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Background Genomic epidemiology studies of gonorrhea in the United States have primarily focused on national surveillance for antibiotic resistance, and patterns of local transmission between demographic groups of resistant and susceptible strains are unknown. Methods We analyzed a convenience sample of genome sequences, antibiotic susceptibility, and patient data from 897 gonococcal isolates cultured at the New York City (NYC) Public Health Laboratory from NYC Department of Health and Mental Hygiene (DOHMH) Sexual Health Clinic (SHC) patients, primarily in 2012–2013. We reconstructed the gonococcal phylogeny, defined transmission clusters using a 10 nonrecombinant single nucleotide polymorphism threshold, tested for clustering of demographic groups, and placed NYC isolates in a global phylogenetic context. Results The NYC gonococcal phylogeny reflected global diversity with isolates from 22/23 of the prevalent global lineages (96%). Isolates clustered on the phylogeny by patient sexual behavior (P < .001) and race/ethnicity (P < .001). Minimum inhibitory concentrations were higher across antibiotics in isolates from men who have sex with men compared to heterosexuals (P < .001) and white heterosexuals compared to black heterosexuals (P < .01). In our dataset, all large transmission clusters (≥10 samples) of N. gonorrhoeae were susceptible to ciprofloxacin, ceftriaxone, and azithromycin, and comprised isolates from patients across demographic groups. Conclusions All large transmission clusters were susceptible to gonorrhea therapies, suggesting that resistance to empiric therapy was not a main driver of spread, even as risk for resistance varied across demographic groups. Further study of local transmission networks is needed to identify drivers of transmission., We used Neisseria gonorrhoeae genomes together with clinical and demographic data to study transmission in New York City. Our results indicate transmission across demographic groups and suggest that antibiotic resistance is not the main driver of gonorrhea spread.

The sexually transmitted infection gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, can cause urethritis, cervicitis, and systemic disease, among other manifestations. N. gonorrhoeae has rapidly rising incidence along with increasing levels of antibiotic resistance to a broad range of drugs including first-line treatments. The rise in resistance has led to fears of untreatable gonorrhea causing substantial disease globally. In this review, we will describe multiple approaches being undertaken to slow and control this spread of resistance. First, a number of old drugs have been repurposed and new drugs are being developed with activity against Neisseria gonorrhoeae. Second, vaccine development, long an important goal, is advancing. Third, new diagnostics promise rapid detection of antibiotic resistance and a shift from empiric to tailored treatment. The deployment of these new tools for addressing the challenge of antibiotic resistance will require careful consideration to provide optimal care for all patients while extending the lifespan of treatment regimens.

Antimicrobial resistance in Neisseria gonorrhoeae is a global health concern. Strains from two internationally circulating sequence types, ST-7363 and ST-1901, have acquired resistance to treatment with third-generation cephalosporins mainly due to the emergence of mosaic penA alleles. These two STs were first detected in Japan; however, when and how the mosaic penA alleles emerged and spread to other countries remains unknown. Here, we addressed the evolution of penA alleles by obtaining complete genomes from three Japanese ST-1901 clinical isolates harboring mosaic penA allele 34 (penA-34) dating from 2005 and generating a phylogenetic representation of 1,075 strains sampled from 37 countries. We also sequenced the genomes of 103 Japanese ST-7363 N. gonorrhoeae isolates from 1996-2005 and reconstructed a phylogeny including 88 previously sequenced genomes. Based on an estimate of the time of emergence of ST-1901 harboring mosaic penA-34 and ST-7363 harboring mosaic penA-10, and >300 additional genome sequences of Japanese strains representing multiple STs isolated in 1996-2015, we suggest that penA-34 in ST-1901 was generated from penA-10 via recombination with another Neisseria species, followed by a second recombination event with a gonococcal strain harboring wildtype penA-1. Following the acquisition of penA-10 in ST-7363, a dominant sub-lineage rapidly acquired fluoroquinolone resistance mutations at GyrA 95 and ParC 87-88, possibly due to independent mutations rather than horizontal gene transfer. Literature data suggest the emergence of these resistance determinants may reflect selection from the standard treatment regimens in Japan at that time. Our findings highlight how recombination and antibiotic use across and within Neisseria species intersect in driving the emergence and spread of drug-resistant gonorrhea.Author summaryAntimicrobial resistance is recognized as one of the greatest threats to human health, and Neisseria gonorrhoeae resistance is classified as one of the most urgent. The two major internationally spreading lineages resistant. to first line drugs likely originated in Japan, but when and how their genetic resistance determinants emerged remain unknown. In this study, we conducted an evolutionary analysis using clinical N. gonorrhoeae isolates from 37 countries, including a historical collection of Japanese isolates, to investigate the emergence of resistance in each of the two major lineages. We showed that the penA allele responsible for resistance to cephalosporins, the first-line treatment for gonorrhea, was possibly generated by two recombination events, one from another Neisseria species and one from another N. gonorrhoeae lineage. We also showed that mutations responsible for resistance to a previously widely used antibiotic treatment occurred twice independently in one of the two major lineages. The emergence of the genetic resistance determinants potentially reflects selection from the standard treatment regimen at that time. Our findings highlight how recombination (horizontal gene transfer) and antibiotic use across and within a bacterial species intersect in driving the emergence and spread of antimicrobial resistance genes and mutations.

Background The mechanisms driving the recent decline in outpatient antibiotic prescribing are unknown. We estimated the extent to which reductions in the number of antibiotic prescriptions filled per outpatient visit (stewardship) and reductions in the monthly rate of outpatient visits (observed disease) for infectious disease conditions each contributed to the decline in outpatient antibiotic prescribing in Massachusetts between 2011 and 2015. Methods Outpatient medical and pharmacy claims from the Massachusetts All-Payer Claims Database were used to estimate rates of antibiotic prescribing and outpatient visits for 20 medical conditions and their contributions to the overall decline in antibiotic prescribing. Trends were compared with those in the National Ambulatory Medical Care Survey (NAMCS). Results Between 2011 and 2015, the January and July antibiotic-prescribing rates per 1000 individuals in Massachusetts declined by 18.9% and 13.6%, respectively. The monthly rate of outpatient visits per 1000 individuals in Massachusetts declined (P Conclusions The decline in antibiotic prescribing in Massachusetts was driven both by a decline in observed disease and improved antibiotic stewardship, in agreement with national trends. A focus on infectious disease prevention should be considered alongside antibiotic stewardship as a means to reduce antibiotic prescribing.

BackgroundWhile Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance, much of the circulating population remains susceptible to historical treatment regimens. Point-of-care diagnostics that report susceptibility could allow for reintroduction of these regimens, but development of such diagnostics has been limited to ciprofloxacin, for which susceptibility can be predicted from a single locus.MethodsWe assembled a dataset of 12,045 N. gonorrhoeae genomes with phenotypic resistance data for tetracycline (n = 3,611) and penicillin (n = 6,935). Using conditional genome wide association studies (GWAS), we sought to define genetic variants associated with susceptibility to penicillin and tetracycline. We evaluated the sensitivity and specificity of these variants for predicting susceptibility and non-resistance in our collection of gonococcal genomes.FindingsIn our conditional penicillin GWAS, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was significantly associated with penicillin susceptibility and had the highest negative effect size of significant variants (p = 5.0 × 10−14, β = -2.5). In combination with the absence of blaTEM, this variant predicted penicillin susceptibility with high specificity (99.8%) and modest sensitivity (36.7%). For tetracycline, the wild type allele at rpsJ codon 57, encoding valine, was significantly associated with tetracycline susceptibility (p = 5.6 × 10−16, β = -1.6) after conditioning on the presence of tetM. The combination of rpsJ codon 57 allele and tetM absence predicted tetracycline susceptibility with high specificity (97.2%) and sensitivity (88.7%).InterpretationAs few as two genetic loci can predict susceptibility to penicillin and tetracycline in N. gonorrhoeae with high specificity. Molecular point-of-care diagnostics targeting these loci have the potential to increase available treatments for gonorrhea.FundingNational Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family FoundationResearch in ContextEvidence before this studyWe searched PubMed with the terms “Neisseria gonorrhoeae” and “diagnostic” or “assay” plus “penicillin” or “tetracycline” for reports in any language published up to July 1, 2021. We additionally searched for “Neisseria gonorrhoeae” and “genome wide association study”. We found that previously proposed molecular diagnostics for penicillin and tetracycline susceptibility either exclusively focused on plasmid-mediated resistance (i.e., targeting blaTEM or tetM) or did not include variants in genes encoding antibiotic targets (e.g., did not include penA or rpsJ). Targets for molecular surveillance have focused on resistance-associated alleles rather than susceptibility-associated alleles. We did not find any previous penicillin or tetracycline conditional genome wide association studies (GWAS) in N. gonorrhoeae.Added value of this studyTo identify targets for molecular diagnostics that predict penicillin and tetracycline susceptibility, we conducted GWAS conditioning on the presence of plasmid-mediated resistance determinants to detect chromosomal loci with the highest association with susceptibility. We discovered a sequence (penA_01) that differentiates susceptible isolates from those with a resistance-associated insertion at codon 345 and from those with mosaic penA alleles, which is associated with penicillin susceptibility. We also found that rpsJ codon 57 was the chromosomal locus contributing the most to tetracycline susceptibility. The combination of these chromosomal loci and the absence of plasmid encoded determinants predicts penicillin and tetracycline susceptibility with high specificity in both a large global collection of N. gonorrhoeae and a validation dataset consisting of recently published genomes from CDC’s Gonococcal Isolate Surveillance Program (GISP) surveillance collected in 2018.Implications of all the available evidenceThe chromosomal loci penA_01 and rpsJ codon 57 in combination with plasmid loci blaTEM and tetM are candidates for the development of point-of-care molecular diagnostics for penicillin and tetracycline susceptibility. The loci may be combined with the currently available ciprofloxacin susceptibility diagnostics to predict susceptibility to multiple antibiotics. Additionally, our study suggests that conditional GWAS focused on variants associated with susceptibility may be a promising approach to identify minimal sets of loci for molecular diagnostics and surveillance.

The analysis of genomes from populations of isolates can help address many questions relevant to the pathogen’s biology and epidemiology and thus inform on clinical and public health practice. These questions include the origins and evolution of the pathogen, its spread through populations, and the genes and genotypes associated with a pathogen’s antimicrobial resistance, virulence, and other phenotypes of interest. Here, I will discuss the application of population genomics to STIs, with a focus on Neisseria gonorrhoeae and with highlights from other sexually transmitted pathogens.

Background Antimicrobial-resistant (AMR) Neisseria gonorrhoeae is an urgent threat to public health, as strains resistant to at least one of the two last-line antibiotics used in empiric therapy of gonorrhoea, ceftriaxone and azithromycin, have spread internationally. Whole genome sequencing (WGS) data can be used to identify new AMR clones and transmission networks and inform the development of point-of-care tests for antimicrobial susceptibility, novel antimicrobials and vaccines. Community-driven tools that provide an easy access to and analysis of genomic and epidemiological data is the way forward for public health surveillance. Methods Here we present a public health-focussed scheme for genomic epidemiology of N. gonorrhoeae at Pathogenwatch (https://pathogen.watch/ngonorrhoeae). An international advisory group of experts in epidemiology, public health, genetics and genomics of N. gonorrhoeae was convened to inform on the utility of current and future analytics in the platform. We implement backwards compatibility with MLST, NG-MAST and NG-STAR typing schemes as well as an exhaustive library of genetic AMR determinants linked to a genotypic prediction of resistance to eight antibiotics. A collection of over 12,000 N. gonorrhoeae genome sequences from public archives has been quality-checked, assembled and made public together with available metadata for contextualization. Results AMR prediction from genome data revealed specificity values over 99% for azithromycin, ciprofloxacin and ceftriaxone and sensitivity values around 99% for benzylpenicillin and tetracycline. A case study using the Pathogenwatch collection of N. gonorrhoeae public genomes showed the global expansion of an azithromycin-resistant lineage carrying a mosaic mtr over at least the last 10 years, emphasising the power of Pathogenwatch to explore and evaluate genomic epidemiology questions of public health concern. Conclusions The N. gonorrhoeae scheme in Pathogenwatch provides customised bioinformatic pipelines guided by expert opinion that can be adapted to public health agencies and departments with little expertise in bioinformatics and lower-resourced settings with internet connection but limited computational infrastructure. The advisory group will assess and identify ongoing public health needs in the field of gonorrhoea, particularly regarding gonococcal AMR, in order to further enhance utility with modified or new analytic methods.

Background Novel therapeutic strategies are urgently needed for Neisseria gonorrhoeae, given its increasing antimicrobial resistance. Treatment of oropharyngeal N. gonorrhoeae infections has proven particularly challenging, with most reported treatment failures of the first-line drug ceftriaxone occurring at this site and lower cure rates in recent trials of new antibiotics reported for oropharyngeal infections compared with other sites of infection. However, the accessibility of the oropharynx to topical therapeutics provides an opportunity for intervention. Local delivery of a therapeutic at a high concentration would enable the use of non-traditional antimicrobial candidates, including biological molecules that exploit underlying chemical sensitivities of N. gonorrhoeae but lack the potency or pharmacokinetic profiles required for effective systemic administration. Methods Two classes of molecules that are thought to limit gonococcal viability in vivo, bile acids and short- and medium-chain fatty acids, were examined for rapid bactericidal activity. Results The bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), but not other bile acid species, exerted extremely rapid bactericidal properties against N. gonorrhoeae, reducing viability more than 100 000-fold after 1 min. The short-chain fatty acids formic acid and hexanoic acid shared this rapid bactericidal activity. All four molecules are effective against a phylogenetically diverse panel of N. gonorrhoeae strains, including clinical isolates with upregulated efflux pumps and resistance alleles to the most widely used classes of existing antimicrobials. DCA and CDCA are both approved therapeutics for non-infectious indications and are well-tolerated by cultured epithelial cells. Conclusions DCA and CDCA are attractive candidates for further development as anti-gonococcal agents.

Surveillance of antimicrobial resistance (AMR) is essential for clinical decision-making and for public health authorities to monitor patterns in resistance and evaluate the effectiveness of interventions and control measures. Existing AMR surveillance is typically based on reports from hospital laboratories and public health laboratories, comprising reports of pathogen frequencies and resistance frequencies among each species detected. Here we propose an improved framework for AMR surveillance, in which the unit of surveillance is patients with specific conditions, rather than biological samples of a particular type. In this ‘case-based’ surveillance, denominators as well as numerators will be clearly defined with clinical relevance and more comparable at the local, national and international level. In locations with sufficient resources, individual-based data on patient characteristics and full antibiotic susceptibility profiles would provide high-quality evidence for monitoring resistant pathogens of clinical importance, clinical treatment of infections and public health responses to outbreaks of infections with resistant bacteria.

Neisseria gonorrhoeae infections have been increasing globally, with prevalence rising across age groups. In this study, we report a case of disseminated gonococcal infection (DGI) involving a prosthetic joint, and we use whole-genome sequencing to characterize resistance genes, putative virulence factors, and the phylogenetic lineage of the infecting isolate. We review the literature on sequence-based prediction of antibiotic resistance and factors that contribute to risk for DGI. We argue for routine sequencing and reporting of invasive gonococcal infections to aid in determining whether an invasive gonococcal infection is sporadic or part of an outbreak and to accelerate understanding of the genetic features of N gonorrhoeae that contribute to pathogenesis.

A gonococcal vaccine could reduce antibiotic prescribing and associated resistance, but the potential gains have not been measured. We estimated the fraction of antibiotic prescribing in the US attributable to gonorrhea. Gonorrhea contributes to an outsized proportion of antibiotic prescriptions in young adults, males, and in the southern and western US. A gonococcal vaccine could substantially reduce antibiotic prescribing in these populations.

Background The first cases of extensively drug resistant gonorrhoea were recorded in the United Kingdom in 2018. There is a public health need for strategies on how to deploy existing and novel antibiotics to minimise the risk of resistance development. As rapid point-of-care tests (POCTs) to predict susceptibility are coming to clinical use, coupling the introduction of an antibiotic with diagnostics that can slow resistance emergence may offer a novel paradigm for maximising antibiotic benefits. Gepotidacin is a novel antibiotic with known resistance and resistance-predisposing mutations. In particular, a mutation that confers resistance to ciprofloxacin acts as the ‘stepping-stone’ mutation to gepotidacin resistance. Aim To investigate how POCTs detecting Neisseria gonorrhoeae resistance mutations for ciprofloxacin and gepotidacin can be used to minimise the risk of resistance development to gepotidacin. Methods We use individual-based stochastic simulations to formally investigate the aim. Results The level of testing needed to reduce the risk of resistance development depends on the mutation rate under treatment and the prevalence of stepping-stone mutations. A POCT is most effective if the mutation rate under antibiotic treatment is no more than two orders of magnitude above the mutation rate without treatment and the prevalence of stepping-stone mutations is 1–13%. Conclusion Mutation frequencies and rates should be considered when estimating the POCT usage required to reduce the risk of resistance development in a given population. Molecular POCTs for resistance mutations and stepping-stone mutations to resistance are likely to become important tools in antibiotic stewardship.

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility., Antibiotic resistance in Neisseria gonorrhoeae is rising, yet sometimes strains emerge that have reverted to susceptibility. Here, the authors find that selective pressures from the host may influence susceptibility through loss-of-function mutations in genes that encode for efflux pumps.

The goal of the Sexually Transmitted Infection Clinical Trial Group’s (STI-CTG) Antimicrobial Resistance (AMR) in Neisseria gonorrhoeae (NG) meeting was to assemble experts from academia, government, non-profit and industry to discuss the current state of research, gaps and challenges in research and technology as well as priorities and new directions to address the contsinued emergence of multi-drug resistant NG infections. Topics discussed at the meeting, that will be the focus of this article, include AMR NG global surveillance initiatives, the use of whole genome sequencing (WGS) and bioinformatics to understand mutations associated with AMR, mechanisms of AMR, and novel antibiotics, vaccines and other methods to treat AMR NG. Key points highlighted during the meeting include: (i) US and International surveillance programs to understand AMR in NG. (ii) The US National Strategy for combating antimicrobial resistant bacteria. (iii) Surveillance needs, challenges and novel technologies. (iv) Plasmid- and chromosomally-mediated mechanisms of AMR in NG, (v) Novel therapeutic (e.g., sialic acid analogs, FH/Fc fusion molecule, monoclonal antibodies, topoisomerase inhibitors, fluoroketolides, LpxC inhibitors) and preventative (e.g., peptide mimic) strategies to combat infection. The way forward will require renewed political will, new funding initiatives and collaborations across academic and commercial research and public health programs.

Background The rise of gonococcal antimicrobial resistance highlights the need for strategies that extend the clinically useful life span of antibiotics. Because there is limited evidence to support the current practice of switching empiric first-line antibiotic when resistance exceeds 5% in the population, our objective was to compare the impact of alternative strategies on the effective life spans of antibiotics and the overall burden of gonorrhea. Methods and findings We developed and calibrated a mathematical model of gonorrhea transmission among men who have sex with men (MSM) in the United States. We calibrated the model to the estimated prevalence of gonorrhea, the rate of gonorrhea cases, and the proportion of cases presenting symptoms among MSM in the US. We used this model to project the effective life span of antibiotics and the number of gonorrhea cases expected under current and alternative surveillance strategies over a 50-year simulation period. We demonstrate that compared to the current practice, a strategy that uses quarterly (as opposed to yearly) surveillance estimates and incorporates both the estimated prevalence of resistance and the trend in the prevalence of resistance to determine treatment guidelines could extend the effective life span of antibiotics by 0.83 years. This is equivalent to successfully treating an additional 80.1 (95% uncertainty interval: [47.7, 111.9]) gonorrhea cases per 100,000 MSM population each year with the first-line antibiotics without worsening the burden of gonorrhea. If the annual number of isolates tested for drug susceptibility is doubled, this strategy could increase the effective life span of antibiotics by 0.94 years, which is equivalent to successfully treating an additional 91.1 (54.3, 127.3) gonorrhea cases per 100,000 MSM population each year without increasing the incidence of gonorrhea. Study limitations include that our conclusions might not be generalizable to other settings because our model describes the transmission of gonorrhea among the US MSM population, and, to better capture uncertainty in the characteristics of current and future antibiotics, we chose to model hypothetical drugs with characteristics similar to the antibiotics commonly used in gonorrhea treatment. Conclusions Our results suggest that use of data from surveillance programs could be expanded to prolong the clinical effectiveness of antibiotics without increasing the burden of the disease. This highlights the importance of maintaining effective surveillance systems and the engagement of policy makers to turn surveillance findings into timely and effective decisions., Reza Yaesoubi and colleagues study possible approaches to delaying the appearance of antimicrobial resistance in treatment of gonorrhea., Author summary Why was this study done? Gonorrhea is the second most common notifiable disease in the United States and has developed resistance to all first-line antibiotics. The selection of antibiotics used for gonorrhea treatment is almost always empiric and based on guideline recommendations. There is limited evidence to support the current practice of switching the first-line antibiotic after resistance to it exceeds 5% in annual surveillance estimates. Our objective was to project how alternative strategies to inform the first-line treatment recommendations impact the life span of antibiotics and the overall burden of gonorrhea. What did the researchers do and find? We developed a mathematical model that describes the key characteristics of gonorrhea transmission among men who have sex with men (MSM) in the United States. Our model estimates the life span of antibiotics and the incidence of gonorrhea under current and alternative strategies for changing first-line empiric antibiotic treatment. We found that compared to the current practice, a strategy that 1) uses quarterly surveillance estimates and 2) incorporates both the estimated prevalence of resistance and the trend in the prevalence of resistance to determine treatment guidelines could extend the effective life span of antibiotics without worsening the burden of gonorrhea. What do these findings mean? This work suggests an opportunity to optimize the use of surveillance systems to slow the spread of antibiotic-resistant strains and control the burden of gonorrhea. This requires enhancing the surveillance systems (e.g., by allowing for more frequent reporting of estimates and a larger number of observations) and the engagement of policy makers to turn surveillance findings into timely decisions. Further studies are needed to investigate the generalizability of these conclusions.

Infections with Neisseria gonorrhoeae, a sexually transmitted pathogen that causes urethritis, cervicitis, and more severe complications, are increasing. Gonorrhea is typically treated with antibiotics; however, N. gonorrhoeae has rapidly acquired resistance to many antibiotic classes, and lineages with reduced susceptibility to the currently recommended therapies are emerging worldwide. In this review, we discuss the contributions of whole genome sequencing (WGS) to our understanding of resistant N. gonorrhoeae. Genomics has illuminated the evolutionary origins and population structure of N. gonorrhoeae and the magnitude of horizontal gene transfer within and between Neisseria species. WGS can be used to predict the susceptibility of N. gonorrhoeae based on known resistance determinants, track the spread of these determinants throughout the N. gonorrhoeae population, and identify novel loci contributing to resistance. WGS has also allowed more detailed epidemiological analysis of transmission of N. gonorrhoeae between individuals and populations than previously used typing methods. Ongoing N. gonorrhoeae genomics will complement other laboratory techniques to understand the biology and evolution of the pathogen, improve diagnostics and treatment in the clinic, and inform public health policies to limit the impact of antibiotic resistance.

Surveillance of drug-resistant bacteria is essential for healthcare providers to deliver effective empirical antibiotic therapy. However, traditional molecular epidemiology does not typically occur on a timescale that could affect patient treatment and outcomes. Here, we present a method called ‘genomic neighbour typing’ for inferring the phenotype of a bacterial sample by identifying its closest relatives in a database of genomes with metadata. We show that this technique can infer antibiotic susceptibility and resistance for both Streptococcus pneumoniae and Neisseria gonorrhoeae. We implemented this with rapid k-mer matching, which, when used on Oxford Nanopore MinION data, can run in real time. This resulted in the determination of resistance within 10 min (91% sensitivity and 100% specificity for S. pneumoniae and 81% sensitivity and 100% specificity for N. gonorrhoeae from isolates with a representative database) of starting sequencing, and within 4 h of sample collection (75% sensitivity and 100% specificity for S. pneumoniae) for clinical metagenomic sputum samples. This flexible approach has wide application for pathogen surveillance and may be used to greatly accelerate appropriate empirical antibiotic treatment., Genomic neighbour typing can be used to infer the antimicrobial susceptibility and resistance of a bacterial sample based on the genomes of closest relatives. Combined with MinION sequencing, it can rapidly determine microbial resistance for clinical samples within 4 h.

The relationship between antibiotic stewardship and population levels of antibiotic resistance remains unclear. In order to better understand shifts in selective pressure due to stewardship, we use publicly available data to estimate the effect of changes in prescribing on exposures to frequently used antibiotics experienced by potentially pathogenic bacteria that are asymptomatically colonizing the microbiome. We quantify this impact under four hypothetical stewardship strategies. In one scenario, we estimate that elimination of all unnecessary outpatient antibiotic use could avert 6% to 48% (IQR: 17% to 31%) of exposures across pairwise combinations of sixteen common antibiotics and nine bacterial pathogens. All scenarios demonstrate that stewardship interventions, facilitated by changes in clinician behavior and improved diagnostics, have the opportunity to broadly reduce antibiotic exposures across a range of potential pathogens. Concurrent approaches, such as vaccines aiming to reduce infection incidence, are needed to further decrease exposures occurring in ‘necessary’ contexts.

Neisseria gonorrhoeaeis an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association on the largest collection ofN. gonorrhoeaeisolates to date (n=4852) to identify loss-of-function (LOF) mutations in the efflux pumpmtrCDEoperon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical isolates relative to urethral isolates (odds ratio (OR) = 3.74, 95% CI [1.98-6.70]). In support of a model in which pump expression incurs a fitness cost in this niche, cervical isolates were also enriched relative to urethral isolates in LOF mutations in themtrCDEactivatormtrA(OR = 8.60, 95% CI [4.96-14.57]) and infarA,a subunit of the FarAB efflux pump (OR = 6.25, 95% CI [3.90-9.83]). In total, approximately 2 in 5 cervical isolates (42.6%) contained a LOF mutation in either the efflux pump componentsmtrCorfarAor the activatormtrA.Our findings extend beyondN. gonorrhoeaeto otherNeisseria:mtrCLOF mutations are rare (N. meningitidisin a collection of 14,798 genomes but enriched in a heterosexual urethritis-associated lineage (8.6%, p = 9.90×10−5), indicating that efflux pump downregulation contributes broadly to the adaptation of pathogenicNeisseriato the female urogenital tract. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.

Surveillance of drug-resistant bacteria is essential for healthcare providers to deliver effective empiric antibiotic therapy. However, traditional molecular epidemiology does not typically occur on a timescale that could impact patient treatment and outcomes. Here we present a method called ‘genomic neighbor typing’ for inferring the phenotype of a bacterial sample by identifying its closest relatives in a database of genomes with metadata. We show that this technique can infer antibiotic susceptibility and resistance for both S. pneumoniae and N. gonorrhoeae. We implemented this with rapid k-mer matching, which, when used on Oxford Nanopore MinION data, can run in real time. This resulted in determination of resistance within ten minutes (sens/spec 91%/100% for S. pneumoniae and 81%/100% N. gonorrhoeae from isolates with a representative database) of sequencing starting, and for clinical metagenomic sputum samples (75%/100% for S. pneumoniae), within four hours of sample collection. This flexible approach has wide application to pathogen surveillance and may be used to greatly accelerate appropriate empirical antibiotic treatment.

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. We treated the libraries with daptomycin to assess whether the strain-dependent differences impact pathways important for survival. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, consistently promote survival under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains., Author summary Antibiotic-resistant Staphylococcus aureus kills thousands of people every year in the United States alone. To stay ahead of the looming threat of multidrug-resistant infections, we must continue to develop new antibiotics and find ways to make our current repertoire of antibiotics more effective, including by finding pairs of compounds that perform best when administered together. In the age of next-generation sequencing, we can now use transposon sequencing to find potential targets for new antibiotics on a genome-wide scale, identified as either essential genes or genes that positively influence survival in the presence of an antibiotic. In this work, we created a compendium of genes that are essential across a range of S. aureus strains, as well as those that are important for growth in the presence of the antibiotic daptomycin. The results will be a resource for researchers working to develop the next generation of antibiotic therapies.

The relationship between antibiotic stewardship and population levels of antibiotic resistance remains unclear. In order to better understand shifts in selective pressure due to stewardship, we use publicly available data to estimate the effect of changes in prescribing on exposures to frequently used antibiotics experienced by potentially pathogenic bacteria that are asymptomatically colonizing the microbiome. We quantify this impact under four hypothetical stewardship strategies. In one scenario, we estimate that elimination of all unnecessary outpatient antibiotic use could avert 6 to 48% (IQR: 17 to 31%) of exposures across pairwise combinations of sixteen common antibiotics and nine bacterial pathogens. All scenarios demonstrate that stewardship interventions, facilitated by changes in clinician behavior and improved diagnostics, have the opportunity to broadly reduce antibiotic exposures across a range of potential pathogens. Concurrent approaches, such as vaccines aiming to reduce infection incidence, are needed to further decrease exposures occurring in “necessary” contexts.

Quantitative assessment of antibiotic-responsive RNA transcripts holds promise for a rapid point-of-care (POC) diagnostic tool for antimicrobial susceptibility testing. These assays aim to distinguish susceptible and resistant isolates by transcriptional differences upon drug exposure. However, an often-overlooked dimension of designing these tests is that the genetic diversity within a species may yield differential transcriptional regulation independent of resistance phenotype. Here, we use a phylogenetically diverse panel of Neisseria gonorrhoeae and transcriptome profiling coupled with reverse transcription-quantitative PCR to test this hypothesis, to identify azithromycin responsive transcripts and evaluate their potential diagnostic value, and to evaluate previously reported diagnostic markers for ciprofloxacin resistance (porB and rpmB). Transcriptome profiling confirmed evidence of genetic distance and population structure impacting transcriptional response to azithromycin. Taking this into account, we found azithromycin-responsive transcripts overrepresented in susceptible strains compared to resistant strains and selected four candidate diagnostic transcripts (rpsO, rplN, omp3, and NGO1079) that were the most significantly differentially regulated between phenotypes across drug exposure. RNA signatures for these markers categorically predicted resistance in 19/20 cases, with the one incorrect categorical assignment for an isolate at the threshold of reduced susceptibility. Finally, we found that porB and rpmB expression were not uniformly diagnostic of ciprofloxacin resistance in a panel of isolates with unbiased phylogenetic sampling. Overall, our results suggest that RNA signatures as a diagnostic tool are promising for future POC diagnostics; however, development and testing should consider representative genetic diversity of the target pathogen.

Diagnostics that minimize the time to selection of an appropriate antibiotic treatment represent an important strategy in addressing the challenge of antimicrobial resistance (AMR). Among this class of diagnostics, the use of pathogen genotype to predict AMR phenotype has been facilitated by advances in rapid sequencing platforms. A longstanding objection to this approach, however, is that the emergence of novel resistance mechanisms will inevitably lead to a decline in the sensitivity of these diagnostics. Here, we show that while the sensitivities of some genetic markers of resistance remain stably high, sensitivities of other markers rapidly decline, as expected, due to the emergence of novel resistance variants. We then present a simple mathematical framework that defines the sampling and phenotypic testing rates needed for early detection of novel resistance variants and thus demonstrate how surveillance can help maintain the sensitivity and utility of sequence-based AMR diagnostics.One sentence summaryTargeted sampling strategies are necessary for early detection of novel resistance mechanisms and sustainability of genotype-based detection of novel resistance mechanisms and sustainability of genotype-based diagnostics.

Background Widespread antimicrobial resistance in Neisseria gonorrhoeae has limited the effective treatment options. Cephalosporins remain one of the few classes of antibiotics recommended for gonococcal infections, but reduced susceptibility to the third-generation cephalosporins, including ceftriaxone, has emerged. Most reduced susceptibility to ceftriaxone is caused by an alternative penA(PBP2) allele. However, the isolates with the among the highest level cephalosporin resistance identified in the US lack this allele and other penA resistance mutations, raising the possibility of cephalosporin resistance not mediated directly through penA. Methods To identify the genetic basis of resistance in these isolates, we employed an undirected transformation strategy, and used molecular microbiology and genetics methods to investigate the mechanism of resistance. Results Here, we show that resistance to ESCs has arisen in clinical isolates multiple times through distinct mutations in the RNA polymerase components rpoB and rpoD. The resistance caused by these changes is not a general tolerance response: these mutations neither changed the growth rate in vitronor altered susceptibility to other classes of antibiotics (including penicillin). These mutations result in large variations in transcription, including in genes coding for penicillin binding proteins (increase in PBP1, decrease in PBPs 3 and 4) and pilus pore. We show that increases in PBP1 protein levels contribute to the rise in CRO MIC, likely through replacement of inhibited PBP2 activity, though other factors are needed to recapitulate the resistance seen in the clinical isolates with rpoB and rpoD mutations. Conclusion Pathways to extended spectrum cephalosporin resistance do not require alterations to penA (PBP2) and can arise through mutations in components of the RNA polymerase holoenzyme. Additional pathways to cephalosporin resistance in goncooccus remain to be identified. These findings have implications for the development of molecular diagnostics and for understanding the mechanistic basis of cephalosporin resistance. Disclosure No significant relationships.

Background Despite ‘best effort’ public health measures, the incidence of gonorrhoea is increasing in many countries. Recently, whole genome sequencing (WGS) has been used to investigate transmission of Neisseria gonorrhoeae, including antimicrobial-resistant (AMR) lineages, but to date, most studies have not combined genomic data with detailed patient-level information on sexual behaviour to define the extent of transmission across population subgroups (‘bridging’). Methods We performed an observational study and undertook WGS and bioinformatic analysis on all cultured clinical isolates of N. gonorrhoeae in the state of Victoria, Australia, from January to December 2017. Antimicrobial susceptibility testing was performed on all isolates, and detailed epidemiological data was obtained, including data on sexual orientation, HIV status, use of HIV pre-exposure prophylaxis (PrEP), sex work, and overseas travel. Epidemiological associations were made with dominant genetic clusters of N. gonorrhoeae. Results A total of 2,186 isolates were sequenced from 2,055 patients, 86·3% of whom were male. We identified eleven dominant genetic clusters, containing thirty or more patients, with the largest cluster comprising 181 patients. There was extensive bridging of clusters between men who have sex with men (MSM) and heterosexuals, with bisexual males identified in seven of the major clusters. Eight major clusters contained HIV-positive and HIV-negative patients (including individuals receiving PrEP). We also identified transmission of a novel azithromycin-resistant clone, associated with a mosaic mtr locus. Conclusion To our knowledge, our study is the first to combine WGS with comprehensive individual-level behavioural risk data, providing verification for transmission of multiple gonococcal lineages within and across distinct sexual networks. Application of these methods in real-time will allow gonorrhoea transmission and antimicrobial resistance to be tracked, with ‘hotspots’ identified for interventions aimed at improving gonorrhoea control. Disclosure No significant relationships.

Objectives Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide. Methods The 2016 WHO reference strains (n = 14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n = 23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing. Results The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n = 14) were presented. Conclusions The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.

Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new anti-gonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm wavelength), an innovative non-pharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no genotoxicity of aBL to the vaginal epithelial cells was observed at the radiant exposure for inactivating N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their co-cultures. No gonococcal resistance to aBL developed after 15 successive cycles of sub-therapeutic aBL inactivation. Taken together, aBL represents a potent potential treatment for antibiotic-resistant gonococcal infection.

Quantitative assessment of antibiotic-responsive RNA transcripts holds promise for a rapid point of care (POC) diagnostic tool for antimicrobial susceptibility testing. These assays aim to distinguish susceptible and resistant isolates by transcriptional differences upon drug exposure. However, an often-overlooked dimension of designing these tests is that the genetic diversity within a species may yield differential transcriptional regulation independent of resistance phenotype. Here, we use a phylogenetically diverse panel of Neisseria gonorrhoeae and transcriptome profiling coupled with RT-qPCR to test this hypothesis, to identify azithromycin responsive transcripts and evaluate their potential diagnostic value, and to evaluate previously reported diagnostic markers for ciprofloxacin resistance (porB and rpmB). Transcriptome profiling confirmed evidence of population structure in transcriptional response to azithromycin. Taking this population structure into account, we found azithromycin-responsive transcripts overrepresented in susceptible strains compared to resistant strains, and selected four candidate diagnostic transcripts (rpsO, rplN, omp3, and NGO1079) that were the most significantly differentially regulated between phenotypes across drug exposure. RNA signatures for these markers categorically predicted resistance in 19/20 cases, with the one incorrect categorical assignment for an isolate at the threshold of reduced susceptibility. Finally, we found that porB and rpmB expression were not diagnostic of ciprofloxacin resistance in a panel of isolates with unbiased phylogenetic sampling. Overall, our results suggest that RNA signatures as a diagnostic tool are promising for future POC diagnostics; however, development and testing should consider representative genetic diversity of the target pathogen.

Antimicrobial resistance in Neisseria gonorrhoeae is a major issue of public health, and there is a critical need for the development of new antigonococcal strategies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL; wavelength, 405 nm), an innovative nonpharmacological approach, for the inactivation of N. gonorrhoeae. Our findings indicated that aBL preferentially inactivated N. gonorrhoeae, including antibiotic-resistant strains, over human vaginal epithelial cells in vitro. Furthermore, no aBL-induced genotoxicity to the vaginal epithelial cells was observed at the radiant exposure used to inactivate N. gonorrhoeae. aBL also effectively inactivated N. gonorrhoeae that had attached to and invaded into the vaginal epithelial cells in their cocultures. No gonococcal resistance to aBL developed after 15 successive cycles of inactivation induced by subtherapeutic exposure to aBL. Endogenous aBL-activatable photosensitizing porphyrins in N. gonorrhoeae were identified and quantified using ultraperformance liquid chromatography, with coproporphyrin being the most abundant species in all N. gonorrhoeae strains studied. Singlet oxygen was involved in aBL inactivation of N. gonorrhoeae. Together, these findings show that aBL represents a potential potent treatment for antibiotic-resistant gonococcal infection.

Bystander selection—the selective pressure for resistance exerted by antibiotics on microbes that are not the target pathogen of treatment—is critical to understanding the total impact of broad-spectrum antibiotic use on pathogenic bacterial species that are often carried asymptomatically. However, to our knowledge, this effect has never been quantified. We quantify bystander selection for resistance for a range of clinically relevant antibiotic–species pairs as the proportion of all antibiotic exposures received by a species for conditions in which that species was not the causative pathogen (“proportion of bystander exposures”). Data sources include the 2010–2011 National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey, the Human Microbiome Project, and additional carriage and etiological data from existing literature. For outpatient prescribing in the United States, we find that this proportion over all included antibiotic classes is over 80% for eight of nine organisms of interest. Low proportions of bystander exposure are often associated with infrequent bacterial carriage or concentrated prescribing of a particular antibiotic for conditions caused by the species of interest. Applying our results, we roughly estimate that pneumococcal conjugate vaccination programs result in nearly the same proportional reduction in total antibiotic exposures of Streptococcus pneumoniae , Staphylococcus aureus , and Escherichia coli , despite the latter two organisms not being targeted by the vaccine. These results underscore the importance of considering antibiotic exposures of bystanders, in addition to the target pathogen, in measuring the impact of antibiotic resistance interventions.

Neisseria gonorrhoeaeis the causative pathogen of the sexually transmitted disease gonorrhea, a disease at risk of becoming untreatable due to increasing antibiotic resistance. There is a critical need for the development of new anti-gonococcal therapies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL), an innovative non-antibiotic approach, for the inactivation of antibiotic-resistantN.gonorrhoeae. Our findings indicated that aBL at 405 nm preferentially inactivated antibiotic-resistantN. gonorrhoeaeover the vaginal epithelial cells. Furthermore, no genotoxicity of aBL to the vaginal epithelial cells was observed at the exposure for inactivatingN. gonorrhoeae. aBL also effectively inactivatedN. gonorrhoeaethat had invaded into the vaginal epithelial cells. No gonococcal resistance to aBL developed after 15 successive cycles of sub-therapeutic inactivation. Endogenous aBL-active photosensitizing chromophores (porphyrins and flavins) inN. gonorrhoeaewere identified and quantified using ultra performance liquid chromatography, with coproporphyrin being the most abundant endogenous porphyrin species. Taken together, aBL at 405 nm represents a potent potential treatment for gonococcal infections.One Sentence SummaryaBL selectively inactivated antibiotic-resistantNeisseria gonorrhoeaenover normal vaginal epithelial cells.

Mosaic interspecifically acquired alleles of the multiple transferable resistance (mtr) efflux pump operon correlate with increased resistance to azithromycin in Neisseria gonorrhoeae in epidemiological studies. However, whether and how these alleles cause resistance is unclear. Here, we use population genomics, transformations, and transcriptional analyses to dissect the relationship between variant mtr alleles and azithromycin resistance. We find that the locus encompassing the mtrR transcriptional repressor and the mtrCDE pump is a hot spot of interspecific recombination introducing alleles from Neisseria meningitidis and Neisseria lactamica into N. gonorrhoeae, with multiple rare haplotypes in linkage disequilibrium at mtrD and the mtr promoter region. Transformations demonstrate that resistance to azithromycin, as well as to other antimicrobial compounds such as polymyxin B and crystal violet, is mediated through epistasis between these two loci and that the full-length mosaic mtrD allele is required. Gene expression profiling reveals the mechanism of resistance in mosaics couples novel mtrD alleles with promoter mutations that increase expression of the pump. Overall, our results demonstrate that epistatic interactions at mtr gained from multiple neisserial species has contributed to increased gonococcal resistance to diverse antimicrobial agents., IMPORTANCE Neisseria gonorrhoeae is the sexually transmitted bacterial pathogen responsible for more than 100 million cases of gonorrhea worldwide each year. The incidence of resistance to the macrolide azithromycin has increased in the past decade; however, a large proportion of the genetic basis of resistance remains unexplained. This study is the first to conclusively demonstrate the acquisition of macrolide resistance through mtr alleles from other Neisseria species, demonstrating that commensal Neisseria bacteria are a reservoir for antibiotic resistance to macrolides, extending the role of interspecies mosaicism in resistance beyond what has been previously described for cephalosporins. Ultimately, our results emphasize that future fine-mapping of genome-wide interspecies mosaicism may be valuable in understanding the pathways to antimicrobial resistance. Our results also have implications for diagnostics and public health surveillance and control, as they can be used to inform the development of sequence-based tools to monitor and control the spread of antibiotic-resistant gonorrhea.

ObjectiveTo identify temporal trends in outpatient antibiotic use and antibiotic prescribing practice among older adults in a high income country.DesignObservational study using United States Medicare administrative claims in 2011-15.SettingMedicare, a US national healthcare program for which 98% of older adults are eligible.Participants4.5 million fee-for-service Medicare beneficiaries aged 65 years old and older.Main outcome measurementsOverall rates of antibiotic prescription claims, rates of potentially appropriate and inappropriate prescribing, rates for each of the most frequently prescribed antibiotics, and rates of antibiotic claims associated with specific diagnoses. Trends in antibiotic use were estimated by multivariable regression adjusting for beneficiaries’ demographic and clinical covariates.ResultsThe number of antibiotic claims fell from 1364.7 to 1309.3 claims per 1000 beneficiaries per year in 2011-14 (adjusted reduction of 2.1% (95% confidence interval 2.0% to 2.2%)), but then rose to 1364.3 claims per 1000 beneficiaries per year in 2015 (adjusted reduction of 0.20% over 2011-15 (0.09% to 0.30%)). Potentially inappropriate antibiotic claims fell from 552.7 to 522.1 per 1000 beneficiaries over 2011-14, an adjusted reduction of 3.9% (3.7% to 4.1%). Individual antibiotics had heterogeneous changes in use. For example, azithromycin claims per beneficiary decreased by 18.5% (18.2% to 18.8%) while levofloxacin claims increased by 27.7% (27.2% to 28.3%). Azithromycin use associated with each of the potentially appropriate and inappropriate respiratory diagnoses decreased, while levofloxacin use associated with each of those diagnoses increased.ConclusionAmong US Medicare beneficiaries, overall antibiotic use and potentially inappropriate use in 2011-15 remained steady or fell modestly, but individual drugs had divergent changes in use. Trends in drug use across indications were stronger than trends in use for individual indications, suggesting that guidelines and concerns about antibiotic resistance were not major drivers of change in antibiotic use.

Structured abstractObjectiveTo identify temporal trends in outpatient antibiotic use and antibiotic prescribing practice among older adults.DesignObservational study using United States Medicare administrative claims during 2011-2015. Trends in antibiotic use were estimated using multivariable regression adjusting for beneficiaries’ demographic and clinical covariates.SettingMedicare.Participants4.6 million Medicare beneficiaries from a nationwide, 20% sample of fee-forservice Medicare beneficiaries ≥65 years old.Main outcome measurementsOverall rates of antibiotic prescription claims, rates of appropriate and inappropriate prescribing, rates for each of the most frequently prescribed antibiotics, and rates of antibiotic claims associated with specific diagnoses.ResultsAntibiotic claims fell from 1362.2 to 1361.6 claims per 1,000 beneficiaries per year during 2011-2015, an overall 0.2% decrease (95% CI 0.07-0.32). Inappropriate antibiotic claims fell from 552 to 533 claims per 1,000 beneficiaries, a 4.1% decrease (CI 3.9-4.3). Individual antibiotics had heterogeneous changes in use. For example, azithromycin claims per beneficiary decreased by 18.4% (CI 18.2-18.7) while levofloxacin claims increased by 28.1% (CI 27.5-28.6). Azithromycin use associated with each of the potentially appropriate and inappropriate respiratory diagnoses we considered decreased, while levofloxacin use associated with each of those diagnoses increased.ConclusionAmong US Medicare beneficiaries, overall antibiotic use and inappropriate use declined modestly, but individual drugs experienced divergent changes in use. Trends in drug use across indications were stronger than trends in use for individual indications, suggesting that guidelines and concerns about antibiotic resistance were not major drivers of change in antibiotic use.

Bystander selection -- the selective pressures exerted by antibiotics on microbial flora that are not the target pathogen of treatment -- is critical to understanding the total impact of broad-spectrum antibiotic use; however, to our knowledge, this effect has never been quantified. Using the 2010-2011 National Ambulatory Medical Care Survey and National Hospital Ambulatory Medical Care Survey (NAMCS/NHAMCS), the Human Microbiome Project, and additional carriage and etiological data from existing literature, we estimate the magnitude of bystander selection for a range of clinically relevant antibiotic-species pairs as the proportion of all exposures of an antibiotic experienced by a species for conditions in which that species was not the causative pathogen (“proportion of bystander exposures”). For outpatient prescribing in the United States, we find that this proportion over all included antibiotics is over 80% for 8 out of 9 organisms of interest. Low proportions of bystander exposure are often associated with infrequent bacterial carriage or a high proportion of antibiotic prescribing focused on conditions caused by the species of interest. Using the proportion of bystander exposures, we roughly estimate that S. aureus and E. coli may benefit from 90.7% and 99.7%, respectively, of the estimated reduction in antibiotic use due to pneumococcal conjugate vaccination, despite not being the pathogen targeted by the vaccine. These results underscore the importance of considering antibiotic exposures to bystanders, in addition to the targeted pathogen, in measuring the impact of antibiotic resistance interventions.Significance StatementThe forces that contribute to changing population prevalence of antibiotic resistance are not well understood. Bystander selection -- the inadvertent pressures imposed by antibiotics on the microbial flora other than the pathogen targeted by treatment -- is hypothesized to be a major factor in the propagation of antibiotic resistance, but its extent has not been characterized. We estimate the proportion of bystander exposures across a range of antibiotics and organisms and describe factors driving variability of these proportions. Impact estimates for antibiotic resistance interventions, including vaccination, are often limited to effects on a target pathogen. However, the reduction of antibiotic treatment for illnesses caused by the target pathogen may have the broader potential to decrease bystander selection pressures for resistance on many other organisms.

The global epidemic of drug-resistant tuberculosis is a catastrophic example of how antimicrobial resistance is undermining the public health gains made possible by combination drug therapy. Recent evidence points to unappreciated bacterial factors that accelerate the emergence of drug resistance. In a genome-wide association study of Mycobacterium tuberculosis isolates from China, we find mutations in the gene encoding the transcription factor prpR enriched in drug-resistant strains. prpR mutations confer conditional drug tolerance to three of the most effective classes of antibiotics by altering propionyl-CoA metabolism. prpR-mediated drug tolerance is carbon-source dependent, and while readily detectable during infection of human macrophages, is not captured by standard susceptibility testing. These data define a previously unrecognized and clinically prevalent class of M. tuberculosis variants that undermine antibiotic efficacy and drive drug resistance.

The rate of infection by methicillin-resistant Staphylococcus aureus (MRSA) has declined over the past decade, but it is unclear whether this represents a decline in S. aureus infections overall. To evaluate the trends in the annual rates of infection by S. aureus subtypes and mean antibiotic resistance, we conducted a 15-year retrospective observational study at two tertiary care institutions in Boston, MA, of 31,753 adult inpatients with S. aureus isolated from clinical specimens. We inferred the gain and loss of methicillin resistance through genome sequencing of 180 isolates from 2016. The annual rates of infection by S. aureus declined from 2003 to 2014 by 4.2% (2.7% to 5.6%), attributable to an annual decline in MRSA of 10.9% (9.3% to 12.6%). Penicillin-susceptible S. aureus (PSSA) increased by 6.1% (4.2% to 8.1%) annually, and rates of methicillin-susceptible penicillin-resistant S. aureus (MSSA) did not change. Resistance in S. aureus decreased from 2000 to 2014 by 0.8 antibiotics (0.7 to 0.8). Within common MRSA clonal complexes, 3/14 MSSA and 2/21 PSSA isolates arose from the loss of resistance-conferring genes. Overall, in two tertiary care institutions in Boston, MA, a decline in S. aureus infections has been accompanied by a shift toward increased antibiotic susceptibility. The rise in PSSA makes penicillin an increasingly viable treatment option.

Introduction Increasing antibiotic resistance limits treatment for gonorrhoea. We examined the extent to which a hypothetical point-of-care (POC) test reporting antibiotic susceptibility profiles could delay emergence of resistance and prolong effectiveness of existing antibiotics. Methods We developed a deterministic compartmental model describing gonorrhoea transmission in a risk-stratified single-sex population with three different antibiotics available to treat infections. Probabilities of resistance emergence on treatment and fitness costs associated with resistance were based on characteristics of fluoroquinolones, azithromycin, and ceftriaxone, as inferred from a previous phylogenomic analysis. We compared strategies in which a POC test was used to guide therapy in varying proportions of cases against the current empiric approach (dual treatment with azithromycin plus ceftriaxone). Results Based on current estimates of gonoccoal susceptibility patterns in the United States, the model indicated that continued empiric dual antibiotic treatment without POC testing resulted in >5% of isolates being resistant to both azithromycin and ceftriaxone within 15 years. When POC testing was used in 10% of identified cases, this time was delayed by 4 years, while time to reach a 1% prevalence of triply-resistant strains was delayed by 5 years. With POC testing in >55% of identified cases, it took over 100 years for dual and triple resistance to exceed 1%, and with POC testing in ≥75% of cases, strains resistant to azithromycin and/or ceftriaxone did not persist in the population. Results were sensitive to assumptions about fitness costs and test sensitivity only when POC test deployment was relatively low ( Conclusion Rapid diagnostics that indicate antibiotic susceptibility have the potential to extend the usefulness of existing antibiotics for treatment of gonorrhoea. More broadly, integration of evidence on fitness costs associated with resistance can enhance strategies for rational antibiotic selection and further delay emergence of resistance.

BackgroundMethicillin resistant S. aureus (MRSA) has been declining over the past decade, but changes in S. aureus overall and the implications for trends in antibiotic resistance remain unclear.ObjectiveTo determine whether the decline in rates of infection by MRSA has been accompanied by changes in rates of infection by methicillin susceptible, penicillin resistant S. aureus (MSSA) and penicillin susceptible S. aureus (PSSA). We test if these dynamics are associated with specific genetic lineages and evaluate gains and losses of resistance at the strain level.MethodsWe conducted a 15 year retrospective observational study at two tertiary care institutions in Boston, MA of 31,589 adult inpatients with S. aureus infections. Surveillance swabs and duplicate specimens were excluded. We also sequenced a sample of contemporary isolates (n = 180) obtained between January 2016 and July 2016. We determined changes in the annual rates of infection per 1,000 inpatient admissions by S. aureus subtype and in the annual mean antibiotic resistance by subtype. We performed phylogenetic analysis to generate a population structure and infer gain and loss of the genetic determinants of resistance.ResultsOf the 43,954 S. aureus infections over the study period, 21,779 were MRSA, 17,565 MSSA and 4,610 PSSA. After multivariate adjustment, annual rates of infection by S. aureus declined from 2003 to 2014 by 2.9% (95% CI, 1.6%-4.3%), attributable to an annual decline in MRSA of 9.1% (95% CI, 6.3%-11.9%) and in MSSA by 2.2% (95% CI, 0.4%-4.0%). PSSA increased over this time period by 4.6% (95% CI, 3.0%-6.3%) annually. Resistance in S. aureus decreased from 2000 to 2014 by 0.86 antibiotics (95% CI, 0.81-0.91). By phylogenetic inference, 5/35 MSSA and 2/20 PSSA isolates in the common MRSA lineages ST5/USA100 and ST8/USA300 arose from the loss of genes conferring resistance.Conclusions and relevanceAt two large tertiary care centers in Boston, MA, S. aureus infections have decreased in rate and have become more susceptible to antibiotics, with a rise in PSSA making penicillin an increasingly viable and important treatment option.

Mathematical modeling suggests that rapid diagnostics that report antibiotic susceptibility have the potential to extend the usefulness of existing antibiotics for treatment of gonorrhea compared with the current guidelines for empiric 2-drug treatment., Background Increasing antibiotic resistance limits treatment options for gonorrhea. We examined the impact of a hypothetical point-of-care (POC) test reporting antibiotic susceptibility profiles on slowing resistance spread. Methods A mathematical model describing gonorrhea transmission incorporated resistance emergence probabilities and fitness costs associated with resistance based on characteristics of ciprofloxacin (A), azithromycin (B), and ceftriaxone (C). We evaluated time to 1% and 5% prevalence of resistant strains among all isolates with the following: (1) empiric treatment (B and C), and treatment guided by POC tests determining susceptibility to (2) A only and (3) all 3 antibiotics. Results Continued empiric treatment without POC testing was projected to result in >5% of isolates being resistant to both B and C within 15 years. Use of either POC test in 10% of identified cases delayed this by 5 years. The 3 antibiotic POC test delayed the time to reach 1% prevalence of triply-resistant strains by 6 years, whereas the A-only test resulted in no delay. Results were less sensitive to assumptions about fitness costs and test characteristics with increasing test uptake. Conclusions Rapid diagnostics reporting antibiotic susceptibility may extend the usefulness of existing antibiotics for gonorrhea treatment, but ongoing monitoring of resistance patterns will be critical.

The sensitivity of genotype-based diagnostics that predict antimicrobial susceptibility is limited by the extent to which they detect genes and alleles that lead to resistance. As novel resistance variants are expected to emerge, such sensitivity is expected to decline unless the new variants are detected and incorporated into the diagnostic. Here, we present a mathematical framework to define how many diagnostic failures may be expected under varying surveillance regimes and thus quantify the surveillance needed to maintain the sensitivity of genotype-based diagnostics., A simple mathematical framework that defines the rates of sampling and phenotypic testing necessary to efficiently detect novel resistance variants and thus maintain the sensitivity of genotype-based antimicrobial resistance diagnostics.

Prediction of antibiotic resistance phenotypes from whole genome sequencing data by machine learning methods has been proposed as a promising platform for the development of sequence-based diagnostics. However, there has been no systematic evaluation of factors that may influence performance of such models, how they might apply to and vary across clinical populations, and what the implications might be in the clinical setting. Here, we performed a meta-analysis of seven large Neisseria gonorrhoeae datasets, as well as Klebsiella pneumoniae and Acinetobacter baumannii datasets, with whole genome sequence data and antibiotic susceptibility phenotypes using set covering machine classification, random forest classification, and random forest regression models to predict resistance phenotypes from genotype. We demonstrate how model performance varies by drug, dataset, resistance metric, and species, reflecting the complexities of generating clinically relevant conclusions from machine learning-derived models. Our findings underscore the importance of incorporating relevant biological and epidemiological knowledge into model design and assessment and suggest that doing so can inform tailored modeling for individual drugs, pathogens, and clinical populations. We further suggest that continued comprehensive sampling and incorporation of up-to-date whole genome sequence data, resistance phenotypes, and treatment outcome data into model training will be crucial to the clinical utility and sustainability of machine learning-based molecular diagnostics., Author summary Machine learning-based prediction of antibiotic resistance from bacterial genome sequences represents a promising tool to rapidly determine the antibiotic susceptibility profile of clinical isolates and reduce the morbidity and mortality resulting from inappropriate and ineffective treatment. However, while there has been much focus on demonstrating the diagnostic potential of these modeling approaches, there has been little assessment of potential caveats and prerequisites associated with implementing predictive models of drug resistance in the clinical setting. Our results highlight significant biological and technical challenges facing the application of machine learning-based prediction of antibiotic resistance as a diagnostic tool. By outlining specific factors affecting model performance, our findings provide a framework for future work on modeling drug resistance and underscore the necessity of continued comprehensive sampling and reporting of treatment outcome data for building reliable and sustainable diagnostics.