Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron.

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Comparative genomics reveals the correlations of stress response genes and bacteriophages in developing antibiotic resistance of Staphylococcus saprophyticus.

IMPORTANCE: Staphylococcus saprophyticus is the second most common bacteria associated with urinary tract infections (UTIs) in women. The antimicrobial treatment regimen for uncomplicated UTI is normally nitrofurantoin, trimethoprim-sulfamethoxazole (TMP-SMX), or a fluoroquinolone without routine susceptibility testing of S. saprophyticus recovered from urine specimens. However, TMP-SMX-resistant S. saprophyticus has been detected recently in UTI patients, as well as in our cohort. Herein, we investigated the understudied resistance patterns of this pathogenic species by linking genomic antibiotic resistance gene (ARG) content to susceptibility phenotypes. We describe ARG associations with known and novel SCCmec configurations as well as phage elements in S. saprophyticus, which may serve as intervention or diagnostic targets to limit resistance transmission. Our analyses yielded a comprehensive database of phenotypic data associated with the ARG sequence in clinical S. saprophyticus isolates, which will be crucial for resistance surveillance and prediction to enable precise diagnosis and effective treatment of S. saprophyticus UTIs.

Uncharacterized and lineage-specific accessory genes within the Proteus mirabilis pan-genome landscape.

Proteus mirabilis is a Gram-negative bacterium recognized for its unique swarming motility and urease activity. A previous proteomic report on four strains hypothesized that, unlike other Gram-negative bacteria, P. mirabilis may not exhibit significant intraspecies variation in gene content. However, there has not been a comprehensive analysis of large numbers of P. mirabilis genomes from various sources to support or refute this hypothesis. We performed comparative genomic analysis on 2,060 Proteus genomes. We sequenced the genomes of 893 isolates recovered from clinical specimens from three large US academic medical centers, combined with 1,006 genomes from NCBI Assembly and 161 genomes assembled from Illumina reads in the public domain. We used average nucleotide identity (ANI) to delineate species and subspecies, core genome phylogenetic analysis to identify clusters of highly related P. mirabilis genomes, and pan-genome annotation to identify genes of interest not present in the model P. mirabilis strain HI4320. Within our cohort, Proteus is composed of 10 named species and 5 uncharacterized genomospecies. P. mirabilis can be subdivided into three subspecies; subspecies 1 represented 96.7% (1,822/1,883) of all genomes. The P. mirabilis pan-genome includes 15,399 genes outside of HI4320, and 34.3% (5,282/15,399) of these genes have no putative assigned function. Subspecies 1 is composed of several highly related clonal groups. Prophages and gene clusters encoding putatively extracellular-facing proteins are associated with clonal groups. Uncharacterized genes not present in the model strain P. mirabilis HI4320 but with homology to known virulence-associated operons can be identified within the pan-genome. IMPORTANCE Gram-negative bacteria use a variety of extracellular facing factors to interact with eukaryotic hosts. Due to intraspecies genetic variability, these factors may not be present in the model strain for a given organism, potentially providing incomplete understanding of host-microbial interactions. In contrast to previous reports on P. mirabilis, but similar to other Gram-negative bacteria, P. mirabilis has a mosaic genome with a linkage between phylogenetic position and accessory genome content. P. mirabilis encodes a variety of genes that may impact host-microbe dynamics beyond what is represented in the model strain HI4320. The diverse, whole-genome characterized strain bank from this work can be used in conjunction with reverse genetic and infection models to better understand the impact of accessory genome content on bacterial physiology and pathogenesis of infection.

Evaluation of the NG-Test CARBA 5 Lateral Flow Assay with an IMP-27-Producing Morganella morganii and Other Morganellaceae.

An isolate of Morganella morganii (MMOR1) that tested susceptible to 3rd/4th-generation cephalosporins and intermediate to meropenem was characterized as positive for NDM and IMP carbapenemases by NG-Test CARBA 5. Our objective was to further investigate this result, given the inconsistent susceptibility profile and unusual epidemiological profile for our region. The MMOR1 isolate was retested for antimicrobial susceptibilities and characterized for carbapenemase production. MMOR1 tested susceptible to ceftazidime, ceftriaxone, cefepime, aztreonam, and ertapenem, and intermediate to meropenem and imipenem. The isolate tested positive by carbapenem inactivation method (CIM) and CIM+EDTA (eCIM) testing, indicating metallo-ß-lactamase production. The isolate tested negative for all carbapenemase genes on Xpert Carba-R, but positive for IMP on repeat testing of NG-Test CARBA 5. Whole-genome sequencing revealed MMOR1 contained blaIMP-27, but no other carbapenemase genes. Additional testing with NG-Test CARBA 5 revealed a false-positive NDM band when the assay was overloaded with test inoculum. Supplementary isolates were tested with an overloaded inoculum (n = 6 M. morganii; n = 1 P. mirabilis; n = 1 IMP-27-producing P. rettgeri; n = 1 IMP-1-producing E. coli; n = 1 K. pneumoniae), and two non-carbapenemase-producing carbapenem non-susceptible M. morganii also generated a false-positive NDM band; though, this was not universal among this species. A dual IMP+/NDM+ M. morganii is an unusual result that should prompt additional investigation, especially in nonendemic regions and when the susceptibility profile is incompatible. IMP-27 is not detected by Xpert Carba-R but is variably detected by NG-Test CARBA 5. The microorganism inoculum used for NG-Test CARBA 5 must be carefully controlled for accurate results. IMPORTANCE The detection of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) is an important function of the clinical microbiology laboratory, where positive identifications have immediate implications for infection control and surveillance strategies in the inpatient setting and can inform appropriate selection of therapy among the various novel anti-CP-CRE agents. NG-Test CARBA 5 is a relatively new lateral flow assay used for detection of carbapenemases in CP-CRE. Here, we describe the characterization of a Morganella morganii isolate that generated a false-positive NDM carbapenemase detection by this assay, and perform bacterial test inoculum experiments with additional isolates to further investigate a cause of false-positive results using the NG-Test CARBA 5. While a lateral flow assay like the NG-Test CARBA 5 is a very desirable test format for clinical laboratories, there are pitfalls to avoid when performing this test and interpreting results, including recognizing an overloaded test assay, which could lead to false-positive results.

Evaluation of Variability in Interpretation of Disk Diffusion Testing for Cefiderocol Using Different Brands of Mueller-Hinton Agar.

BACKGROUND: Cefiderocol is a new antibiotic used to treat infections with antibiotic resistant Gram-negative bacilli. The impact of differences between Mueller-Hinton agar (MHA) brands on susceptibility testing is underexplored. Compounding the implementation of cefiderocol susceptibility testing is a lack of harmonization between different regulatory body breakpoint criteria. METHODS: We performed Kirby-Bauer disk diffusion using BD, Hardy, and Remel MHA, in addition to broth microdilution for Acinetobacter baumannii (n = 25), Enterobacterales (n = 25), Stenotrophomonas maltophilia (n = 24), and Pseudomonas aeruginosa (n = 23). We analyzed disk diffusion diameters and minimum inhibitory concentrations using interpretive criteria from the Clinical and Laboratory Standards Institute (CLSI), US Food and Drug Administration (FDA), and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). RESULTS: Breakpoint criteria impacted interpretation of susceptibly testing results, for example with the broth microdilution we found 8% (2/25) of A. baumannii isolates change interpretation between CLSI and EUCAST and 32% (8/25) change between CLSI and FDA, 12% (3/25) of Enterobacterales change between CLSI and EUCAST, 13% (3/23) of P. aeruginosa interpretations change between CLSI and FDA, and 4% (1/25) S. maltophilia change between CLSI and FDA. There was a significant difference between the zone disk diffusion diameters for P. aeruginosa and S. maltophilia between Hardy and BD; which changed interpretation (using CLSI criteria) for 8.7% (2/23) for P. aeruginosa but 0% (0/24) for S. maltophilia. CONCLUSIONS: Breakpoint criteria impact cefiderocol susceptibility testing interpretation for broth microdilution and disk diffusion. Choice of MHA brand can also affect result interpretation.

Outpatient Clonal Propagation and Rapid Regional Establishment of an Emergent Carbapenem-Resistant Acinetobacter baumannnii Lineage Sequence Type 499Pas.

Eliminating carbapenem-resistant Acinetobacter baumannii (CRAb) disease requires comprehensive knowledge of how this noncommensal organism propagates among at-risk hosts. We molecularly characterized an ongoing surge of CRAb cases among patients in a Midwest US healthcare system, which coincided with sustained reductions in hospital-acquired CRAb infections and falloffs of cases associated with distinctly more resistant antibiotypes. Genome sequencing revealed surge isolates belonged to an emergent Pasteur scheme sequence type 499 and comprised multiple contemporaneous clonal clusters. Detailed query of health records revealed no consistent hospital source but instead identified various outpatient healthcare settings linked to cluster cases. We show that CRAb can rapidly establish a regional presence even without gains in breadth of antibiotic resistance and negligible contribution from sustained intrahospital transmission. As CRAb lineages may sidestep control efforts via outpatient epidemiological niches, our approach can be implemented to investigate outpatient CRAb propagation and inform subsequent local surveillance outside hospital settings.

E. coli catheter-associated urinary tract infections are associated with distinctive virulence and biofilm gene determinants.

Urinary catheterization facilitates urinary tract colonization by E. coli and increases infection risk. Here, we aimed to identify strain-specific characteristics associated with the transition from colonization to infection in catheterized patients. In a single-site study population, we compared E. coli isolates from patients with catheter-associated asymptomatic bacteriuria (CAASB) to those with catheter-associated urinary tract infection (CAUTI). CAUTI isolates were dominated by a phylotype B2 subclade containing the multidrug-resistant ST131 lineage relative to CAASB isolates, which were phylogenetically more diverse. A distinctive combination of virulence-associated genes was present in the CAUTI-associated B2 subclade. Catheter-associated biofilm formation was widespread among isolates and did not distinguish CAUTI from CAASB strains. Preincubation with CAASB strains could inhibit catheter colonization by multiple ST131 CAUTI isolates. Comparative genomic analysis identified a group of variable genes associated with high catheter biofilm formation present in both CAUTI and CAASB strains. Among these, ferric citrate transport (Fec) system genes were experimentally associated with enhanced catheter biofilm formation using reporter and fecA deletion strains. These results are consistent with a variable role for catheter biofilm formation in promoting CAUTI by ST131-like strains or resisting CAUTI by lower-risk strains that engage in niche exclusion.

Genomic Surveillance of Clinical Pseudomonas aeruginosa Isolates Reveals an Additive Effect of Carbapenemase Production on Carbapenem Resistance.

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of ß-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. ß-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all ß-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-ß-lactamases (MBLs) or extended-spectrum ß-lactamases (ESBLs) in the blaGES, blaVIM, and blaNDM families, and mutations in the porin gene oprD. These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and ß-lactam/ß-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of ß-lactamase genes.

Multiplatform Assessment of Saliva for SARS-CoV-2 Molecular Detection in Symptomatic Healthcare Personnel and Patients Presenting to the Emergency Department.

BACKGROUND: Saliva has garnered great interest as an alternative specimen type for molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Data are limited on the relative performance of different molecular methods using saliva specimens and the relative sensitivity of saliva to nasopharyngeal (NP) swabs. METHODS: To address the gap in knowledge, we enrolled symptomatic healthcare personnel (n = 250) from Barnes-Jewish Hospital/Washington University Medical Center and patients presenting to the Emergency Department with clinical symptoms compatible with coronavirus disease 2019 (COVID-19; n = 292). We collected paired saliva specimens and NP swabs. The Lyra SARS-CoV-2 assay (Quidel) was evaluated on paired saliva and NP samples. Subsequently we compared the Simplexa COVID-19 Direct Kit (Diasorin) and a modified SalivaDirect (Yale) assay on a subset of positive and negative saliva specimens. RESULTS: The positive percent agreement (PPA) between saliva and NP samples using the Lyra SARS-CoV-2 assay was 63.2%. Saliva samples had higher SARS-CoV-2 cycle threshold values compared to NP swabs (P < 0.0001). We found a 76.47% (26/34) PPA for Simplexa COVID-19 Direct Kit on saliva and a 67.6% (23/34) PPA for SalivaDirect compared to NP swab results. CONCLUSION: These data demonstrate molecular assays have variability in performance for detection of SARS-CoV-2 in saliva.

Evaluation of PCR cycle threshold values by patient population with the quidel lyra SARS-CoV-2 assay.

The Lyra SARS-CoV-2 assay was the primary method for molecular testing performed at Barnes-Jewish Healthcare System in St. Louis, Missouri during the initial COVID-19 surge from mid-March to late-April 2020. We performed a retrospective analysis of 1,043 positive Lyra SARS-CoV-2 results during these 36 days to investigate associations between cycle threshold (CT)  value and patient characteristics. Total RNA were extracted from NP or OP swabs using either the EasyMag or KingFisher automated extraction systems and quantified with RotorGene Q (Qiagen) or Applied Biosystems 7500 Fast Dx thermocyclers respectively. Notably, we found lower a significant median lower CT for samples tested on the KingFisher-ABI 7500 fastDX (KF/ABI) system compared to the EasyMag/RotorGene (EM/RGQ) platform. Since 77.5% of our tests were ran on the EM/RGQ pipeline we then perform additional analysis on these values and found that C T values in outpatient care settings compared to samples obtained in the emergency department or inpatient had significantly lower C T values. These collective findings suggests a difference in viral load amongst various patient populations.

IL-7 Immunotherapy in a Nonimmunocompromised Patient With Intractable Fungal Wound Sepsis.

A nonimmunocompromised patient developed life-threatening soft tissue infection with Trichosporon asahii, Fusarium, and Saksenaea that progressed despite maximum antifungal therapies and aggressive debridement. Interleukin-7 immunotherapy resulted in clinical improvement, fungal clearance, reversal of lymphopenia, and improved T-cell function. Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening fungal infections.

A Visual Interactive Narrative Intervention (VINI) for aphasia education: Can digital applications administer augmented input to educate stroke survivors with aphasia?

OBJECTIVE: Persons with aphasia (PWA) face additional barriers to proper healthcare due to inadequate patient education by health professionals unequipped to use augmentative and alternative communication (AAC). The current study examines a digital application that evokes and sustains health information processing through AAC specifically aimed at increasing comprehension with augmented input (AI). METHODS: A digital application designed to educate PWA about their health condition was compared to a video-recorded doctor providing oral-only education. Sixteen PWA received both education interventions in a crossover manner. Health information processing was assessed through heart rate (HR) and skin conductance levels (SCL), which were collected continually during each administration of education interventions. RESULTS: PWA demonstrated greater cognitive processing of health information via HR and SCL indices during the digital application compared to the typical oral-only education intervention. The oral-only intervention led PWA to disengage with health information. CONCLUSION: By combining visuographic materials and adapted language into a customizable narrative structure, digital applications can utilize AI to educate PWA about basic health information (i.e., diagnosis and prognosis). PRACTICE IMPLICATIONS: The current study's AAC requires minimal training and can be used as an aided support in conjunction with other techniques that increase PWA's access to health information.

Genomic Prediction of Antimicrobial Resistance: Ready or Not, Here It Comes!

BACKGROUND: Next-generation sequencing (NGS) technologies are being used to predict antimicrobial resistance. The field is evolving rapidly and transitioning out of the research setting into clinical use. Clinical laboratories are evaluating the accuracy and utility of genomic resistance prediction, including methods for NGS, downstream bioinformatic pipeline components, and the clinical settings in which this type of testing should be offered. CONTENT: We describe genomic sequencing as it pertains to predicting antimicrobial resistance in clinical isolates and samples. We elaborate on current methodologies and workflows to perform this testing and summarize the current state of genomic resistance prediction in clinical settings. To highlight this aspect, we include 3 medically relevant microorganism exemplars: Mycobacterium tuberculosis, Staphylococcus aureus, and Neisseria gonorrhoeae. Last, we discuss the future of genomic-based resistance detection in clinical microbiology laboratories. SUMMARY: Antimicrobial resistance prediction by genomic approaches is in its infancy for routine patient care. Genomic approaches have already added value to the current diagnostic testing landscape in specific circumstances and will play an increasingly important role in diagnostic microbiology. Future advancements will shorten turnaround time, reduce costs, and improve our analysis and interpretation of clinically actionable results.

Breakpoint beware: reliance on historical breakpoints for Enterobacteriaceae leads to discrepancies in interpretation of susceptibility testing for carbapenems and cephalosporins and gaps in detection of carbapenem-resistant organisms.

Carbapenem-resistant Enterobacteriaceae (CRE) are an important public health and infection prevention threat. CRE are typically detected via phenotypic antimicrobial susceptibility testing (AST), for which interpretive standards were modified in recent years. Our objective was to measure the impact of breakpoint changes on AST interpretation for CRE. Zone sizes from disk diffusion AST for Enterobacteriaceae isolates recovered from clinical cultures over a 1-year period (n = 10,183) and CRE from clinical and environmental sources from the USA and Pakistan (n = 342) were evaluated. Results were interpreted according to historical (CLSI M100-S19) and current (CLSI M100-S29) breakpoints. Interpretive errors were calculated according to the FDA definitions. Using current breakpoints as the reference standard, 56 (17%) very major (false susceptibility) errors occurred for cefepime and 13 (45%) very major errors for meropenem interpretation using historical breakpoints in clinical isolates of Enterobacteriaceae, corresponding to 12 carbapenemase-producing CRE that would have been missed during the 1-year period. For confirmed blaKPC CP-CRE clinical and environmental isolates (n = 149), the very major error rate for historic breakpoints was 8%, 30%, 63%, and 0% for cefepime, meropenem, imipenem, and ertapenem, respectively. For blaKPC isolates, the use of historical breakpoints would have led to 42 (28%) reports of false susceptibility to meropenem. Failure to adopt updated AST breakpoints may lead to reports of false susceptibility for antimicrobials commonly used to treat Gram-negative infections and preclude recognition of CRE. Such errors could negatively impact patient care and hamper infection control and public health efforts.

Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces.

Bacterial pathogens that infect patients also contaminate hospital surfaces. These contaminants impact hospital infection control and epidemiology, prompting quantitative examination of their transmission dynamics. Here we investigate spatiotemporal and phylogenetic relationships of multidrug resistant (MDR) bacteria on intensive care unit surfaces from two hospitals in the United States (US) and Pakistan collected over one year. MDR bacteria isolated from 3.3% and 86.7% of US and Pakistani surfaces, respectively, include common nosocomial pathogens, rare opportunistic pathogens, and novel taxa. Common nosocomial isolates are dominated by single lineages of different clones, are phenotypically MDR, and have high resistance gene burdens. Many resistance genes (e.g., blaNDM, blaOXA carbapenamases), are shared by multiple species and flanked by mobilization elements. We identify Acinetobacter baumannii and Enterococcus faecium co-association on multiple surfaces, and demonstrate these species establish synergistic biofilms in vitro. Our results highlight substantial MDR pathogen burdens in hospital built-environments, provide evidence for spatiotemporal-dependent transmission, and demonstrate potential mechanisms for multi-species surface persistence.

Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan.

OBJECTIVES: Linezolid is an important therapeutic option for the treatment of infections caused by VRE. Linezolid is a synthetic antimicrobial and resistance to this antimicrobial agent remains relatively rare. As a result, data on the comparative genomics of linezolid resistance determinants in Enterococcus faecium are relatively sparse. METHODS: To address this knowledge gap in E. faecium, we deployed phenotypic antibiotic susceptibility testing and Illumina WGS on hospital surface (environmental) and clinical isolates from the USA and Pakistan. RESULTS: We found complete concordance between isolate source country and mechanism of linezolid resistance, with all the US isolates possessing a 23S rRNA gene mutation and the Pakistan isolates harbouring two to three acquired antibiotic resistance genes. These resistance genes include the recently elucidated efflux-pump genes optrA and poxtA and a novel cfr-like variant. Although there was no difference in the linezolid MIC between the US and Pakistan isolates, there was a significant difference in the geometric mean of the MIC between the Pakistan isolates that had two versus three of the acquired antibiotic resistance genes. In five of the Pakistan E. faecium that possessed all three of the resistance genes, we found no difference in the local genetic context of poxtA and the cfr-like gene, but we identified different genetic contexts surrounding optrA. CONCLUSIONS: These results demonstrate that E. faecium from different geographical regions employ alternative strategies to counter selective pressure of increasing clinical linezolid use.

In Silico Analysis of Gardnerella Genomospecies Detected in the Setting of Bacterial Vaginosis.

BACKGROUND: Gardnerella vaginalis is implicated as one of the causative agents of bacterial vaginosis, but it can also be isolated from the vagina of healthy women. Previous efforts to study G. vaginalis identified 4 to 6 clades, but average nucleotide identity analysis indicates that G. vaginalis may be multiple species. Recently, Gardnerella was determined to be 13 genomospecies, with Gardnerella piottii, Gardnerella leopoldii, and Gardnerella swidsinkii delineated as separate species. METHODS: We accessed 103 publicly available genomes annotated as G. vaginalis. We performed comprehensive taxonomic and phylogenomic analysis to quantify the number of species called G. vaginalis, the similarity of their core genes, and their burden of their accessory genes. We additionally analyzed publicly available metatranscriptomic data sets of bacterial vaginosis to determine whether the newly delineated genomospecies are present, and to identify putative conserved features of Gardnerella pathogenesis. RESULTS: Gardnerella could be classified into 8 to 14 genomospecies depending on the in silico classification tools used. Consensus classification identified 9 different Gardnerella genomospecies, here annotated as GS01 through GS09. The genomospecies could be readily distinguished by the phylogeny of their shared genes and burden of accessory genes. All of the new genomospecies were identified in metatranscriptomes of bacterial vaginosis. CONCLUSIONS: Multiple Gardnerella genomospecies operating in isolation or in concert with one another may be responsible for bacterial vaginosis. These results have important implications for future efforts to understand the evolution of the Gardnerella genomospecies, host-pathogen interactions of the genomospecies during bacterial vaginosis, diagnostic assay development for bacterial vaginosis, and metagenomic investigations of the vaginal microbiota.

Genotypic and Phenotypic Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae: a Cross-Sectional Study of Isolates Recovered from Routine Urine Cultures in a High-Incidence Setting.

The objectives of this study were to perform genomic and phenotypic characterization of antimicrobial resistance in Neisseria gonorrhoeae isolates recovered from urine samples from patients in St. Louis, MO, USA. Sixty-four clinical isolates were banked over a 2-year period and subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion (penicillin, tetracycline, cefuroxime, and ciprofloxacin) and gradient diffusion (tetracycline, doxycycline, azithromycin, ceftriaxone, cefixime, ciprofloxacin, gemifloxacin, and delafloxacin). The medical records for the patients were evaluated to determine the demographics, location, and prescribed treatment regimen. Isolate draft genomes were assembled from Illumina shotgun sequencing data, and resistance determinants were identified by ResFinder and PointFinder. Of the 64 isolates, 97% were nonsusceptible to penicillin, with resistant isolates all containing the blaTEM-1b gene; 78 and 81% of isolates were nonsusceptible to tetracycline and doxycycline, respectively, with resistant isolates all containing the tet(M) gene. One isolate was classified as non-wild-type to azithromycin, and all isolates were susceptible to ceftriaxone; 89% of patients received this combination of drugs as first-line therapy. Six percent of isolates were resistant to ciprofloxacin, with most resistant isolates containing multiple gyrA and parC mutations. Correlation between disk and gradient diffusion AST devices was high for tetracycline and ciprofloxacin (R2 > 99% for both). The rates of N. gonorrhoeae antibiotic resistance in St. Louis are comparable to current rates reported nationally, except ciprofloxacin resistance was less common in our cohort. Strong associations between specific genetic markers and phenotypic susceptibility testing hold promise for the utility of genotype-based diagnostic assays to guide directed antibiotic therapy.IMPORTANCENeisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is most commonly diagnosed using a DNA-based detection method that does not require growth and isolation of N. gonorrhoeae in the laboratory. This is problematic because the rates of antibiotic resistance in N. gonorrhoeae are increasing, but without isolating the organism in the clinical laboratory, antibiotic susceptibility testing cannot be performed on strains recovered from clinical specimens. We observed an increase in the frequency of urine cultures growing N. gonorrhoeae after we implemented a total laboratory automation system for culture in our clinical laboratory. Here, we report on the rates of resistance to multiple historically used, first-line, and potential future-use antibiotics for 64 N. gonorrhoeae isolates. We found that the rates of antibiotic resistance in our isolates were comparable to national rates. Additionally, resistance to specific antibiotics correlated closely with the presence of genetic resistance genes, suggesting that DNA-based tests could also be designed to guide antibiotic therapy for treating gonorrhea.