Finding, characterizing, and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive, and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19,933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3,565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.Originality-Significance Statement This work is applicable to a wide range of research. It helps to define conditions under which hybridization capture is useful regarding not only antimicrobial resistance specifically, but also more generally how to assess the ongoing utility of existing bait sets - giving objective criteria for when and by what strategies baits should be updated. We also provide a method for quantifying and comparing antimicrobial resistance genes (ARGs) similar to what is used for RNAseq experiments. This approach improves comparison of ARGs across environments. Thus, the work provides an improved foundation for ARG future studies, while cutting across traditional areas of microbiology and extending beyond.Competing Interest StatementThe EHS DNA lab provides oligonucleotide aliquots and library preparation services at cost, including some oligonucleotides and services used in this manuscript (baddna.uga.edu). JE and AD were employed by, and thereby have financial interest in, Daicel Arbor Biosciences, who provided the in-solution capture reagents used in this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

BACKGROUND: Since 2014, Neisseria gonorrhoeae (Ng) azithromycin (AZM) susceptibility has declined in the United States, but high-level azithromycin resistance (HL-AZMR) has been infrequent and sporadic. We describe a cluster of 14 Ng isolates with HL-AZMR identified in Indianapolis over 13 months. METHODS: Ng culture specimens (genital and extragenital) were collected from attendees of the Bell Flower Clinic. Isolates underwent antimicrobial susceptibility testing (AST) by Etest . AZM minimum inhibitory concentrations 256 g/ml were classified as HL-AZMR. Local disease intervention specialists interviewed patients whose isolates demonstrated HL-AZMR and conducted partner services. Relatedness of isolates was investigated by genomic analyses. RESULTS: During 2017-2018, 1,016 Ng isolates collected at the Bell Flower Clinic underwent AST. Fourteen isolates (1.4%) from 12 men collected over 13 months demonstrated HL-AZMR; all were cephalosporin-susceptible. Of the 12 men, nine were white and reported male sex partners. Nine of the men were able to be re-tested; all were cured with 250mg ceftriaxone plus 1g azithromycin. Two men named each other as partners; no other partners in common were reported. Genomic analysis demonstrated close relatedness of the HL-AZMR isolates and a novel combination of a mosaic-mtrR promoter along with 23S rRNA mutations that appear to have emerged from circulating strains. CONCLUSIONS: The close genetic relatedness with limited epidemiological linkages between patients highlights the challenges of gonorrhea partner investigations and suggests undetected local transmission. Local AST, rapid public health action, and epidemiologic investigations combined with genomic analysis provides a multi-pronged approach to understanding an STD outbreak.

BACKGROUND: Since 2014, Neisseria gonorrhoeae (Ng) azithromycin (AZM) susceptibility has declined in the United States, but high-level azithromycin resistance (HL-AZMR) has been infrequent and sporadic. We describe a cluster of 14 Ng isolates with HL-AZMR identified in Indianapolis over 13 months. METHODS: Ng culture specimens (genital and extragenital) were collected from attendees of the Bell Flower Clinic. Isolates underwent antimicrobial susceptibility testing (AST) by Etest ®. AZM minimum inhibitory concentrations ≥256 µg/ml were classified as HL-AZMR. Local disease intervention specialists interviewed patients whose isolates demonstrated HL-AZMR and conducted partner services. Relatedness of isolates was investigated by genomic analyses. RESULTS: During 2017-2018, 1,016 Ng isolates collected at the Bell Flower Clinic underwent AST. Fourteen isolates (1.4%) from 12 men collected over 13 months demonstrated HL-AZMR; all were cephalosporin-susceptible. Of the 12 men, nine were white and reported male sex partners. Nine of the men were able to be re-tested; all were cured with 250 mg ceftriaxone plus 1g azithromycin. Two men named each other as partners; no other partners in common were reported. Genomic analysis demonstrated close relatedness of the HL-AZMR isolates and a novel combination of a mosaic-mtrR promoter along with 23S rRNA mutations that appear to have emerged from circulating strains. CONCLUSIONS: The close genetic relatedness with limited epidemiological linkages between patients highlights the challenges of gonorrhea partner investigations and suggests undetected local transmission. Local AST, rapid public health action, and epidemiologic investigations combined with genomic analysis provides a multi-pronged approach to understanding an STD outbreak.

We would like to extend our gratitude to Drs. Deng and Klausner for their interest in our article, in which we phylogenetically characterized gonococcal isolates collected through national sentinel surveillance and circulating in the United States, between 2014 and 2016 [1]. We identified 2 major subpopulations of strains associated with reduced susceptibility to either azithromycin (multilocus sequence typing [MLST] ST9363) or cephalosporins (MLST ST1901), and we detailed the evolution of several strains that possessed mutations that were not observed in the United States in 2000–2013. | | Antimicrobial resistance (AMR) in the gonococcus represents a major public health threat, chiefly due to its rapidly evolving nature and the increasingly limited number of available treatment options. Although routine surveillance of AMR in circulating strains is essential in monitoring this threat, an equally important practice involves the promotion of antibiotic stewardship in limiting the spread of resistance. It is notable that the Centers for Disease Control and Prevention’s Gonococcal Isolate Surveillance Project, a historic sentinel surveillance program, has tracked AMR trends for over 30 years and, more recently, included whole-genome sequencing (WGS) of a subset of isolates [2]. As a result, the implementation of WGS has generated massive quantities of genomic data that can be used in tandem with data from traditional antimicrobial susceptibility testing to examine AMR trends. In addition, the release of these data to the public repositories has facilitated the ability of other investigators to conduct a myriad of follow-up studies.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

BACKGROUND: Given the lack of new antimicrobials or a vaccine, understanding the evolutionary dynamics of Neisseria gonorrhoeae is a significant public and global health priority. We investigated the emergence and spread of gonococcal strains with decreased susceptibility to cephalosporins and azithromycin using detailed genomic analyses of gonococcal isolates collected in the United States from 2014 to 2016. METHODS: We sequenced the genomes of 649 isolates collected through the Gonococcal Isolate Surveillance Project (GISP). We examined the genetic relatedness of isolates and assessed associations between clades and various genotypic and phenotypic combinations. RESULTS: We identified a large and clonal lineage of strains (MLST ST9363) associated with elevated azithromycin MICs (AZI em), characterized by a mosaic mtr locus (C-substitution in the mtrR promoter, mosaic mtrR and mtrD). Mutations in 23S rRNA were sporadically distributed among AZI em strains. Another clonal group (MLST ST1901) possessed seven unique PBP2 patterns, and it shared common mutations in other genes associated with cephalosporin resistance. CONCLUSIONS: Whole genome sequencing methods can enhance monitoring of antimicrobial resistant gonococcal strains by identifying gonococcal populations containing mutations of concern. These methods could inform the development of point-of-care diagnostic tests designed to determine the specific antibiotic susceptibility profile of a gonococcal infection within a patient.

BACKGROUND: Recognizing the importance of having a broad exploration of how cultural perspectives may shape thinking about ethical considerations, the Centers for Disease Control and Prevention (CDC) funded four regional meetings in Africa, Asia, Latin America, and the Eastern Mediterranean to explore these perspectives relevant to pandemic influenza preparedness and response. The meetings were attended by 168 health professionals, scientists, academics, ethicists, religious leaders, and other community members representing 40 countries in these regions. METHODS: We reviewed the meeting reports, notes and stories and mapped outcomes to the key ethical challenges for pandemic influenza response described in the World Health Organization's (WHO's) guidance, Ethical Considerations in Developing a Public Health Response to Pandemic Influenza: transparency and public engagement, allocation of resources, social distancing, obligations to and of healthcare workers, and international collaboration. RESULTS: The important role of transparency and public engagement were widely accepted among participants. However, there was general agreement that no "one size fits all" approach to allocating resources can address the variety of economic, cultural and other contextual factors that must be taken into account. The importance of social distancing as a tool to limit disease transmission was also recognized, but the difficulties associated with this measure were acknowledged. There was agreement that healthcare workers often have competing obligations and that government has a responsibility to assist healthcare workers in doing their job by providing appropriate training and equipment. Finally, there was agreement about the importance of international collaboration for combating global health threats. CONCLUSION: Although some cultural differences in the values that frame pandemic preparedness and response efforts were observed, participants generally agreed on the key ethical principles discussed in the WHO's guidance. Most significantly the input gathered from these regional meetings pointed to the important role that procedural ethics can play in bringing people and countries together to respond to the shared health threat posed by a pandemic influenza despite the existence of cultural differences.

Individualized risk assessments during HIV testing are an integral component of prevention counseling, a currently recommended behavioral intervention for patients in high-risk settings. Additionally, aggregate risk assessment data are the source of aggregate behavioral statistics that inform prevention programs and allocation of resources. Consequently, inaccurate or incomplete risk behavior disclosure during test counseling may impact the efficacy of the counseling intervention, as well as bias aggregate behavioral statistics. To quantify client-reported accuracy during the risk assessment and identify barriers and facilitators to risk behavior disclosure, we interviewed young men accessing HIV testing services in a southeastern United States city using mixed methodology. Data were collected from August 2007 to April 2008. Based on data collected via an audio and computer-assisted self-interview (n=203), over 30% of men reported that they were not accurate during the risk assessment. Participants reported numerous interpersonal facilitators to complete disclosure. During qualitative interviews (n=25), participants revealed that many did not understand the purpose of the risk assessment. Findings suggest that risk assessments completed during HIV test counseling may be incomplete. Modifications to the risk assessment process, including better explaining the role of the risk assessment in prevention counseling, may increase the validity of the data. Mary Ann Liebert, Inc.

Human immunodeficiency virus (HIV) prevention programs and agencies are fighting growing rates of infection with decreasing resources. Identification of gaps in HIV prevention services can help inform prevention funding and program policies. To describe HIV prevention needs in a southern U.S. state, we conducted face-to-face interviews with prevention agencies and persons considered by others in their community to be "influential informants" of the community's HIV prevention services in a sample of counties in North Carolina. Using county as the unit of analysis (n = 10), we investigated differences in gaps by community characteristics, such as disparities in sexually transmitted disease rates. Lack of programs and problems with service program coordination/cooperation were reported frequently by rural counties. The most commonly reported barrier to meeting the needs of persons at risk for HIV was funding, followed by stigma. Findings from this study can inform local and regional planners on how to efficiently target prevention programs, including programs aimed at reducing racial and geographic disparities in sexually transmitted diseases, such as HIV.

OBJECTIVES: We assessed agreement of reported gender of sex partners in 2 statewide HIV databases linked by client identifiers. METHODS: Counseling, testing, and referral (CTR) records on all men aged 18 to 30 years who tested newly positive for HIV in North Carolina between 2000 and 2005 were matched to data abstracted from partner counseling and referral services (PCRS) records. We compared client-reported gender of sex partners at the time of testing (CTR records) with those reported during postdiagnosis partner notification (PCRS records). RESULTS: PCRS records appeared to be a more complete measure of the gender of sex partners. Of the 212 men who told their HIV test counselor that they had only had female sex partner or partners in their lifetime, 62 (29.2%) provided contact information for male sex partner(s) during partner notification. CONCLUSIONS: During the test counseling risk assessment, many men did not fully report the gender of their sex partners; this suggests that CTR data may not fully capture clients' risk behaviors.