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ABSTRACT Escherichia coli is the leading cause of urinary tract infections (UTIs) in children and adults. The gastrointestinal tract is the primary reservoir of uropathogenic E. coli, which can be acquired from a variety of environmental exposures, including retail meat. In the current study, we used a novel statistical-genomic approach to estimate the proportion of pediatric UTIs caused by foodborne zoonotic E. coli strains. E. coli urine isolates were collected from DC residents aged 2 months to 17 years from the Children’s National Medical Center Laboratory, 2013–2014. During the same period, E. coli isolates were collected from retail poultry products purchased from 15 sites throughout DC. A total of 52 urine and 56 poultry isolates underwent whole-genome sequencing, core genome phylogenetic analysis, and host-origin prediction by a Bayesian latent class model that incorporated data on the presence of mobile genetic elements (MGEs) among E. coli isolates from multiple vertebrate hosts. A total of 56 multilocus sequence types were identified among the isolates. Five sequence types—ST10, ST38, ST69, ST117, and ST131—were observed among both urine and poultry isolates. Using the Bayesian latent class model, we estimated that 19% (10/52) of the clinical E. coli isolates in our population were foodborne zoonotic strains. These data suggest that a substantial portion of pediatric UTIs in the Washington DC region may be caused by E. coli strains originating in food animals and likely transmitted via contaminated poultry meat.IMPORTANCEEscherichia coli UTIs are a heavy public health burden and can have long-term negative health consequences for pediatric patients. E. coli has an extremely broad host range, including humans, chickens, turkeys, pigs, and cattle. E. coli derived from food animals is a frequent contaminant of retail meat products, but little is known about the risk these strains pose to pediatric populations. Quantifying the proportion of pediatric UTIs caused by food-animal-derived E. coli, characterizing the highest-risk strains, and identifying their primary reservoir species could inform novel intervention strategies to reduce UTI burden in this vulnerable population. Our results suggest that retail poultry meat may be an important vehicle for pediatric exposure to zoonotic E. coli strains capable of causing UTIs. Vaccinating poultry against the highest-risk strains could potentially reduce poultry colonization, poultry meat contamination, and downstream pediatric infections.

Abstract Antibiotic-resistant infections are a global concern, especially those caused by multidrug-resistant (MDR) bacteria, defined as those resistant to more than three drug classes. The animal agriculture industry contributes to the antimicrobial resistant foodborne illness burden via contaminated retail meat. In the United States, retail meat is shipped across the country. Therefore, understanding geospatial factors that influence MDR bacterial contamination is vital to protect consumers and inform interventions. Using data available from the United States Food and Drug Administration’s National Antimicrobial Resistance Monitoring System (NARMS), we describe retail meat shipping distances using processor and retailer locations and investigated this distance as a risk factor for MDR bacteria meat contamination using log-binomial regression. Meat samples collected during 2012–2014 totaled 11,243, of which 4791 (42.61%) were contaminated with bacteria and 835 (17.43%) of those bacteria were MDR. All examined geospatial factors were associated with MDR bacteria meat contamination. After adjustment for year and meat type, we found higher prevalence of MDR contamination among meat processed in the south (relative adjusted prevalence ratio [aPR] 1.35; 95% CI 1.06–1.73 when compared to the next-highest region), sold in Maryland (aPR 1.12; 95% CI 0.95–1.32 when compared to the next-highest state), and shipped from 194 to 469 miles (aPR 1.59; 95% CI 1.31–1.94 when compared to meats that traveled < 194 miles). However, sensitivity analyses revealed that New York sold the meat with the highest prevalence of MDR Salmonella contamination (4.84%). In this secondary analysis of NARMS data, both geographic location where products were sold and the shipping distance were associated with microbial contamination on retail meat.

Summary: Background: Escherichia coli sequence type 131 (ST131), specifically its fluoroquinolone-resistant H30R clade (ST131-H30R), is a global multidrug-resistant pathogen. The gut microbiome's role in ST131-H30R intestinal carriage is undefined. Methods: Veterans and their household members underwent longitudinal fecal swab surveillance for ST131 in 2014–2018. The fecal microbiome was characterized by 16S rRNA qPCR and sequencing. We evaluated associations between ST131-H30R carriage and gut microbiome at baseline by random forest models to identify the most informative gut bacterial phyla and genera attributes for ST131 and ST131-H30R carriage status. Next, we assessed longitudinal associations between fecal microbiome and ST131-H30R carriage using a mixed-effects logistic regression with longitudinal measures. Findings: Of the 519 participants, 78 were carriers of ST131, among whom 49 had ST131-H30R. At the baseline timepoint, H30R-positive participants had higher proportional abundances of Actinobacteria phylum (mean: 4.9% vs. 3.1%) than ST131-negative participants. H30R-positive participants also had higher abundances of Collinsella (mean: 2.3% vs. 1.1%) and lower abundances of Alistipes (mean: 2.1% vs. 2.6%) than ST131-negative participants. In the longitudinal analysis, Collinsella abundance correlated positively with ST131-H30R carriage status and negatively with the loss of ST131-H30R. Conversely, Alistipes corresponded with the loss and persistent absence of ST131-H30R even in the presence of a household exposure. Interpretation: Abundances of specific fecal bacteria correlated with ST131-H30R carriage, persistence, and loss, suggesting their potential as targets for microbiome-based strategies to reduce carriage of ST131-H30R, a significant risk factor for invasive infections. Funding: This work was supported in part by National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R21AI117654 and UM1AI104681 and the Office of Research and Development, Department of Veterans Affairs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs.

Abstract Dolosigranulum pigrum—a lactic acid bacterium that is increasingly recognized as an important member of the nasal microbiome. Currently, there are limited rapid and low-cost options for confirming D. pigrum isolates and detecting D. pigrum in clinical specimens. Here we describe the design and validation of a novel PCR assay targeting D. pigrum that is both sensitive and specific. We designed a PCR assay targeting murJ, a single-copy core species gene identified through the analysis of 21 D. pigrum whole genome sequences. The assay achieved 100% sensitivity and 100% specificity against D. pigrum and diverse bacterial isolates and an overall 91.1% sensitivity and 100% specificity using nasal swabs, detecting D. pigrum at a threshold of 1.0 × 104 D. pigrum 16S rRNA gene copies per swab. This assay adds a reliable and rapid D. pigrum detection tool to the microbiome researcher toolkit investigating the role of generalist and specialist bacteria in the nasal environment.

A one-health perspective may provide new and actionable information about Escherichia coli transmission. E. coli colonizes a broad range of vertebrates, including humans and food-production animals, and is a leading cause of bladder, kidney, and bloodstream infections in humans. Substantial evidence supports foodborne transmission of pathogenic E. coli strains from food animals to humans. However, the relative contribution of foodborne zoonotic E. coli (FZEC) to the human extraintestinal disease burden and the distinguishing characteristics of such strains remain undefined. Using a comparative genomic analysis of a large collection of contemporaneous, geographically-matched clinical and meat-source E. coli isolates (n = 3111), we identified 17 source-associated mobile genetic elements – predominantly plasmids and bacteriophages – and integrated them into a novel Bayesian latent class model to predict the origins of clinical E. coli isolates. We estimated that approximately 8 % of human extraintestinal E. coli infections (mostly urinary tract infections) in our study population were caused by FZEC. FZEC strains were equally likely to cause symptomatic disease as non-FZEC strains. Two FZEC lineages, ST131-H22 and ST58, appeared to have particularly high virulence potential. Our findings imply that FZEC strains collectively cause more urinary tract infections than does any single non-E. coli uropathogenic species (e.g., Klebsiella pneumoniae). Our novel approach can be applied in other settings to identify the highest-risk FZEC strains, determine their sources, and inform new one-health strategies to decrease the heavy public health burden imposed by extraintestinal E. coli infections.

Transmission of livestock-associated Staphylococcus aureus clonal complex 9 (LA-SA CC9) between pigs raised on industrial hog operations (IHOs) and humans in the United States is poorly understood. We analyzed whole-genome sequences from 32 international S. aureus CC9 isolates and 49 LA-SA CC9 isolates from IHO pigs and humans who work on or live near IHOs in 10 pig-producing counties in North Carolina, USA. Bioinformatic analysis of sequence data from the 81 isolates demonstrated 3 major LA-SA CC9 clades. North Carolina isolates all fell within a single clade (C3). High-resolution phylogenetic analysis of C3 revealed 2 subclades of intermingled IHO pig and human isolates differing by 0–34 single-nucleotide polymorphisms. Our findings suggest that LA-SA CC9 from pigs and humans share a common source and provide evidence of transmission of antimicrobial-resistant LA-SA CC9 between IHO pigs and humans who work on or live near IHOs in North Carolina.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) can acquire phage-encoded immune modulators, such as the immune evasion cluster (IEC), which protects bacteria from components of the human innate immune system, and the enzyme TarP, which protects against antibody-mediated immune recognition. We used whole-genome sequencing and epidemiologic investigations to study the effects of IEC- and tarP-harboring phages on household transmission of LA-MRSA in North Denmark Region during 2004–2011. We reviewed information about all patients throughout Denmark who experienced LA-MRSA infection during 2007–2018 to determine whether IEC is associated with increased spread into the general population. Horizontal acquisition of IEC in the human host was associated with increased household transmission of LA-MRSA and spillover into the community and healthcare settings, whereas we found no evidence to suggest that IEC-positive LA-MRSA isolates have become self-sustainable in the general population. By contrast, TarP did not seem to influence household transmission of LA-MRSA.

ABSTRACT Escherichia coli sequence type 131 (ST131) is a major cause of urinary and bloodstream infections. Its association with extended-spectrum β-lactamases (ESBLs) significantly complicates treatment. Its best-described component is the rapidly expanding H30Rx clade, containing allele 30 of the type 1 fimbrial adhesin gene fimH. This lineage appears to have emerged in the United States and spread around the world in part due to the acquisition of the ESBL-encoding blaCTX-M-15 gene and resistance to fluoroquinolones. However, non-H30 ST131 sublineages with other acquired CTX-M-type resistance genes are also emerging. Based on whole-genome analyses, we describe here the presence of an (fimH) H27 E. coli ST131 sublineage that has recently caused an outbreak of community-acquired bacteremia and recurrent urinary tract infections (UTIs) in Denmark. This sublineage has acquired both a virulence plasmid (pAA) that defines the enteroaggregative E. coli (EAEC) diarrheagenic pathotype and multiple genes associated with extraintestinal E. coli (ExPEC); combined, these traits have made this particular ST131 sublineage successful at colonizing its human host and causing recurrent UTI. Moreover, using a historic World Health Organization (WHO) E. coli collection and publicly available genome sequences, we identified a global H27 EAEC ST131 sublineage that dates back as far as 1998. Most H27 EAEC ST131 isolates harbor pAA or pAA-like plasmids, and our analysis strongly implies a single ancestral acquisition among these isolates. These findings illustrate both the profound plasticity of this important pathogenic E. coli ST131 H27 sublineage and genetic acquisitions of EAEC-specific virulence traits that likely confer an enhanced ability to cause intestinal colonization. IMPORTANCE E. coli ST131 is an important extraintestinal pathogenic lineage. A signature characteristic of ST131 is its ability to asymptomatically colonize the gastrointestinal tract and then opportunistically cause extraintestinal infections, such as cystitis, pyelonephritis, and urosepsis. In this study, we identified an ST131 H27 sublineage that has acquired the enteroaggregative diarrheagenic phenotype, spread across multiple continents, and caused multiple outbreaks of community-acquired ESBL-associated bloodstream infections in Denmark. The strain’s ability to both cause diarrhea and innocuously colonize the human gastrointestinal tract may facilitate its dissemination and establishment in the community.

Abstract Background We sought to determine if the prevalence of antibiotic-resistant Escherichia coli differed across retail poultry products and among major production categories, including organic, “raised without antibiotics”, and conventional. Results We collected all available brands of retail chicken and turkey—including conventional, “raised without antibiotic”, and organic products—every two weeks from January to December 2012. In total, E. coli was recovered from 91% of 546 turkey products tested and 88% of 1367 chicken products tested. The proportion of samples contaminated with E. coli was similar across all three production categories. Resistance prevalence varied by meat type and was highest among E. coli isolates from turkey for the majority of antibiotics tested. In general, production category had little effect on resistance prevalence among E. coli isolates from chicken, although resistance to gentamicin and multidrug resistance did vary. In contrast, resistance prevalence was significantly higher for 6 of the antibiotics tested—and multidrug resistance—among isolates from conventional turkey products when compared to those labelled organic or “raised without antibiotics”. E. coli isolates from chicken varied strongly in resistance prevalence among different brands within each production category. Conclusion The high prevalence of resistance among E. coli isolates from conventionally-raised turkey meat suggests greater antimicrobial use in conventional turkey production as compared to “raised without antibiotics” and organic systems. However, among E. coli from chicken meat, resistance prevalence was more strongly linked to brand than to production category, which could be caused by brand-level differences during production and/or processing, including variations in antimicrobial use.

ABSTRACT Avian-pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) presumed to be zoonotic and to represent an external reservoir for extraintestinal infections in humans, including uropathogenic E. coli (UPEC) causing urinary tract infections. Comparative genomics has previously been applied to investigate whether APEC and human ExPEC are distinct entities. Even so, whole-genome-based studies are limited, and large-scale comparisons focused on single sequence types (STs) are not available yet. In this study, comparative genomic analysis was performed on 323 APEC and human ExPEC genomes belonging to sequence type 95 (ST95) to investigate whether APEC and human ExPEC are distinct entities. Our study showed that APEC of ST95 did not constitute a unique ExPEC branch and was genetically diverse. A large genetic overlap between APEC and certain human ExPEC was observed, with APEC located on multiple branches together with closely related human ExPEC, including nearly identical APEC and human ExPEC. These results illustrate that certain ExPEC clones may indeed have the potential to cause infection in both poultry and humans. Previously described ExPEC-associated genes were found to be encoded on ColV plasmids. These virulence-associated plasmids seem to be crucial for ExPEC strains to cause avian colibacillosis and are strongly associated with strains of the mixed APEC/human ExPEC clusters. The phylogenetic analysis revealed two distinct branches consisting of exclusively closely related human ExPEC which did not carry the virulence-associated plasmids, emphasizing a lower avian virulence potential of human ExPEC in relation to an avian host. IMPORTANCE APEC causes a range of infections in poultry, collectively called colibacillosis, and is the leading cause of mortality and is associated with major economic significance in the poultry industry. A growing number of studies have suggested APEC as an external reservoir of human ExPEC, including UPEC, which is a reservoir. ExPEC belonging to ST95 is considered one of the most important pathogens in both poultry and humans. This study is the first in-depth whole-genome-based comparison of ST95 E. coli which investigates both the core genomes as well as the accessory genomes of avian and human ExPEC. We demonstrated that multiple lineages of ExPEC belonging to ST95 exist, of which the majority may cause infection in humans, while only part of the ST95 cluster seem to be avian pathogenic. These findings further support the idea that urinary tract infections may be a zoonotic infection.

ABSTRACT The spread of livestock-associated methicillin-resistant Staphylococcus aureus clonal complex 398 (LA-MRSA CC398) within the Danish pig production system has been linked to an increased number of human infections. Yet, the population structure and transmission dynamics of this important pathogen remain poorly understood. In this study, whole-genome sequences from 371 LA-MRSA CC398 isolates collected between 2004 and 2015 were subjected to bioinformatic analyses. The isolates originated from Danish pig farms (n = 209) and people having livestock contact (n = 79). In addition, whole-genome sequence data from 82 isolates representing an international reference collection and 83 isolates from Danish patients were included in the analysis. The results demonstrated that the increasing prevalence of LA-MRSA CC398 in Danish pigs and patients was caused by clonal expansion of three dominant lineages. The results also showed that these lineages were enriched for the tetracycline resistance gene tet(K) and other determinants conferring resistance to some of the most frequently used antimicrobials in Danish pigs. The association between pig movements and the spread of LA-MRSA CC398 was assessed in a Poisson regression analysis of 17,009 pig movements into 273 farms with known LA-MRSA CC398 status. The results demonstrated that animal movements have played a critical role in the dissemination of LA-MRSA CC398 within the Danish pig production system, although other transmission routes may also have contributed. Consistent with this scenario, the genetic relatedness of isolates from different farms was positively correlated with the number of animal movements between the farms. IMPORTANCE Livestock-associated methicillin-resistant Staphylococcus aureus clonal complex CC398 (LA-MRSA CC398) is resistant to nearly all β-lactams and several non-β-lactam antimicrobials. Over the last decade, it has become widespread in pig farms across Europe and is now an important cause of human infections in countries with previously low levels of MRSA, such as the Netherlands and Denmark. The hitherto uncontrolled spread of LA-MRSA CC398 underscores an urgent need to understand its epidemiology in order to develop evidence-based interventions. This study demonstrates that pig movements between farms in combination with increased bacterial resistance to specific antibiotics and heavy metals were important drivers of the rapid spread of LA-MRSA CC398 in the Danish pig production system. These findings should be taken into consideration when researchers and policy makers evaluate and decide on actions and policies to limit the spread of LA-MRSA CC398 and other pathogens in food animals.

ABSTRACT Escherichia coli sequence type 131 (ST131) has emerged rapidly to become the most prevalent extraintestinal pathogenic E. coli clones in circulation today. Previous investigations appeared to exonerate retail meat as a source of human exposure to ST131; however, these studies focused mainly on extensively multidrug-resistant ST131 strains, which typically carry allele 30 of the fimH type 1 fimbrial adhesin gene (ST131-H30). To estimate the frequency of extraintestinal human infections arising from foodborne ST131 strains without bias toward particular sublineages or phenotypes, we conducted a 1-year prospective study of E. coli from meat products and clinical cultures in Flagstaff, Arizona. We characterized all isolates by multilocus sequence typing, fimH typing, and core genome phylogenetic analyses, and we screened isolates for avian-associated ColV plasmids as an indication of poultry adaptation. E. coli was isolated from 79.8% of the 2,452 meat samples and 72.4% of the 1,735 culture-positive clinical samples. Twenty-seven meat isolates were ST131 and belonged almost exclusively (n = 25) to the ST131-H22 lineage. All but 1 of the 25 H22 meat isolates were from poultry products, and all but 2 carried poultry-associated ColV plasmids. Of the 1,188 contemporaneous human clinical E. coli isolates, 24 were ST131-H22, one-quarter of which occurred in the same high-resolution phylogenetic clades as the ST131-H22 meat isolates and carried ColV plasmids. Molecular clock analysis of an international ST131-H22 genome collection suggested that ColV plasmids have been acquired at least six times since the 1940s and that poultry-to-human transmission is not limited to the United States. IMPORTANCE E. coli ST131 is an important extraintestinal pathogen that can colonize the gastrointestinal tracts of humans and food animals. Here, we combined detection of accessory traits associated with avian adaptation (ColV plasmids) with high-resolution phylogenetics to quantify the portion of human infections caused by ST131 strains of food animal origin. Our results suggest that one ST131 sublineage—ST131-H22—has become established in poultry populations around the world and that meat may serve as a vehicle for human exposure and infection. ST131-H22 is just one of many E. coli lineages that may be transmitted from food animals to humans. Additional studies that combine detection of host-associated accessory elements with phylogenetics may allow us to quantify the total fraction of human extraintestinal infections attributable to food animal E. coli strains.

ABSTRACT Carbadox is an antibiotic used to control dysentery and promote growth in swine in the United States; however, the drug also causes tumors and birth defects in laboratory animals. Despite this and because the drug has no analogs in human medicine, it is not considered “medically important” and can be used in livestock without veterinarian oversight. In their recent study, T. A. Johnson et al. (mBio 8:e00709-17, 2017, https://doi.org/10.1128/mBio.00709-17 ) demonstrated that carbadox has profound effects on the swine gut microbiome, including the induction of transducing phage carrying tetracycline, aminoglycoside, and beta-lactam resistance genes. In swine production, carbadox can be used in conjunction with other antibiotics (e.g., oxytetracycline) that could fuel the emergence of strains carrying phage-encoded resistance determinants. Johnson et al.’s findings underscore the potential unforeseen consequences of using antibiotics in livestock production and call into question our current methods for classifying whether or not a veterinary drug has relevance to human health.

ABSTRACT Sexual transmission of HIV requires exposure to the virus and infection of activated mucosal immune cells, specifically CD4+ T cells or dendritic cells. The foreskin is a major site of viral entry in heterosexual transmission of HIV. Although the probability of acquiring HIV from a sexual encounter is low, the risk varies even after adjusting for known HIV risk factors. The genital microbiome may account for some of the variability in risk by interacting with the host immune system to trigger inflammatory responses that mediate the infection of mucosal immune cells. We conducted a case-control study of uncircumcised participants nested within a randomized-controlled trial of male circumcision in Rakai, Uganda. Using penile (coronal sulcus) swabs collected by study personnel at trial enrollment, we characterized the penile microbiome by sequencing and real-time PCR and cytokine levels by electrochemiluminescence assays. The absolute abundances of penile anaerobes at enrollment were associated with later risk of HIV seroconversion, with a 10-fold increase in Prevotella, Dialister, Finegoldia, and Peptoniphilus increasing the odds of HIV acquisition by 54 to 63%, after controlling for other known HIV risk factors. Increased abundances of anaerobic bacteria were also correlated with increased cytokines, including interleukin-8, which can trigger an inflammatory response that recruits susceptible immune cells, suggesting a mechanism underlying the increased risk. These same anaerobic genera can be shared between heterosexual partners and are associated with increased HIV acquisition in women, pointing to anaerobic dysbiosis in the genital microbiome and an accompanying inflammatory response as a novel, independent, and transmissible risk factor for HIV infection. IMPORTANCE We found that uncircumcised men who became infected by HIV during a 2-year clinical trial had higher levels of penile anaerobes than uncircumcised men who remained HIV negative. We also found that having higher levels of penile anaerobes was also associated with higher production of immune factors that recruit HIV target cells to the foreskin, suggesting that anaerobes may modify HIV risk by triggering inflammation. These anaerobes are known to be shared by heterosexual partners and are associated with HIV risk in women. Therefore, penile anaerobes may be a sexually transmissible risk factor for HIV, and modifying the penile microbiome could potentially reduce HIV acquisition in both men and women.

Staphylococcus aureus strain sequence type (ST) 398 has emerged during the last decade, largely among persons who have contact with swine or other livestock. Although colonization with ST398 is common in livestock workers, infections are not frequently documented. We report recurrent ST398-IIa infection in an Iowa farmer in contact with swine and cattle.

ABSTRACT The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B− plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B− plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring blaCTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

ABSTRACT Escherichia coli sequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration of blaCTX-M within subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, the blaCTX-M-14/14-like group. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. IMPORTANCE Escherichia coli, perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specific E. coli lineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.

ABSTRACT Bacterial vaginosis (BV) is a common vaginal bacterial imbalance associated with risk for HIV and poor gynecologic and obstetric outcomes. Male circumcision reduces BV-associated bacteria on the penis and decreases BV in female partners, but the link between penile microbiota and female partner BV is not well understood. We tested the hypothesis that having a female partner with BV increases BV-associated bacteria in uncircumcised men. We characterized penile microbiota composition and density (i.e., the quantity of bacteria per swab) by broad-coverage 16S rRNA gene-based sequencing and quantitative PCR (qPCR) in 165 uncircumcised men from Rakai, Uganda. Associations between penile community state types (CSTs) and female partner's Nugent score were assessed. We found seven distinct penile CSTs of increasing density (CST1 to 7). CST1 to 3 and CST4 to 7 were the two major CST groups. CST4 to 7 had higher prevalence and abundance of BV-associated bacteria, such as Mobiluncus and Dialister, than CST1 to 3. Men with CST4 to 7 were significantly more likely to have a female partner with a high Nugent score (P = 0.03). Men with two or more extramarital partners were significantly more likely to have CST4 to 7 than men with only marital partners (CST4 to 7 prevalence ratio, 1.84; 95% confidence interval [CI], 1.16 to 2.92). Female partner Nugent BV is significantly associated with penile microbiota. Our data support the exchange of BV-associated bacteria through intercourse, which may explain BV recurrence and persistence. IMPORTANCE Bacterial vaginosis (BV) is sexually associated but not considered a sexually transmitted disease. Our findings suggest that the uncircumcised penis is an important niche for BV-associated genital anaerobes. In addition, we found a link between extramarital sexual relationships and BV-associated bacteria in men, which parallels earlier findings of the association between sexual activity and BV in women. This suggests the sexual transmissibility of BV-associated bacteria. Reducing bacterial exchange by barrier methods and managing carriage of BV-associated bacteria in men may decrease BV persistence and recurrence in women.

ABSTRACT For centuries, cholera has been one of the most feared diseases. The causative agent Vibrio cholerae is a waterborne Gram-negative enteric pathogen eliciting a severe watery diarrheal disease. In October 2010, the seventh pandemic reached Haiti, a country that had not experienced cholera for more than a century. By using whole-genome sequence typing and mapping strategies of 116 serotype O1 strains from global sources, including 44 Haitian genomes, we present a detailed reconstructed evolutionary history of the seventh pandemic with a focus on the Haitian outbreak. We catalogued subtle genomic alterations at the nucleotide level in the genome core and architectural rearrangements from whole-genome map comparisons. Isolates closely related to the Haitian isolates caused several recent outbreaks in southern Asia. This study provides evidence for a single-source introduction of cholera from Nepal into Haiti followed by rapid, extensive, and continued clonal expansion. The phylogeographic patterns in both southern Asia and Haiti argue for the rapid dissemination of V. cholerae across the landscape necessitating real-time surveillance efforts to complement the whole-genome epidemiological analysis. As eradication efforts move forward, phylogeographic knowledge will be important for identifying persistent sources and monitoring success at regional levels. The results of molecular and epidemiological analyses of this outbreak suggest that an indigenous Haitian source of V. cholerae is unlikely and that an indigenous source has not contributed to the genomic evolution of this clade. IMPORTANCE In this genomic epidemiology study, we have applied high-resolution whole-genome-based sequence typing methodologies on a comprehensive set of genome sequences that have become available in the aftermath of the Haitian cholera epidemic. These sequence resources enabled us to reassess the degree of genomic heterogeneity within the Vibrio cholerae O1 serotype and to refine boundaries and evolutionary relationships. The established phylogenomic framework showed how outbreak isolates fit into the global phylogeographic patterns compared to a comprehensive globally and temporally diverse strain collection and provides strong molecular evidence that points to a nonindigenous source of the 2010 Haitian cholera outbreak and refines epidemiological standards used in outbreak investigations for outbreak inclusion/exclusion following the concept of genomic epidemiology. The generated phylogenomic data have major public health relevance in translating sequence-based information to assist in future diagnostic, epidemiological, surveillance, and forensic studies of cholera.

ABSTRACT Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. IMPORTANCE With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

ABSTRACT The Escherichia coli sequence type 131 (ST131) clone is notorious for extraintestinal infections, fluoroquinolone resistance, and extended-spectrum beta-lactamase (ESBL) production, attributable to a CTX-M-15-encoding mobile element. Here, we applied pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing to reconstruct the evolutionary history of the ST131 clone. PFGE-based cluster analyses suggested that both fluoroquinolone resistance and ESBL production had been acquired by multiple ST131 sublineages through independent genetic events. In contrast, the more robust whole-genome-sequence-based phylogenomic analysis revealed that fluoroquinolone resistance was confined almost entirely to a single, rapidly expanding ST131 subclone, designated H30-R. Strikingly, 91% of the CTX-M-15-producing isolates also belonged to a single, well-defined clade nested within H30-R, which was named H30-Rx due to its more extensive resistance. Despite its tight clonal relationship with H30Rx, the CTX-M-15 mobile element was inserted variably in plasmid and chromosomal locations within the H30-Rx genome. Screening of a large collection of recent clinical E. coli isolates both confirmed the global clonal expansion of H30-Rx and revealed its disproportionate association with sepsis (relative risk, 7.5; P < 0.001). Together, these results suggest that the high prevalence of CTX-M-15 production among ST131 isolates is due primarily to the expansion of a single, highly virulent subclone, H30-Rx. IMPORTANCE We applied an advanced genomic approach to study the recent evolutionary history of one of the most important Escherichia coli strains in circulation today. This strain, called sequence type 131 (ST131), causes multidrug-resistant bladder, kidney, and bloodstream infections around the world. The rising prevalence of antibiotic resistance in E. coli is making these infections more difficult to treat and is leading to increased mortality. Past studies suggested that many different ST131 strains gained resistance to extended-spectrum cephalosporins independently. In contrast, our research indicates that most extended-spectrum-cephalosporin-resistant ST131 strains belong to a single highly pathogenic subclone, called H30-Rx. The clonal nature of H30-Rx may provide opportunities for vaccine or transmission prevention-based control strategies, which could gain importance as H30-Rx and other extraintestinal pathogenic E. coli subclones become resistant to our best antibiotics.

Amplification fragment-length polymorphism (AFLP) analysis has proven to be a powerful tool for developing a large number of reliable genetic markers across a wide variety of organisms. Often it is desirable to further characterize these markers by obtaining internal and flanking sequence information. Here, we present a systematic approach for obtaining such information from AFLP markers. AFLP fragments can be isolated from dried polyacryamide sequencing gels (that have been stored for extended periods of time), amplified using PCR and subjected to sequence analysis. Outwardly oriented locus-specific primers are designed from the internal sequence and used in conjunction with adapter primers to amplify unknown regions that flank the internal sequence from up to 22 different restriction-ligation (R-L) reactions. This often results in multiple reactions yielding products of appropriate size and specificity for direct sequencing without the need for a nested PCR, extensive gel purification or subcloning. The detailed protocol is presented with PCR results from a variable AFLP fragment from Bacillus anthracis.

ABSTRACT Male circumcision reduces female-to-male HIV transmission. Hypothesized mechanisms for this protective effect include decreased HIV target cell recruitment and activation due to changes in the penis microbiome. We compared the coronal sulcus microbiota of men from a group of uncircumcised controls (n = 77) and from a circumcised intervention group (n = 79) at enrollment and year 1 follow-up in a randomized circumcision trial in Rakai, Uganda. We characterized microbiota using16S rRNA gene-based quantitative PCR (qPCR) and pyrosequencing, log response ratio (LRR), Bayesian classification, nonmetric multidimensional scaling (nMDS), and permutational multivariate analysis of variance (PerMANOVA). At baseline, men in both study arms had comparable coronal sulcus microbiota; however, by year 1, circumcision decreased the total bacterial load and reduced microbiota biodiversity. Specifically, the prevalence and absolute abundance of 12 anaerobic bacterial taxa decreased significantly in the circumcised men. While aerobic bacterial taxa also increased postcircumcision, these gains were minor. The reduction in anaerobes may partly account for the effects of circumcision on reduced HIV acquisition. IMPORTANCE The bacterial changes identified in this study may play an important role in the HIV risk reduction conferred by male circumcision. Decreasing the load of specific anaerobes could reduce HIV target cell recruitment to the foreskin. Understanding the mechanisms that underlie the benefits of male circumcision could help to identify new intervention strategies for decreasing HIV transmission, applicable to populations with high HIV prevalence where male circumcision is culturally less acceptable.

ABSTRACT Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

ABSTRACT Cholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio cholerae isolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. cholerae isolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. cholerae isolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks. IMPORTANCE Cholera is one of the ancient classical diseases and particularly prone to cause major outbreaks following major natural disasters, such as earthquakes and hurricanes, where the normal separation between sewage and drinking water is destroyed. This was the case following the 2010 earthquake in Haiti. Rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. Sequencing the genomes of bacteria can give detailed information on whether isolates from different sites share a common origin. We used this technology to sequence isolates of Vibrio cholerae from Nepal, identify single-nucleotide polymorphisms (SNPs), and compare these high-resolution genotypes to the complete genome sequences of isolates from the Haiti outbreak. We provide support for the hypothesis that the isolates were brought to Haiti from Nepal.

Retail poultry products are known sources of antibiotic-resistant Escherichia coli, a major human health concern. Consumers have a range of choices for poultry, including conventional, organic, kosher, and raised without antibiotics (RWA) – designations that are perceived to indicate differences in quality and safety. However, whether these categories vary in the frequency of contamination with antibiotic-resistant E. coli is unknown. We examined the occurrence of antibiotic-resistant E. coli on raw chicken marketed as conventional, organic, kosher and RWA. From April – June 2012, we purchased 213 samples of raw chicken from 15 locations in the New York City metropolitan area. We screened E. coli isolates from each sample for resistance to 12 common antibiotics. Although the organic and RWA labels restrict the use of antibiotics, the frequency of antibiotic-resistant E. coli tended to be only slightly lower for RWA, and organic chicken was statistically indistinguishable from conventional products that have no restrictions. Kosher chicken had the highest frequency of antibiotic-resistant E. coli, nearly twice that of conventional products, a result that belies the historical roots of kosher as a means to ensure food safety. These results indicate that production methods influence the frequency of antibiotic-resistant E. coli on poultry products available to consumers. Future research to identify the specific practices that cause the high frequency of antibiotic-resistant E. coli in kosher chicken could promote efforts to reduce consumer exposure to this potential pathogen.

Retail poultry products are known sources of antibiotic-resistant Escherichia coli, a major human health concern. Consumers have a range of choices for poultry, including conventional, organic, kosher, and raised without antibiotics (RWA)-designations that are perceived to indicate differences in quality and safety. However, whether these categories vary in the frequency of contamination with antibiotic-resistant E. coli is unknown. We examined the occurrence of antibiotic-resistant E. coli on raw chicken marketed as conventional, organic, kosher and RWA. From April – June 2012, we purchased 213 samples of raw chicken from 15 locations in the New York City metropolitan area. We screened E. coli isolates from each sample for resistance to 12 common antibiotics. Although the organic and RWA labels restrict the use of antibiotics, the frequency of antibiotic-resistant E. coli tended to be only slightly lower for RWA, and organic chicken was statistically indistinguishable from conventional products that have no restrictions. Kosher chicken had the highest frequency of antibiotic-resistant E. coli, nearly twice that of conventional products, a result that belies the historical roots of kosher as a means to ensure food safety. These results indicate that production methods influence the frequency of antibiotic-resistant E. coli on poultry products available to consumers. Future research to identify the specific practices that cause the high frequency of antibiotic-resistant E. coli in kosher chicken could promote efforts to reduce consumer exposure to this potential pathogen.