such as Pacific Islanders, remain underrepresented in research. We investigated the microbial composition, structure, and function of these anatomic microbiomes using metagenomic shotgun sequencing among a Fijian cohort. We found, primarily among C. trachomatis-infected women, unique microbial profiles in endocervical, vaginal, and rectal microbiomes with an increased diversity and more complex microbial pathways in endocervical than vaginal microbiomes. Similarities in microbiome composition across sites for some women suggested intragenitorectal transmission. These novel insights into genitorectal microbiomes and their purported function require prospective studies to better define Pacific Islander microbiomes and microbial/microbiome interactions that promote C. trachomatis infection.

ABSTRACT Phage therapy has been proposed as a possible alternative treatment for infections caused by the ubiquitous bacterial pathogen Staphylococcus aureus. However, successful therapy requires understanding the genetic basis of host range—the subset of strains in a species that could be killed by a particular phage. We searched diverse sets of S. aureus public genome sequences against a database of genes suggested from prior studies to influence host range to look for patterns of variation across the species. We found that genes encoding biosynthesis of molecules that were targets of S. aureus phage adsorption to the outer surface of the cell were the most conserved in the pangenome. Putative phage resistance genes that were core components of the pangenome genes had similar nucleotide diversity, ratio of nonsynonymous to synonymous substitutions, and functionality (measured by delta-bitscore) to other core genes. However, phage resistance genes that were not part of the core genome were significantly less consistent with the core genome phylogeny than all noncore genes in this set, suggesting more frequent movement between strains by horizontal gene transfer. Only superinfection immunity genes encoded by temperate phages inserted in the genome correlated with experimentally determined temperate phage resistance. Taken together, these results suggested that, while phage adsorption genes are heavily conserved in the S. aureus species, HGT may play a significant role in strain-specific evolution of host range patterns. IMPORTANCE Staphylococcus aureus is a widespread, hospital- and community-acquired pathogen that is commonly antibiotic resistant. It causes diverse diseases affecting both the skin and internal organs. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent, such as phage therapy, in which viruses specific to infecting bacteria clear infection. S. aureus phage host range not only determines whether phage therapy will be successful by killing bacteria but also horizontal gene transfer through transduction of host genetic material by phages. In this work, we comprehensively reviewed existing literature to build a list of S. aureus phage resistance genes and searched our database of almost 43,000 S. aureus genomes for these genes to understand their patterns of evolution, finding that prophages’ superinfection immunity correlates best with phage resistance and HGT. These findings improved our understanding of the relationship between known phage resistance genes and phage host range in the species.

ABSTRACT Staphylococcus aureus is a prominent nosocomial pathogen that causes several life-threatening diseases, such as pneumonia and bacteremia. S. aureus modulates the expression of its arsenal of virulence factors through sensing and integrating responses to environmental signals. The agr (accessory gene regulator) quorum sensing (QS) system is a major regulator of virulence phenotypes in S. aureus. There are four agr specificity groups each with a different autoinducer peptide sequence encoded by the agrD gene. Although agr is critical for the expression of many toxins, paradoxically, S. aureus strains often have nonfunctional agr activity due to loss-of-function mutations in the four-gene agr operon. To understand patterns in agr variability across S. aureus, we undertook a species-wide genomic investigation. We developed a software tool (AgrVATE; https://github.com/VishnuRaghuram94/AgrVATE) for typing and detecting frameshift mutations in the agr operon. In an analysis of over 40,000 S. aureus genomes, we showed a close association between agr type and S. aureus clonal complex. We also found a strong linkage between agrBDC alleles (encoding the peptidase, autoinducing peptide itself, and peptide sensor, respectively) but not agrA (encoding the response regulator). More than 5% of the genomes were found to have frameshift mutations in the agr operon. While 52% of these frameshifts occurred only once in the entire species, we observed cases where the recurring mutations evolved convergently across different clonal lineages with no evidence of long-term phylogenetic transmission, suggesting that strains with agr frameshifts were evolutionarily short-lived. Overall, genomic analysis of agr operon suggests evolution through multiple processes with functional consequences that are not fully understood. IMPORTANCE Staphylococcus aureus is a globally pervasive pathogen that produces a plethora of toxic molecules that can harm host immune cells. Production of these toxins is mainly controlled by an active agr quorum-sensing system, which senses and responds to bacterial cell density. However, there are many reports of S. aureus strains with genetic changes leading to impaired agr activity that are often found during chronic bloodstream infections and may be associated with increased disease severity. We developed an open-source software called AgrVATE to type agr systems and identify mutations. We used AgrVATE for a species-wide genomic survey of S. aureus, finding that more than 5% of strains in the public database had nonfunctional agr systems. We also provided new insights into the evolution of these genetic mutations in the agr system. Overall, this study contributes to our understanding of a common but relatively understudied means of virulence regulation in S. aureus.

Vancomycin-intermediate Staphylococcus aureus (VISA) typically arises through accumulation of chromosomal mutations that alter cell-wall thickness and global regulatory pathways. Genome-based prediction of VISA requires understanding whether strain background influences patterns of mutation that lead to resistance. We used an iterative method to experimentally evolve three important methicillin-resistant S. aureus (MRSA) strain backgrounds—(CC1, CC5 and CC8 (USA300)) to generate a library of 120 laboratory selected VISA isolates. At the endpoint, isolates had vancomycin MICs ranging from 4 to 10 μg/mL. We detected mutations in more than 150 genes, but only six genes (already known to be associated with VISA from prior studies) were mutated in all three background strains (walK, prs, rpoB, rpoC, vraS, yvqF). We found evidence of interactions between loci (e.g., vraS and yvqF mutants were significantly negatively correlated) and rpoB, rpoC, vraS and yvqF were more frequently mutated in one of the backgrounds. Increasing vancomycin resistance was correlated with lower maximal growth rates (a proxy for fitness) regardless of background. However, CC5 VISA isolates had higher MICs with fewer rounds of selection and had lower fitness costs than the CC8 VISA isolates. Using multivariable regression, we found that genes differed in their contribution to overall MIC depending on the background. Overall, these results demonstrated that VISA evolved through mutations in a similar set of loci in all backgrounds, but the effect of mutation in common genes differed with regard to fitness and contribution to resistance in different strains.

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Background There are about 15 million Americans working full-time on evening, night, or rotating shifts. Between 48% and 81.9% of those working rotating or night shifts report abdominal pain, constipation, diarrhea and other symptoms of functional bowel disorders. The basis for this high prevalence of functional bowel disorders, including irritable bowel syndrome (IBS), among shift workers is unknown. Animal studies, however, suggest that circadian disruption, similar to that in shift workers, may contribute to the development of GI complaints among shift workers by altering the composition and normal diurnal rhythmicity of the resident intestinal microbes. Therefore, the present study was designed to determine if there were differences in (1) composition and diversity of the microbiome of night shift workers compared to day shift workers; and (2) the composition and diversity of the microbiome among shift workers experiencing functional bowel symptoms compared to shift workers who did not experience functional bowel symptoms. Methods Fifty-one full time staff nurses who worked either 12-hour day or night shifts completed demographic information, and the Rome III IBS module. They also collected two samples of gut microbiota before the beginning and at the end of their last work shift on day 14, using validated field-tested methods consistent with the Human Microbiome Project. After DNA extraction, 16S rRNA sequencing and assignment to the genus level was completed, samples were then compared to determine if there were (1) differences in the diversity and profile of the microbiome by shift type; (2) if there were differences in the microbiome by time of day for collection; and (3) whether there were differences in the diversity and profile of the microbiome of nurses with IBS and those without IBS. Results There were no differences in alpha or beta diversity of gut microbiota when specimens from day and night shift nurses were compared. There were however marginal differences in beta diversity when specimens collected at the beginning and end of the shifts were compared, with seven OTUs being differentially abundant when collected from day shift workers in the evening. There were also three OTUs to be differentially abundant in participants reporting IBS symptoms.

ObjectiveTo evaluate the association between the early pregnancy vaginal microbiome and spontaneous preterm birth (sPTB) and early term birth (sETB) among African American women.MethodsVaginal samples collected in early pregnancy (8-14 weeks’ gestation) from 436 women enrolled in the Emory University African American Vaginal, Oral, and Gut Microbiome in Pregnancy Study underwent 16S rRNA gene sequencing of the V3-V4 region, taxonomic classification, and community state type (CST) assignment. We compared vaginal CST and abundance of taxa for women whose pregnancy ended in sPTB (N = 44) or sETB (N= 84) to those who delivered full term (N = 231).ResultsNearly half of the women had a vaginal microbiome classified as CST IV (Diverse CST), while one-third had CST III (L. iners dominated) and just 16% had CST I, II, or V (non-iners Lactobacillus dominated). Compared to vaginal CST I, II, or V (non-iners Lactobacillus dominated), both CST III (L. iners dominated) and CST IV (Diverse) were associated with sPTB with an adjusted odds ratio (95% confidence interval) of 4.1 (1.1, infinity) and 7.7 (2.2, infinity), respectively, in multivariate logistic regression. In contrast, no vaginal CST was associated with sETB. The linear decomposition model (LDM) based on amplicon sequence variant (ASV) relative abundance found a significant overall effect of the vaginal microbiome on sPTB (p=0.034) but not sETB (p=0.320), whereas the LDM based on presence/absence of ASV found no overall effect on sPTB (p=0.328) but a significant effect on sETB (p=0.030). In testing for ASV-specific effects, the LDM found that no ASV was significantly associated with sPTB considering either relative abundance or presence/absence data after controlling for multiple comparisons (FDR 10%), although in marginal analysis the relative abundance of Gardnerella vaginalis (p=0.011), non-iners Lactobacillus (p=0.016), and Mobiluncus curtisii (p=0.035) and the presence of Atopobium vaginae (p=0.049), BVAB2 (p=0.024), Dialister microaerophilis (p=0.011), and Prevotella amnii (p=0.044) were associated with sPTB. The LDM identified the higher abundance of 7 ASVs and the presence of 13 ASVs, all commonly residents of the gut, as associated with sETB at FDR < 10%.ConclusionsIn this cohort of African American women, an early pregnancy vaginal CST III or IV was associated with an increased risk of sPTB but not sETB. The relative abundance and presence of distinct taxa within the early pregnancy vaginal microbiome was associated with either sPTB or sETB.

Chlamydia trachomatisC. trachomatisC. trachomatis

Staphylococcus aureus

ABSTRACT Sequencing of bacterial genomes using Illumina technology has become such a standard procedure that often data are generated faster than can be conveniently analyzed. We created a new series of pipelines called Bactopia, built using Nextflow workflow software, to provide efficient comparative genomic analyses for bacterial species or genera. Bactopia consists of a data set setup step (Bactopia Data Sets [BaDs]), which creates a series of customizable data sets for the species of interest, the Bactopia Analysis Pipeline (BaAP), which performs quality control, genome assembly, and several other functions based on the available data sets and outputs the processed data to a structured directory format, and a series of Bactopia Tools (BaTs) that perform specific postprocessing on some or all of the processed data. BaTs include pan-genome analysis, computing average nucleotide identity between samples, extracting and profiling the 16S genes, and taxonomic classification using highly conserved genes. It is expected that the number of BaTs will increase to fill specific applications in the future. As a demonstration, we performed an analysis of 1,664 public Lactobacillus genomes, focusing on Lactobacillus crispatus, a species that is a common part of the human vaginal microbiome. Bactopia is an open source system that can scale from projects as small as one bacterial genome to ones including thousands of genomes and that allows for great flexibility in choosing comparison data sets and options for downstream analysis. Bactopia code can be accessed at https://www.github.com/bactopia/bactopia. IMPORTANCE It is now relatively easy to obtain a high-quality draft genome sequence of a bacterium, but bioinformatic analysis requires organization and optimization of multiple open source software tools. We present Bactopia, a pipeline for bacterial genome analysis, as an option for processing bacterial genome data. Bactopia also automates downloading of data from multiple public sources and species-specific customization. Because the pipeline is written in the Nextflow language, analyses can be scaled from individual genomes on a local computer to thousands of genomes using cloud resources. As a usage example, we processed 1,664 Lactobacillus genomes from public sources and used comparative analysis workflows (Bactopia Tools) to identify and analyze members of the L. crispatus species.

ABSTRACT Staphylococcus aureus has recently overtaken Pseudomonas aeruginosa as the most commonly recognized bacterial pathogen that infects the respiratory tracts of individuals with the genetic disease cystic fibrosis (CF) in the United States. Most studies of S. aureus in CF patient lung infections have focused on a few isolates, often exclusively laboratory-adapted strains, and how they are killed by P. aeruginosa. Less is known about the diversity of S. aureus CF patient lung isolates in terms of both their virulence and their interaction with P. aeruginosa. To begin to address this gap, we recently sequenced 64 clinical S. aureus isolates and a reference isolate, JE2. Here, we analyzed the antibiotic resistance genotypes, sequence types, clonal complexes, spa types, agr types, and presence/absence of other known virulence factor genes of these isolates. We hypothesized that virulence phenotypes of S. aureus, namely, toxin production and the mucoid phenotype, would be lost in these isolates due to adaptation in the CF patient lung. In contrast to these expectations, we found that most isolates can lyse both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), suggesting that these phenotypes were not lost during adaptation to the CF lung. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus isolates from CF patients, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health. IMPORTANCE Staphylococcus aureus is now the most frequently detected recognized pathogen in the lungs of individuals who have cystic fibrosis (CF) in the United States, followed closely by Pseudomonas aeruginosa. When these pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus. Here, we present an in-depth study of 64 S. aureus clinical isolates from CF patients, for which we investigated genetic diversity utilizing whole-genome sequencing, virulence phenotypes, and interactions with P. aeruginosa. We found that S. aureus isolated from CF lungs are phylogenetically diverse; most retain known virulence factors and vary in their interactions with P. aeruginosa (i.e., they range from being highly sensitive to P. aeruginosa to completely tolerant to it). Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

Background The delta-toxin (δ-toxin) of Staphylococcus aureus is the only hemolysin shown to cause mast cell degranulation and is linked to atopic dermatitis, a chronic inflammatory skin disease. We sought to characterize variation in δ-toxin production across S. aureus strains and identify genetic loci potentially associated with differences between strains. Methods A set of 124 S. aureus strains was genome-sequenced and δ-toxin levels in stationary phase supernatants determined by high performance liquid chromatography (HPLC). SNPs and kmers were associated with differences in toxin production using four genome-wide association study (GWAS) methods. Transposon mutations in candidate genes were tested for their δ-toxin levels. We constructed XGBoost models to predict toxin production based on genetic loci discovered to be potentially associated with the phenotype. Results The S. aureus strain set encompassed 40 sequence types (STs) in 23 clonal complexes (CCs). δ-toxin production ranged from barely detectable levels to >90,000 units, with a median of >8,000 units. CC30 had significantly lower levels of toxin production than average while CC45 and CC121 were higher. MSSA (methicillin sensitive) strains had higher δ-toxin production than MRSA (methicillin resistant) strains. Through multiple GWAS approaches, 45 genes were found to be potentially associated with toxicity. Machine learning models using loci discovered through GWAS as features were able to predict δ-toxin production (as a high/low binary phenotype) with a precision of .875 and specificity of .990 but recall of .333. We discovered that mutants in the carA gene, encoding the small chain of carbamoyl phosphate synthase, completely abolished toxin production and toxicity in Caenorhabditis elegans. Conclusions The amount of stationary phase production of the toxin is a strain-specific phenotype likely affected by a complex interaction of number of genes with different levels of effect. We discovered new candidate genes that potentially play a role in modulating production. We report for the first time that the product of the carA gene is necessary for δ-toxin production in USA300. This work lays a foundation for future work on understanding toxin regulation in S. aureus and prediction of phenotypes from genomic sequences.

Abstract Background USA300 methicillin-resistant Staphylococcus aureus (MRSA) is a community- and hospital-acquired pathogen that frequently causes infections but also can survive on the human body asymptomatically as a part of the normal microbiota. We devised a comparative genomic strategy to track colonizing USA300 at different body sites after an initial infection. We sampled ST8 S. aureus from subjects at the site of a first known MRSA infection. Within 60 days of this infection and again 12 months later, each subject was tested for asymptomatic colonization in the nose, throat and perirectal region. 93 S. aureus strains underwent whole genome shotgun sequencing. Results Among 28 subjects at the initial sampling time, we isolated S. aureus from the nose, throat and perirectal sites from 15, 11 and 15 of them, respectively. Twelve months later we isolated S. aureus from 9 subjects, with 6, 3 and 3 strains from the nose, throat and perirectal area, respectively. Genome sequencing revealed that 23 patients (ages 0–66 years) carried USA300 intra-subject lineages (ISLs), defined as having an index infection isolate and closely related colonizing strains. Pairwise distance between strains in different ISLs was 48 to 162 single nucleotide polymorphisms (SNPs) across the core regions of the chromosome, whereas within the same ISL it was 0 to 26 SNPs. Strains in ISLs from the same subject differed in plasmid and prophage content, and contained deletions that removed the mecA-containing SCCmec and ACME regions. Five strains contained frameshift mutations in agr toxin-regulating genes. Persistence of an ISL was not associated with clinical or demographic subject characteristics. We inferred that colonization with the ISL occurred about 18 weeks before the first assessment of asymptomatic colonization. Conclusions Clonal lineages of USA300 may continue to colonize people at one or more anatomic sites up to a year after an initial infection and experience loss of the SCCmec, loss and gain of other mobile genetic elements, and mutations in the agr operon.

Abstract Although rare in the U.S., outbreaks due to Neisseria meningitidis do occur. Rapid, early outbreak detection is important for timely public health response. In this study, we characterized U.S. meningococcal isolates (N = 201) from 15 epidemiologically defined outbreaks (2009–2015) along with temporally and geographically matched sporadic isolates using multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), and six whole genome sequencing (WGS) based methods. Recombination-corrected maximum likelihood (ML) and Bayesian phylogenies were reconstructed to identify genetically related outbreak isolates. All WGS analysis methods showed high degree of agreement and distinguished isolates with similar or indistinguishable PFGE patterns, or the same strain genotype. Ten outbreaks were caused by a single strain; 5 were due to multiple strains. Five sporadic isolates were phylogenetically related to 2 outbreaks. Analysis of 9 outbreaks using timed phylogenies identified the possible origin and estimated the approximate time that the most recent common ancestor emerged for outbreaks analyzed. U.S. meningococcal outbreaks were caused by single- or multiple-strain introduction, with organizational outbreaks mainly caused by a clonal strain and community outbreaks by divergent strains. WGS can infer linkage of meningococcal cases when epidemiological links are uncertain. Accurate identification of outbreak-associated cases requires both WGS typing and epidemiological data.

Abstract Background The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes – the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans – its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature’s Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. Results We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. Conclusions This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

Abstract Background Adverse birth and neonatal outcomes disproportionately affect African American women and infants compared to those of other races/ethnicities. While significant research has sought to identify underlying factors contributing to these disparities, current understanding remains limited, constraining prevention, early diagnosis, and treatment. With the development of next generation sequencing techniques, the contribution of the vaginal microbiome to adverse maternal and neonatal outcomes has come under consideration. However, most microbiome in pregnancy studies include few African American women, do not consider the potential contribution of non-vaginal microbiome sites, and do not consider the effects of sociodemographic or behavioral factors on the microbiome. Methods We conceived our on-going, 5-year longitudinal study, Biobehavioral Determinants of the Microbiome and Preterm Birth in Black Women, as an intra-race study to enable the investigation of risk and protective factors within the disparate group. We aim to recruit over 500 pregnant African American women, enrolling them into the study at 8–14 weeks of pregnancy. Participants will be asked to complete questionnaires and provide oral, vaginal, and gut microbiome samples at enrollment and again at 24–30 weeks. Chart review will be used to identify pregnancy outcomes, infections, treatments, and complications. DNA will be extracted from the microbiome samples and sequencing of the V3 and V4 regions of the 16S rRNA gene will be conducted. Processing and mapping will be completed with QIIME and operational taxonomic units (OTUs) will be mapped to Greengenes version 13_8. Community state types (CSTs) and diversity measures at each site and time will be identified and considered in light of demographic, psychosocial, clinical, and biobehavioral variables. Discussion This rich data set will allow future consideration of risk and protective factors, between and within groups of women, providing the opportunity to uncover the roots of the persistent health disparity experienced by African American families.

Objective A growing body of research has investigated the human microbiota and pregnancy outcomes, especially preterm birth. Most studies of the prenatal microbiota have focused on the vagina, with fewer investigating other body sites during pregnancy. Although pregnancy involves profound hormonal, immunological and metabolic changes, few studies have investigated either shifts in microbiota composition across pregnancy at different body sites or variation in composition at any site that may be explained by maternal characteristics. The purpose of this study was to investigate: (1) the stability of the vaginal, oral, and gut microbiota from early (8–14 weeks) through later (24–30 weeks) pregnancy among African American women according to measures of socioeconomic status, accounting for prenatal antibiotic use; (2) whether measures of socioeconomic status are associated with changes in microbiota composition over pregnancy; and (3) whether exposure to prenatal antibiotics mediate any observed associations between measures of socioeconomic status and stability of the vaginal, oral, and gut microbiota across pregnancy. Methods We used paired vaginal, oral, or gut samples available for 16S rRNA gene sequencing from two time points in pregnancy (8–14 and 24–30 weeks) to compare within-woman changes in measures of alpha diversity (Shannon and Chao1) and beta-diversity (Bray–Curtis dissimilarity) among pregnant African American women (n = 110). Multivariable linear regression was used to examine the effect of level of education and prenatal health insurance as explanatory variables for changes in diversity, considering antibiotic exposure as a mediator, adjusting for age, obstetrical history, and weeks between sampling. Results For the oral and gut microbiota, there were no significant associations between measures of socioeconomic status or prenatal antibiotic use and change in Shannon or Chao1 diversity. For the vaginal microbiota, low level of education (high school or less) was associated with an increase in Shannon and Chao1 diversity over pregnancy, with minimal attenuation when controlling for prenatal antibiotic use. Conversely, for within-woman Bray–Curtis dissimilarity for early compared to later pregnancy, low level of education and prenatal antibiotics were associated with greater dissimilarity for the oral and gut sites, with minimal attenuation when controlling for prenatal antibiotics, and no difference in dissimilarity for the vaginal site. Conclusions Measures of maternal socioeconomic status are variably associated with changes in diversity across pregnancy for the vaginal, oral, and gut microbiota, with minimal attenuation by prenatal antibiotic exposure. Studies that evaluate stability of the microbiota across pregnancy in association with health outcomes themselves associated with socioeconomic status (such as preterm birth) should incorporate measures of socioeconomic status to avoid finding spurious relationships.

Background It is possible to detect bacterial species in shotgun metagenome datasets through the presence of only a few sequence reads. However, false positive results can arise, as was the case in the initial findings of a recent New York City subway metagenome project. False positives are especially likely when two closely related are present in the same sample. Bacillus anthracis, the etiologic agent of anthrax, is a high-consequence pathogen that shares >99% average nucleotide identity with Bacillus cereus group (BCerG) genomes. Our goal was to create an analysis tool that used k-mers to detect B. anthracis, incorporating information about the coverage of BCerG in the metagenome sample. Methods Using public complete genome sequence datasets, we identified a set of 31-mer signatures that differentiated B. anthracis from other members of the B. cereus group (BCerG), and another set which differentiated BCerG genomes (including B. anthracis) from other Bacillus strains. We also created a set of 31-mers for detecting the lethal factor gene, the key genetic diagnostic of the presence of anthrax-causing bacteria. We created synthetic sequence datasets based on existing genomes to test the accuracy of a k-mer based detection model. Results We found 239,503 B. anthracis-specific 31-mers (the Ba31 set), 10,183 BCerG 31-mers (the BCerG31 set), and 2,617 lethal factor k-mers (the lef31 set). We showed that false positive B. anthracis k-mers—which arise from random sequencing errors—are observable at high genome coverages of B. cereus. We also showed that there is a “gray zone” below 0.184× coverage of the B. anthracis genome sequence, in which we cannot expect with high probability to identify lethal factor k-mers. We created a linear regression model to differentiate the presence of B. anthracis-like chromosomes from sequencing errors given the BCerG background coverage. We showed that while shotgun datasets from the New York City subway metagenome project had no matches to lef31 k-mers and hence were negative for B. anthracis, some samples showed evidence of strains very closely related to the pathogen. Discussion This work shows how extensive libraries of complete genomes can be used to create organism-specific signatures to help interpret metagenomes. We contrast “specialist” approaches to metagenome analysis such as this work to “generalist” software that seeks to classify all organisms present in the sample and note the more general utility of a k-mer filter approach when taxonomic boundaries lack clarity or high levels of precision are required.

Low-cost Illumina sequencing of clinically-important bacterial pathogens has generated thousands of publicly available genomic datasets. Analyzing these genomes and extracting relevant information for each pathogen and the associated clinical phenotypes requires not only resources and bioinformatic skills but organism-specific knowledge. In light of these issues, we created Staphopia, an analysis pipeline, database and application programming interface, focused on Staphylococcus aureus, a common colonizer of humans and a major antibiotic-resistant pathogen responsible for a wide spectrum of hospital and community-associated infections. Written in Python, Staphopia’s analysis pipeline consists of submodules running open-source tools. It accepts raw FASTQ reads as an input, which undergo quality control filtration, error correction and reduction to a maximum of approximately 100× chromosome coverage. This reduction significantly reduces total runtime without detrimentally affecting the results. The pipeline performs de novo assembly-based and mapping-based analysis. Automated gene calling and annotation is performed on the assembled contigs. Read-mapping is used to call variants (single nucleotide polymorphisms and insertion/deletions) against a reference S. aureus chromosome (N315, ST5). We ran the analysis pipeline on more than 43,000 S. aureus shotgun Illumina genome projects in the public European Nucleotide Archive database in November 2017. We found that only a quarter of known multi-locus sequence types (STs) were represented but the top 10 STs made up 70% of all genomes. methicillin-resistant S. aureus (MRSA) were 64% of all genomes. Using the Staphopia database we selected 380 high quality genomes deposited with good metadata, each from a different multi-locus ST, as a non-redundant diversity set for studying S. aureus evolution. In addition to answering basic science questions, Staphopia could serve as a potential platform for rapid clinical diagnostics of S. aureus isolates in the future. The system could also be adapted as a template for other organism-specific databases.

In this study we developed a genome-based method for detecting Staphylococcus aureus subtypes from metagenome shotgun sequence data. We used a binomial mixture model and the coverage counts at >100,000 known S. aureus SNP (single nucleotide polymorphism) sites derived from prior comparative genomic analysis to estimate the proportion of 40 subtypes in metagenome samples. We were able to obtain >87% sensitivity and >94% specificity at 0.025X coverage for S. aureus. We found that 321 and 149 metagenome samples from the Human Microbiome Project and metaSUB analysis of the New York City subway, respectively, contained S. aureus at genome coverage >0.025. In both projects, CC8 and CC30 were the most common S. aureus clonal complexes encountered. We found evidence that the subtype composition at different body sites of the same individual were more similar than random sampling and more limited evidence that certain body sites were enriched for particular subtypes. One surprising finding was the apparent high frequency of CC398, a lineage often associated with livestock, in samples from the tongue dorsum. Epidemiologic analysis of the HMP subject population suggested that high BMI (body mass index) and health insurance are possibly associated with S. aureus carriage but there was limited power to identify factors linked to carriage of even the most common subtype. In the NYC subway data, we found a small signal of geographic distance affecting subtype clustering but other unknown factors influence taxonomic distribution of the species around the city.

Chlamydia trachomatis is a global cause of blinding trachoma and sexually transmitted infections (STIs). We used comparative genomics of the family Chlamydiaceae to select conserved housekeeping genes for C. trachomatis multilocus sequencing, characterizing 19 reference and 68 clinical isolates from 6 continental/subcontinental regions. There were 44 sequence types (ST). Identical STs for STI isolates were recovered from different regions, whereas STs for trachoma isolates were restricted by continent. Twenty-nine of 52 alleles had nonuniform distributions of frequencies across regions (p

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) USA300 is a successful S. aureus clone in the United States and a common cause of skin and soft tissue infections (SSTIs). We performed whole-genome sequencing (WGS) of 146 USA300 MRSA isolates from SSTIs and colonization cultures obtained from an investigation conducted from 2008 to 2010 in Chicago and Los Angeles households that included an index case with an S. aureus SSTI. Identifying unique single nucleotide polymorphisms (SNPs) and analyzing whole-genome phylogeny, we characterized isolates to understand transmission dynamics, genetic relatedness, and microevolution of USA300 MRSA within the households. We also compared the 146 USA300 MRSA isolates from our study with the previously published genome sequences of the USA300 MRSA isolates from San Diego (n = 35) and New York City (n = 277). We found little genetic variation within the USA300 MRSA household isolates from Los Angeles (mean number of SNPs ± standard deviation, 17.6 ± 35; π nucleotide diversity, 3.1 × 10−5) or from Chicago (mean number of SNPs ± standard deviation, 12 ± 19; π nucleotide diversity, 3.1 × 10−5). The isolates within a household clustered into closely related monophyletic groups, suggesting the introduction into and transmission within each household of a single common USA300 ancestral strain. From a Bayesian evolutionary reconstruction, we inferred that USA300 persisted within households for 2.33 to 8.35 years prior to sampling. We also noted that fluoroquinolone-resistant USA300 clones emerged around 1995 and were more widespread in Los Angeles and New York City than in Chicago. Our findings strongly suggest that unique USA300 MRSA isolates are transmitted within households that contain an individual with an SSTI. Decolonization of household members may be a critical component of prevention programs to control USA300 MRSA spread in the United States. IMPORTANCE USA300, a virulent and easily transmissible strain of methicillin-resistant Staphylococcus aureus (MRSA), is the predominant community-associated MRSA clone in the United States. It most commonly causes skin infections but also causes necrotizing pneumonia and endocarditis. Strategies to limit the spread of MRSA in the community can only be effective if we understand the most common sources of transmission and the microevolutionary processes that provide a fitness advantage to MRSA. We performed a whole-genome sequence comparison of 146 USA300 MRSA isolates from Chicago and Los Angeles. We show that households represent a frequent site of transmission and a long-term reservoir of USA300 strains; individuals within households transmit the same USA300 strain among themselves. Our study also reveals that a large proportion of the USA300 isolates sequenced are resistant to fluoroquinolone antibiotics. The significance of this study is that if households serve as long-term reservoirs of USA300, household MRSA eradication programs may result in a uniquely effective control method.

ABSTRACT The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP−). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP−. Whole-genome sequence analysis of 146 CP− USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates. IMPORTANCE The USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP−) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP+) phenotype is unlikely in vivo or in vitro since it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP− and provide new insight into the evolution of the USA300 and USA500 lineages.

Neisseria gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection (STI) of major importance. As a result of antibiotic resistance, there are now limited options for treating patients. We collected draft genome sequence data and associated metadata data on 76 N. gonorrhoeae strains from around the globe and searched for known determinants of antibiotics resistance within the strains. The population structure and evolutionary forces within the pathogen population were analyzed. Our results indicated a cosmopolitan gonoccocal population mainly made up of five subgroups. The estimated ratio of recombination to mutation (r/m = 2.2) from our data set indicates an appreciable level of recombination occurring in the population. Strains with resistance phenotypes to more recent antibiotics (azithromycin and cefixime) were mostly found in two of the five population subgroups.

Abstract Following publication of the original article [1], the authors pointed out that the Methods included one step that is no longer necessary but which was inadvertently carried over from an earlier protocol.

ABSTRACT Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966T, and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966T. The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966T and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. IMPORTANCE Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.

ABSTRACT Chlamydia psittaci is an obligate intracellular bacterium. Interest in Chlamydia stems from its high degree of virulence as an intestinal and pulmonary pathogen across a broad range of animals, including humans. C. psittaci human pulmonary infections, referred to as psittacosis, can be life-threatening, which is why the organism was developed as a bioweapon in the 20th century and is listed as a CDC biothreat agent. One remarkable recent result from comparative genomics is the finding of frequent homologous recombination across the genome of the sexually transmitted and trachoma pathogen Chlamydia trachomatis. We sought to determine if similar evolutionary dynamics occurred in C. psittaci. We analyzed 20 C. psittaci genomes from diverse strains representing the nine known serotypes of the organism as well as infections in a range of birds and mammals, including humans. Genome annotation revealed a core genome in all strains of 911 genes. Our analyses showed that C. psittaci has a history of frequently switching hosts and undergoing recombination more often than C. trachomatis. Evolutionary history reconstructions showed genome-wide homologous recombination and evidence of whole-plasmid exchange. Tracking the origins of recombinant segments revealed that some strains have imported DNA from as-yet-unsampled or -unsequenced C. psittaci lineages or other Chlamydiaceae species. Three ancestral populations of C. psittaci were predicted, explaining the current population structure. Molecular clock analysis found that certain strains are part of a clonal epidemic expansion likely introduced into North America by South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots. IMPORTANCE Chlamydia psittaci is classified as a CDC biothreat agent based on its association with life-threatening lung disease, termed psittacosis, in humans. Because of the recent remarkable findings of frequent recombination across the genome of the human sexually transmitted and ocular trachoma pathogen Chlamydia trachomatis, we sought to determine if similar evolutionary dynamics occur in C. psittaci. Twenty C. psittaci genomes were analyzed from diverse strains that may play a pathogenic role in human disease. Evolution of the strains revealed genome-wide recombination occurring at a higher rate than for C. trachomatis. Certain strains were discovered to be part of a recent epidemic clonal expansion originating in South America. These strains may have been introduced into the United States from South American bird traders, suggesting that psittacosis is a recently emerged disease originating in New World parrots. Our analyses indicate that C. psittaci strains have a history of frequently switching hosts and undergoing recombination.

ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium that causes a diversity of severe and debilitating diseases worldwide. Sporadic and ongoing outbreaks of lymphogranuloma venereum (LGV) strains among men who have sex with men (MSM) support the need for research on virulence factors associated with these organisms. Previous analyses have been limited to single genes or genomes of laboratory-adapted reference strain L2/434 and outbreak strain L2b/UCH-1/proctitis. We characterized an unusual LGV strain, termed L2c, isolated from an MSM with severe hemorrhagic proctitis. L2c developed nonfusing, grape-like inclusions and a cytotoxic phenotype in culture, unlike the LGV strains described to date. Deep genome sequencing revealed that L2c was a recombinant of L2 and D strains with conserved clustered regions of genetic exchange, including a 78-kb region and a partial, yet functional, toxin gene that was lost with prolonged culture. Indels (insertions/deletions) were discovered in an ftsK gene promoter and in the tarp and hctB genes, which encode key proteins involved in replication, inclusion formation, and histone H1-like protein activity, respectively. Analyses suggest that these indels affect gene and/or protein function, supporting the in vitro and disease phenotypes. While recombination has been known to occur for C. trachomatis based on gene sequence analyses, we provide the first whole-genome evidence for recombination between a virulent, invasive LGV strain and a noninvasive common urogenital strain. Given the lack of a genetic system for producing stable C. trachomatis mutants, identifying naturally occurring recombinants can clarify gene function and provide opportunities for discovering avenues for genomic manipulation. IMPORTANCE Lymphogranuloma venereum (LGV) is a prevalent and debilitating sexually transmitted disease in developing countries, although there are significant ongoing outbreaks in Australia, Europe, and the United States among men who have sex with men (MSM). Relatively little is known about LGV virulence factors, and only two LGV genomes have been sequenced to date. We isolated an LGV strain from an MSM with severe hemorrhagic proctitis that was morphologically unique in tissue culture compared with other LGV strains. Bioinformatic and statistical analyses identified the strain as a recombinant of L2 and D strains with highly conserved clustered regions of genetic exchange. The unique culture morphology and, more importantly, disease phenotype could be traced to the genes involved in recombination. The findings have implications for bacterial species evolution and, in the case of ongoing LGV outbreaks, suggest that recombination is a mechanism for strain emergence that results in significant disease pathology.