A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase.

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 ß' 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 ß and ß' 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. IMPORTANCE: The 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.

The Burden and Impact of Early Post-transplant Multidrug-Resistant Organism Detection Among Renal Transplant Recipients, 2005-2021.

Background: Reducing the burden of multidrug-resistant organism (MDRO) colonization and infection among renal transplant recipients (RTRs) may improve patient outcomes. We aimed to assess whether the detection of an MDRO or a comparable antibiotic-susceptible organism (CSO) during the early post-transplant (EPT) period was associated with graft loss and mortality among RTRs. Methods: We conducted a retrospective cohort study of RTRs transplanted between 2005 and 2021. EPT positivity was defined as a positive bacterial culture within 30 days of transplant. The incidence and prevalence of EPT MDRO detection were calculated. The primary outcome was a composite of 1-year allograft loss or mortality following transplant. Multivariable Cox hazard regression, competing risk, propensity score-weighted sensitivity, and subgroup analyses were performed. Results: Among 3507 RTRs, the prevalence of EPT MDRO detection was 1.3% (95% CI, 0.91%-1.69%) with an incidence rate per 1000 EPT-days at risk of 0.42 (95% CI, 0.31-0.57). Among RTRs who met survival analysis inclusion criteria (n = 3432), 91% (3138/3432) had no positive EPT cultures and were designated as negative controls, 8% (263/3432) had a CSO detected, and 1% (31/3432) had an MDRO detected in the EPT period. EPT MDRO detection was associated with the composite outcome (adjusted hazard ratio [aHR], 3.29; 95% CI, 1.21-8.92) and death-censored allograft loss (cause-specific aHR, 7.15; 95% CI, 0.92-55.5; subdistribution aHR, 7.15; 95% CI, 0.95-53.7). A similar trend was seen in the subgroup and sensitivity analyses. Conclusions: MDRO detection during the EPT period was associated with allograft loss, suggesting the need for increased strategies to optimize prevention of MDRO colonization and infection.

Experimentally evolved Staphylococcus aureus shows increased survival in the presence of Pseudomonas aeruginosa by acquiring mutations in the amino acid transporter, GltT.

When cultured together under standard laboratory conditions Pseudomonas aeruginosa has been shown to be an effective inhibitor of Staphylococcus aureus. However, P. aeruginosa and S. aureus are commonly observed in coinfections of individuals with cystic fibrosis (CF) and in chronic wounds. Previous work from our group revealed that S. aureus isolates from CF infections are able to persist in the presence of P. aeruginosa strain PAO1 with a range of tolerances with some isolates being eliminated entirely and others maintaining large populations. In this study, we designed a serial transfer, evolution experiment to identify mutations that allow S. aureus to survive in the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa PAO1. After eight coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompeted wild-type S. aureus when glutamate was absent from chemically defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two are cultured together.

Factors associated with foreign body infection in methicillin-resistant Staphylococcus aureus bacteremia.

Background: We 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). Methods: All 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. Results: In 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 <18 months (OR=3.3 [1.1-10], p=0.031); and positive blood culture ≥48h (OR=16.7 [4.3-65.7], p<0.001). The most prevalent sequence type was ST8 (39%), followed by ST5 (29%), and ST105 (20%) with no significant difference between patients with or without foreign body infection. Only 39% of MRSA isolates formed a moderate/strong biofilm. No significant difference was observed between patients with foreign body infection and those without foreign body infection. In multivariable analysis, subjects infected with a MRSA isolate producing moderate/strong in vitro biofilm were more likely to have a history of MRSA infection in the last year (OR=3.41 [1.23-9.43]), interval between foreign body implantation and MRSA BSI <18 months (OR=3.1 [1.05-9.2]) and ST8 (OR=10.64 [2-57.3]). Conclusion: Most factors associated with foreign body infection in MRSA BSI were also characteristic of persistent infections. Biofilm-forming isolates were not associated with a higher risk of foreign-body infection but appeared to be associated with MRSA genetic lineage, especially ST8.

Incomplete immunity in a natural animal-microbiota interaction selects for higher pathogen virulence.

Incomplete immunity in recovered hosts is predicted to favor more virulent pathogens upon re-infection in the population.1 The microbiota colonizing animals can generate a similarly long-lasting, partial immune response, allowing for infection but dampened disease severity.2 We tracked the evolutionary trajectories of a widespread pathogen (Pseudomonas aeruginosa), experimentally passaged through populations of nematodes immune-primed by a natural microbiota member (P. berkeleyensis). This bacterium can induce genes regulated by a mitogen-activated protein kinase (MAPK) signaling pathway effective at conferring protection against pathogen-induced death despite infection.3 Across host populations, this incomplete immunity selected for pathogens more than twice as likely to kill as those evolved in non-primed (i.e., naive) or immune-compromised (mutants with a knockout of the MAPK ortholog) control populations. Despite the higher virulence, pathogen molecular evolution in immune-primed hosts was slow and constrained. In comparison, evolving pathogens in immune-compromised hosts were characterized by substantial genomic differentiation and attenuated virulence. These findings directly attribute the incomplete host immunity induced from microbiota as a significant force shaping the virulence and evolutionary dynamics of novel infectious diseases.

Average Nucleotide Identity based Staphylococcus aureus strain grouping allows identification of strain-specific genes in the pangenome.

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 core gene Average Nucleotide Identity (ANI) 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 associated with prophages, strain-concentrated with the vSaß genome island and rare genes (<10% frequency) concentrated near the origin of replication. Antibiotic genes were enriched in the strain-diffuse class, while virulence genes were distributed between strain-diffuse, strain-concentrated, core and rare classes. This study shows how different patterns of gene movement help create strains as distinct subspecies entities and provide insight into the diverse histories of important S. aureus functions.

The role of hypermutation and collateral sensitivity in antimicrobial resistance diversity of Pseudomonas aeruginosa populations in cystic fibrosis lung infection.

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.

Unique microbial diversity, community composition, and networks among Pacific Islander endocervical and vaginal microbiomes with and without Chlamydia trachomatis infection in Fiji.

IMPORTANCE: Chlamydia 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.

Comparison of genomic diversity between single and pooled Staphylococcus aureus colonies isolated from human colonization cultures.

The most common approach to sampling the bacterial populations within an infected or colonized host is to sequence genomes from a single colony obtained from a culture plate. However, it is recognized that this method does not capture the genetic diversity in the population. Sequencing a mixture of several colonies (pool-seq) is a better approach to detect population heterogeneity, but it is more complex to analyse due to different types of heterogeneity, such as within-clone polymorphisms, multi-strain mixtures, multi-species mixtures and contamination. Here, we compared 8 single-colony isolates (singles) and pool-seq on a set of 2286 Staphylococcus aureus culture samples to identify features that can distinguish pure samples, samples undergoing intraclonal variation and mixed strain samples. The samples were obtained by swabbing 3 body sites on 85 human participants quarterly for a year, who initially presented with a methicillin-resistant S. aureus skin and soft-tissue infection (SSTI). We compared parameters such as sequence quality, contamination, allele frequency, nucleotide diversity and pangenome diversity in each pool to those for the corresponding singles. Comparing singles from the same culture plate, we found that 18% of sample collections contained mixtures of multiple multilocus sequence types (MLSTs or STs). We showed that pool-seq data alone could predict the presence of multi-ST populations with 95% accuracy. We also showed that pool-seq could be used to estimate the number of intra-clonal polymorphic sites in the population. Additionally, we found that the pool may contain clinically relevant genes such as antimicrobial resistance markers that may be missed when only examining singles. These results highlight the potential advantage of analysing genome sequences of total populations obtained from clinical cultures rather than single colonies.

Integrative analysis of microbial 16S gene and shotgun metagenomic sequencing data improves statistical efficiency.

Background: The most widely used technologies for profiling microbial communities are 16S marker-gene sequencing and shotgun metagenomic sequencing. Interestingly, many microbiome studies have performed both sequencing experiments on the same cohort of samples. The two sequencing datasets often reveal consistent patterns of microbial signatures, highlighting the potential for an integrative analysis to improve power of testing these signatures. However, differential experimental biases, partially overlapping samples, and differential library sizes pose tremendous challenges when combining the two datasets. Currently, researchers either discard one dataset entirely or use different datasets for different objectives. Methods: In this article, we introduce the first method of this kind, named Com-2seq, that combines the two sequencing datasets for testing differential abundance at the genus and community levels while overcoming these difficulties. The new method is based on our LOCOM model (Hu et al., 2022), which employs logistic regression for testing taxon differential abundance while remaining robust to experimental bias. To benchmark the performance of Com-2seq, we introduce two ad hoc approaches: applying LOCOM to pooled taxa count data and combining LOCOM p-values from analyzing each dataset separately. Results: Our simulation studies indicate that Com-2seq substantially improves statistical efficiency over analysis of either dataset alone and works better than the two ad hoc approaches. An application of Com-2seq to two real microbiome studies uncovered scientifically plausible findings that would have been missed by analyzing individual datasets. Conclusions: Com-2seq performs integrative analysis of 16S and metagenomic sequencing data, which improves statistical efficiency and has the potential to accelerate the search of microbial communities and taxa that are involved in human health and diseases.

Experimentally Evolved Staphylococcus aureus Survives in the Presence of Pseudomonas aeruginosa by Acquiring Mutations in the Amino Acid Transporter, GltT.

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacterial pathogens isolated from cystic fibrosis (CF) related lung infections. When both of these opportunistic pathogens are found in a coinfection, CF patients tend to have higher rates of pulmonary exacerbations and experience a more rapid decrease in lung function. When cultured together under standard laboratory conditions, it is often observed that P. aeruginosa effectively inhibits S. aureus growth. Previous work from our group revealed that S. aureus from CF infections have isolate-specific survival capabilities when cocultured with P. aeruginosa. In this study, we designed a serial transfer evolution experiment to identify mutations that allow S. aureus to adapt to the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa strain, PAO1. After 8 coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompete wild-type S. aureus when glutamate is absent from chemically-defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two bacteria are cultured together.

Comparison of genomic diversity between single and pooled Staphylococcus aureus colonies isolated from human colonisation cultures.

The most common approach to sampling the bacterial populations within an infected or colonised host is to sequence genomes from a single colony obtained from a culture plate. However, it is recognized that this method does not capture the genetic diversity in the population. An alternative is to sequence a mixture containing multiple colonies ("pool-seq"), but this has the disadvantage that it is a non-homogeneous sample, making it difficult to perform specific experiments. We compared differences in measures of genetic diversity between eight single-colony isolates (singles) and pool-seq on a set of 2286 S. aureus culture samples. The samples were obtained by swabbing three body sites on 85 human participants quarterly for a year, who initially presented with a methicillin-resistant S. aureus skin and soft-tissue infection (SSTI). We compared parameters such as sequence quality, contamination, allele frequency, nucleotide diversity and pangenome diversity in each pool to the corresponding singles. Comparing singles from the same culture plate, we found that 18% of sample collections contained mixtures of multiple Multilocus sequence types (MLSTs or STs). We showed that pool-seq data alone could predict the presence of multi-ST populations with 95% accuracy. We also showed that pool-seq could be used to estimate the number of polymorphic sites in the population. Additionally, we found that the pool may contain clinically relevant genes such as antimicrobial resistance markers that may be missed when only examining singles. These results highlight the potential advantage of analysing genome sequences of total populations obtained from clinical cultures rather than single colonies.

The Role of Hypermutation and Collateral Sensitivity in Antimicrobial Resistance Diversity of Pseudomonas aeruginosa Populations in Cystic Fibrosis Lung Infection.

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.

Integrative analysis of microbial 16S gene and shotgun metagenomic sequencing data improves statistical efficiency.

The most widely used technologies for profiling microbial communities are 16S marker-gene sequencing and shotgun metagenomic sequencing. Interestingly, many microbiome studies have performed both sequencing experiments on the same cohort of samples. The two sequencing datasets often reveal consistent patterns of microbial signatures, highlighting the potential for an integrative analysis to improve power of testing these signatures. However, differential experimental biases, partially overlapping samples, and differential library sizes pose tremendous challenges when combining the two datasets. Currently, researchers either discard one dataset entirely or use different datasets for different objectives. In this article, we introduce the first method of this kind, named Com-2seq, that combines the two sequencing datasets for the objective of testing differential abundance at the genus and community levels while overcoming these difficulties. We demonstrate that Com-2seq substantially improves statistical efficiency over analysis of either dataset alone and works better than two ad hoc approaches.

Transcriptional activation of ompA in Neisseria gonorrhoeae mediated by the XRE family member protein NceR.

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.

Detecting patterns of accessory genome coevolution in Staphylococcus aureus using data from thousands of genomes.

Bacterial genomes exhibit widespread horizontal gene transfer, resulting in highly variable genome content that complicates the inference of genetic interactions. In this study, we develop a method for detecting coevolving genes from large datasets of bacterial genomes based on pairwise comparisons of closely related individuals, analogous to a pedigree study in eukaryotic populations. We apply our method to pairs of genes from the Staphylococcus aureus accessory genome of over 75,000 annotated gene families using a database of over 40,000 whole genomes. We find many pairs of genes that appear to be gained or lost in a coordinated manner, as well as pairs where the gain of one gene is associated with the loss of the other. These pairs form networks of rapidly coevolving genes, primarily consisting of genes involved in virulence, mechanisms of horizontal gene transfer, and antibiotic resistance, particularly the SCCmec complex. While we focus on gene gain and loss, our method can also detect genes that tend to acquire substitutions in tandem, or genotype-phenotype or phenotype-phenotype coevolution. Finally, we present the R package DeCoTUR that allows for the computation of our method.

Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

Introduction: Chlamydia 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. Methods: We 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. Results: The 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. Discussion: The 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.

Pre-epidemic evolution of the MRSA USA300 clade and a molecular key for classification.

Introduction: USA300 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. Methods: Here, 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. Results: Phylogenetic 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.

Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis.

Chlamydia 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. We 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. The 78 C. trachomatis genomes from participants were from two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorecta"l 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. The absence of large numbers of fixed SNPs between C. trachomatis strains within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant variation in the 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. Importance: Chlamydia trachomatis is a bacterial pathogen that causes millions of sexually transmitted infections (STIs) annually across the globe. Because C. trachomatis lives inside human cells, it has historically been hard to study. We know little about how the bacterium spreads between body sites. Here, samples from 26 study participants who had simultaneous infections in their vagina, rectum and endocervix were genetically analyzed using an improved method to extract C. trachomatis DNA directly from clinical samples for genome sequencing. By analyzing patterns of mutations in the genomes, we found that 21 participants shared very similar C. trachomatis strains in all three anatomic sites, suggesting recent infection and spread. For five participants two C. trachomatis strains were evident, indicating multiple infections. This study is significant in that improved enrichment methods for genome sequencing provides robust data to genetically trace patterns of C. trachomatis infection and transmission within an individual for epidemiologic and pathogenesis interrogations.

Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains.

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.