ABSTRACT
Borrelia miyamotoi is an emerging tickborne pathogen that has been associated with central nervous system infections in immunocompromised patients, albeit infrequently. We describe a case-patient in Minnesota, USA, who had meningeal symptoms of 1 month duration. B. miyamotoi infection was diagnosed by Gram staining on cerebrospinal fluid and confirmed by sequencing.
Subject(s)
Borrelia , Meningoencephalitis , Humans , Borrelia/isolation & purification , Borrelia/genetics , Minnesota/epidemiology , Meningoencephalitis/microbiology , Meningoencephalitis/diagnosis , Male , Borrelia Infections/diagnosis , Borrelia Infections/microbiology , Borrelia Infections/drug therapy , Borrelia Infections/complications , Anti-Bacterial Agents/therapeutic use , Middle Aged , Acute Disease , FemaleABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited. METHODS: We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples. RESULTS: We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January. CONCLUSIONS: We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.
Subject(s)
COVID-19 , SARS-CoV-2 , Diagnostic Techniques and Procedures , Humans , Los Angeles/epidemiology , Retrospective StudiesABSTRACT
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused global disruption of human health and activity. Being able to trace the early outbreak of SARS-CoV-2 within a locality can inform public health measures and provide insights to contain or prevent viral transmission. Investigation of the transmission history requires efficient sequencing methods and analytic strategies, which can be generally useful in the study of viral outbreaks. METHODS: The County of Los Angeles (hereafter, LA County) sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history, we carried out surveillance viral genome sequencing to determine 142 viral genomes from unique patients seeking care at the University of California, Los Angeles (UCLA) Health System. 86 of these genomes were from samples collected before April 19, 2020. RESULTS: We found that the early outbreak in LA County, as in other international air travel hubs, was seeded by multiple introductions of strains from Asia and Europe. We identified a USA-specific strain, B.1.43, which was found predominantly in California and Washington State. While samples from LA County carried the ancestral B.1.43 genome, viral genomes from neighboring counties in California and from counties in Washington State carried additional mutations, suggesting a potential origin of B.1.43 in Southern California. We quantified the transmission rate of SARS-CoV-2 over time, and found evidence that the public health measures put in place in LA County to control the virus were effective at preventing transmission, but might have been undermined by the many introductions of SARS-CoV-2 into the region. CONCLUSION: Our work demonstrates that genome sequencing can be a powerful tool for investigating outbreaks and informing the public health response. Our results reinforce the critical need for the USA to have coordinated inter-state responses to the pandemic.
Subject(s)
COVID-19 , COVID-19/epidemiology , Disease Outbreaks , Genomics , Humans , Los Angeles/epidemiology , SARS-CoV-2/geneticsABSTRACT
Candida auris is an emerging multidrug-resistant yeast. We describe an ongoing C. auris outbreak that began in October 2019 in Los Angeles, California, USA. We used genomic analysis to determine that isolates from 5 of 6 patients belonged to clade III; 4 isolates were closely related.
Subject(s)
Candida , Candidiasis , Antifungal Agents , Genomics , Humans , Los Angeles , Microbial Sensitivity TestsABSTRACT
Aerococcus urinae is increasingly recognized as a potentially significant urinary tract bacterium. A. urinae has been isolated from urine collected from both males and females with a wide range of clinical conditions, including urinary tract infection (UTI), urgency urinary incontinence (UUI), and overactive bladder (OAB). A. urinae is of particular clinical concern because it is highly resistant to many antibiotics and, when undiagnosed, can cause invasive and life-threatening bacteremia, sepsis, or soft tissue infections. Previous genomic characterization studies have examined A. urinae strains isolated from patients experiencing UTI episodes. Here, we analyzed the genomes of A. urinae strains isolated as part of the urinary microbiome from patients with UUI or OAB. Furthermore, we report that certain A. urinae strains exhibit aggregative in vitro phenotypes, including flocking, which can be modified by various growth medium conditions. Finally, we performed in-depth genomic comparisons to identify pathways that distinguish flocking and nonflocking strains.IMPORTANCEAerococcus urinae is a urinary bacterium of emerging clinical interest. Here, we explored the ability of 24 strains of A. urinae isolated from women with lower urinary tract symptoms to display aggregation phenotypes in vitro We sequenced and analyzed the genomes of these A. urinae strains. We performed functional genomic analyses to determine whether the in vitro hyperflocking aggregation phenotype displayed by certain A. urinae strains was related to the presence or absence of certain pathways. Our findings demonstrate that A. urinae strains have different propensities to display aggregative properties in vitro and suggest a potential association between phylogeny and flocking.
Subject(s)
Aerococcus/genetics , Genome, Bacterial , Gram-Positive Bacterial Infections/microbiology , Lower Urinary Tract Symptoms/microbiology , Aerococcus/classification , Aerococcus/drug effects , Aerococcus/physiology , Anti-Bacterial Agents/pharmacology , Biofilms , Female , Humans , Male , Microbial Sensitivity Tests , PhylogenyABSTRACT
BACKGROUND: Since the discovery of the bladder microbiome (urobiome), interest has grown in learning whether urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for women with common forms of urinary incontinence. OBJECTIVE: This study aimed to test the hypothesis that the bladder urobiome differs among women in the control cohort and women affected by urinary incontinence by assessing associations between urinary incontinence status and the cultured urobiome. STUDY DESIGN: With institutional review board oversight, urine specimens from 309 adult women were collected through transurethral catheterization. These women were categorized into 3 cohorts (continent control, stress urinary incontinence [SUI], and urgency urinary incontinence [UUI]) based on their responses to the validated Pelvic Floor Distress Inventory (PFDI) questionnaire. Among 309 women, 150 were in the continent control cohort, 50 were in the SUI cohort, and 109 were in the UUI cohort. Symptom severity was assessed by subscale scoring with the Urinary Distress Inventory (UDI), subscale of the Pelvic Floor Distress Inventory. Microbes were assessed by expanded quantitative urine culture protocol, which detects the most common bladder microbes (bacteria and yeast). Microbes were identified to the species level by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Alpha diversity indices were calculated for culture-positive samples and compared across the 3 cohorts. The correlations of UDI scores, alpha diversity indices, and species abundance were estimated. RESULTS: Participants had a mean age of 53 years (range 22-90); most were whites (65%). Women with urinary incontinence were slightly older (control, 47; SUI, 54; UUI, 61). By design, UDI symptom scores differed (control, 8.43 [10.1]; SUI, 97.95 [55.36]; UUI, 93.71 [49.12]; P<.001). Among 309 participants, 216 (70%) had expanded quantitative urine culture-detected bacteria; furthermore, the urinary incontinence cohorts had a higher detection frequency than the control cohort (control, 57%; SUI, 86%; UUI, 81%; P<.001). In addition, the most frequently detected species among the cohorts were as follows: continent control, Lactobacillus iners (12.7%), Streptococcus anginosus (12.7%), L crispatus (10.7%), and L gasseri (10%); SUI, S anginosus (26%), L iners (18%), Staphylococcus epidermidis (18%), and L jensenii (16%); and UUI, S anginosus (30.3%), L gasseri (22%), Aerococcus urinae (18.3%), and Gardnerella vaginalis (17.4%). However, only Actinotignum schaalii (formerly Actinobaculum schaalii), A urinae, A sanguinicola, and Corynebacterium lipophile group were found at significantly higher mean abundances in 1 of the urinary incontinence cohorts when compared with the control cohort (Wilcoxon rank sum test; P<.02), and no individual genus differed significantly between the 2 urinary incontinence cohorts. Both urinary incontinence cohorts had increased alpha diversity similar to continent control cohort with indices of species richness, but not evenness, strongly associated with urinary incontinence. CONCLUSION: In adult women, the composition of the culturable bladder urobiome is associated with urinary incontinence, regardless of common incontinence subtype. Detection of more unique living microbes was associated with worsening incontinence symptom severity. Culturable species richness was significantly greater in the urinary incontinence cohorts than in the continent control cohort.
Subject(s)
Biodiversity , Microbiota , Urinary Bladder/microbiology , Urinary Incontinence, Stress/microbiology , Urinary Incontinence, Urge/microbiology , Actinomycetaceae/isolation & purification , Adult , Aerococcus/isolation & purification , Aged , Aged, 80 and over , Corynebacterium/isolation & purification , Cross-Sectional Studies , Female , Gardnerella vaginalis/isolation & purification , Humans , Lactobacillus/isolation & purification , Lactobacillus crispatus/isolation & purification , Lactobacillus gasseri/isolation & purification , Middle Aged , Staphylococcus epidermidis/isolation & purification , Streptococcus anginosus/isolation & purification , Young AdultABSTRACT
BACKGROUND: Previous work has shown that the vaginal microbiome decreases in Lactobacillus predominance and becomes more diverse after menopause. It has also been shown that estrogen therapy restores Lactobacillus dominance in the vagina and that topical estrogen is associated with overactive bladder symptom improvement. We now know that the bladder contains a unique microbiome and that increased bladder microbiome diversity is associated with overactive bladder. However, there is no understanding of how quickly each pelvic floor microbiome responds to estrogen or if those changes are associated with symptom improvement. OBJECTIVE: This study aimed to determine if estrogen treatment of postmenopausal women with overactive bladder decreases urobiome diversity. STUDY DESIGN: We analyzed data from postmenopausal participants in 2 trials (NCT02524769 and NCT02835846) who chose vaginal estrogen as the primary overactive bladder treatment and used 0.5 g of conjugated estrogen (Premarin cream; Pfizer, New York City, NY) twice weekly for 12 weeks. Baseline and 12-week follow-up data included the Overactive Bladder questionnaire, and participants provided urine samples via catheter, vaginal swabs, perineal swabs, and voided urine samples. Microbes were detected by an enhanced culture protocol. Linear mixed models were used to estimate microbiome changes over time. Urinary antimicrobial peptide activity was assessed by a bacterial growth inhibition assay and correlated with relative abundance of members of the urobiome. RESULTS: In this study, 12 weeks of estrogen treatment resulted in decreased microbial diversity within the vagina (Shannon, P=.047; Richness, P=.043) but not in the other niches. A significant increase in Lactobacillus was detected in the bladder (P=.037) but not in the vagina (P=.33), perineum (P=.56), or voided urine (P=.28). The change in Lactobacillus levels in the bladder was associated with modest changes in urgency incontinence symptoms (P=.02). The relative abundance of the genus Corynebacterium correlated positively with urinary antimicrobial peptide activity after estrogen treatment. CONCLUSION: Estrogen therapy may change the microbiome of different pelvic floor niches. The vagina begins to decrease in diversity, and the bladder experiences a significant increase in Lactobacillus levels; the latter is correlated with a modest improvement in the symptom severity subscale of the Overactive Bladder questionnaire.
Subject(s)
Estrogens, Conjugated (USP)/therapeutic use , Estrogens/therapeutic use , Lactobacillus/isolation & purification , Microbiota , Urinary Bladder, Overactive/drug therapy , Urinary Bladder/microbiology , Urine/microbiology , Actinomyces/isolation & purification , Administration, Intravaginal , Aged , Antimicrobial Cationic Peptides/urine , Biodiversity , Chromatography, High Pressure Liquid , Corynebacterium/isolation & purification , Female , Humans , Middle Aged , Postmenopause , Streptococcus/isolation & purification , Treatment Outcome , Urinary Bladder, Overactive/physiopathology , Urinary Incontinence, Urge/physiopathologyABSTRACT
INTRODUCTION AND HYPOTHESIS: The current etiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is poorly understood and multifactorial. Recent studies suggest the female urinary microbiota (FUM) contribute to IC/PBS symptoms. This study was designed to determine if the FUM, analyzed using mid-stream voided urine samples, differs between IC/PBS patients and controls. METHODS: This prospective case-controlled study compared the voided FUM of women with symptoms of urinary frequency, urgency, and bladder pain for > 6 months with the voided FUM of healthy female controls without pain. Bacterial identification was performed using 16S rRNA gene sequencing and EQUC, a validated enhanced urine culture approach. Urotype was defined by a genus present at > 50% relative abundance. If no genus was present above this threshold, the urotype was classified as 'mixed.' Group comparisons were performed for urotype and diversity measures. RESULTS: A mid-stream voided specimen was collected from 21 IC/PBS patients and 20 asymptomatic controls. The two groups had similar demographics. Urotypes did not differ between cohorts as assessed by either EQUC or 16S rRNA gene sequencing. We detected no significant differences between cohorts by alpha diversity. Cohorts also were not distinct using principle component analysis or hierarchical clustering. Detection by EQUC of bacterial species considered uropathogenic was high in both cohorts, but detection of these uropathogenic species did not differ between groups (p = 0.10). CONCLUSIONS: Enhanced culture and DNA sequencing methods provide evidence that IC/PBS symptoms may not be related to differences in the FUM, at least not its bacterial components. Future larger studies are needed to confirm this preliminary finding.
Subject(s)
Bacteria/isolation & purification , Cystitis, Interstitial/microbiology , Microbiota , Urethra/microbiology , Urinary Bladder/microbiology , Adult , Case-Control Studies , Female , Humans , Middle Aged , Prospective StudiesABSTRACT
Urinary tract infection (UTI) is clinically important, given that it is one of the most common bacterial infections in adult women. However, the current understanding of UTI remains based on a now disproven concept that the urinary bladder is sterile. Thus, current standards for UTI diagnosis have significant limitations that may reduce the opportunity to improve patient care. Using data from our work and numerous other peer-reviewed studies, we identified four major limitations to the contemporary UTI description: the language of UTI, UTI diagnostic testing, the Escherichia coli-centric view of UTI, and the colony-forming units (CFU) threshold-based diagnosis. Contemporary methods and technology, combined with continued rigorous clinical research can be used to correct these limitations.
Subject(s)
Colony Count, Microbial/methods , Urinary Tract Infections/diagnosis , Adult , Aged , Escherichia coli/isolation & purification , Female , Humans , Middle Aged , Urinary Bladder/microbiology , Urinary Tract Infections/microbiology , Urine/microbiologyABSTRACT
Enhanced quantitative urine culture (EQUC) detects live microorganisms in the vast majority of urine specimens reported as "no growth" by the standard urine culture protocol. Here, we evaluated an expanded set of EQUC conditions (expanded-spectrum EQUC) to identify an optimal version that provides a more complete description of uropathogens in women experiencing urinary tract infection (UTI)-like symptoms. One hundred fifty adult urogynecology patient-participants were characterized using a self-completed validated UTI symptom assessment (UTISA) questionnaire and asked "Do you feel you have a UTI?" Women responding negatively were recruited into the no-UTI cohort, while women responding affirmatively were recruited into the UTI cohort; the latter cohort was reassessed with the UTISA questionnaire 3 to 7 days later. Baseline catheterized urine samples were plated using both standard urine culture and expanded-spectrum EQUC protocols: standard urine culture inoculated at 1 µl onto 2 agars incubated aerobically; expanded-spectrum EQUC inoculated at three different volumes of urine onto 7 combinations of agars and environments. Compared to expanded-spectrum EQUC, standard urine culture missed 67% of uropathogens overall and 50% in participants with severe urinary symptoms. Thirty-six percent of participants with missed uropathogens reported no symptom resolution after treatment by standard urine culture results. Optimal detection of uropathogens could be achieved using the following: 100 µl of urine plated onto blood (blood agar plate [BAP]), colistin-nalidixic acid (CNA), and MacConkey agars in 5% CO2 for 48 h. This streamlined EQUC protocol achieved 84% uropathogen detection relative to 33% detection by standard urine culture. The streamlined EQUC protocol improves detection of uropathogens that are likely relevant for symptomatic women, giving clinicians the opportunity to receive additional information not currently reported using standard urine culture techniques.
Subject(s)
Bacteria/isolation & purification , Bacterial Infections/diagnosis , Bacterial Load , Bacteriological Techniques/methods , Urinary Tract Infections/diagnosis , Urine/microbiology , Adult , Aged , Aged, 80 and over , Bacterial Infections/microbiology , Female , Humans , Middle Aged , Sensitivity and Specificity , Surveys and Questionnaires , Urinary Tract Infections/microbiology , Young AdultABSTRACT
INTRODUCTION AND HYPOTHESIS: Many adult women have resident urinary bacteria (urinary microbiome/microbiota). In adult women affected by urinary urgency incontinence (UUI), the etiologic and/or therapeutic role of the urinary microbiome/microbiota remains unknown. We hypothesized that microbiome/microbiota characteristics would relate to clinically relevant treatment response to UUI medication per os. METHODS: Adult women initiating medication treatment orally for UUI and a comparator group of unaffected women were recruited in a tertiary care health-care system. All participants provided baseline clinical data and urine samples. Women with UUI were given 5 mg solifenacin, with potential dose escalation to 10 mg for inadequate UUI symptom control at 4 weeks. Additional data and urine samples were collected from women with UUI at 4 and 12 weeks. The samples were assessed using 16S ribosomal RNA (rRNA) gene sequencing and enhanced quantitative urine culturing. The primary outcome was treatment response as measured by the validated Patient Global Symptom Control (PGSC) questionnaire. Clinically relevant UUI symptom control was defined as a 4 or 5 score on the PGSC. RESULTS: Diversity and composition of the urinary microbiome/microbiota of women with and without UUI differed at baseline. Women with UUI had more bacteria and a more diverse microbiome/microbiota. The clinical response to solifenacin in UUI participants was related to baseline microbiome/microbiota, with responders more likely to have fewer bacteria and a less diverse community at baseline. Nonresponders had a more diverse community that often included bacteria not typically found in responders. CONCLUSIONS: Knowledge of an individual's urinary microbiome/microbiota may help refine UUI treatment. Complementary tools, DNA sequencing, and expanded urine culture provide information about bacteria that appear to be related to UUI incontinence status and treatment response in this population of adult women.
Subject(s)
Bacteriuria/microbiology , Microbiota , Muscarinic Antagonists/therapeutic use , RNA, Ribosomal, 16S/analysis , Solifenacin Succinate/therapeutic use , Urinary Incontinence, Urge/drug therapy , Urinary Incontinence, Urge/microbiology , Urinary Tract/microbiology , Actinomyces/isolation & purification , Administration, Oral , Adult , Aged , Case-Control Studies , Colony Count, Microbial , Corynebacterium/isolation & purification , Female , Humans , Lactobacillus/isolation & purification , Middle Aged , Muscarinic Antagonists/administration & dosage , Prospective Studies , Solifenacin Succinate/administration & dosage , Streptococcus/isolation & purification , Treatment OutcomeABSTRACT
Our previous study showed that bacterial genomes can be identified using 16S rRNA sequencing in urine specimens of both symptomatic and asymptomatic patients who are culture negative according to standard urine culture protocols. In the present study, we used a modified culture protocol that included plating larger volumes of urine, incubation under varied atmospheric conditions, and prolonged incubation times to demonstrate that many of the organisms identified in urine by 16S rRNA gene sequencing are, in fact, cultivable using an expanded quantitative urine culture (EQUC) protocol. Sixty-five urine specimens (from 41 patients with overactive bladder and 24 controls) were examined using both the standard and EQUC culture techniques. Fifty-two of the 65 urine samples (80%) grew bacterial species using EQUC, while the majority of these (48/52 [92%]) were reported as no growth at 10(3) CFU/ml by the clinical microbiology laboratory using the standard urine culture protocol. Thirty-five different genera and 85 different species were identified by EQUC. The most prevalent genera isolated were Lactobacillus (15%), followed by Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%), and Staphylococcus (6.9%). Other genera commonly isolated include Aerococcus, Gardnerella, Bifidobacterium, and Actinobaculum. Our current study demonstrates that urine contains communities of living bacteria that comprise a resident female urine microbiota.
Subject(s)
Bacteria/isolation & purification , Microbiological Techniques/methods , Specimen Handling/methods , Urinary Bladder/microbiology , Urine/microbiology , Adult , Bacteria/classification , Female , Humans , Sensitivity and SpecificityABSTRACT
Background: Multistep laboratory testing is recommended for the diagnosis of Clostridioides difficile infection (CDI). The aim of this study was to present the impact of multistep CDI diagnostic testing in an academic hospital system and evaluate the toxin B gene polymerase chain reaction (PCR) cycle threshold (Ct) values of PCR-positive tests. Methods: In October 2022, our system began reflex testing all PCR-positive stool samples with the C. DIFF QUIK CHEK COMPLETE (Techlab), an enzyme immunoassay-based test with results for the glutamate dehydrogenase antigen (GDH) and C difficile toxin A/B. Hospital-onset (HO) CDI and CDI antibiotic use before and after testing were tracked. Ct values were obtained from the Infectious Diseases Diagnostic Laboratory. Receiver operating curve analysis was used to examine the sensitivity and specificity for identifying GDH+/toxin+ and GDH-/toxin- at various Ct thresholds. Results: The HO-CDI rate decreased from 0.352 cases per 1000 patient-days to 0.115 cases per 1000 patient-days post-reflex testing (P < .005). Anti-CDI antibiotics use decreased, but the decrease was not commensurate with CDI rates following reflex testing. PCR+/GDH+/toxin+ samples had a lower mean Ct value than PCR+/GDH-/toxin- samples (23.3 vs 33.5, P < .0001). A Ct value of 28.65 could distinguish between those 2 groups. Fifty-four percent of PCR+/GDH+/toxin- samples had a Ct value below that cut-off, suggesting the possibility of CDI with a negative toxin test. Conclusions: Reflex testing for a laboratory diagnosis of CDI results in rapid, systemwide decreases in the rate of HO-CDI. Additional research is needed to distinguish CDI from C difficile colonization in patients with discordant testing.
ABSTRACT
Next-generation sequencing (NGS) technologies have become increasingly available for use in the clinical microbiology diagnostic environment. There are three main applications of these technologies in the clinical microbiology laboratory: whole genome sequencing (WGS), targeted metagenomics sequencing and shotgun metagenomics sequencing. These applications are being utilized for initial identification of pathogenic organisms, the detection of antimicrobial resistance mechanisms and for epidemiologic tracking of organisms within and outside hospital systems. In this review, we analyze these three applications and provide a comprehensive summary of how these applications are currently being used in public health, basic research, and clinical microbiology laboratory environments. In the public health arena, WGS is being used to identify and epidemiologically track food borne outbreaks and disease surveillance. In clinical hospital systems, WGS is used to identify multi-drug-resistant nosocomial infections and track the transmission of these organisms. In addition, we examine how metagenomics sequencing approaches (targeted and shotgun) are being used to circumvent the traditional and biased microbiology culture methods to identify potential pathogens directly from specimens. We also expand on the important factors to consider when implementing these technologies, and what is possible for these technologies in infectious disease diagnosis in the next 5 years.
Subject(s)
Anti-Infective Agents , Communicable Diseases , Communicable Diseases/diagnosis , Communicable Diseases/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Metagenomics , Whole Genome SequencingABSTRACT
OBJECTIVES: This study aimed to assess whether the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Epsilon variant (B.1.429/427) is more virulent, leading to more hospitalization and more severe disease requiring intensive care unit (ICU) admission. METHODS: SARS-CoV-2 genomic surveillance was performed on respiratory samples from 231 unique patients, collected at a single large health system in Southern California between November 2020 and March 2021 during the winter surge. RESULTS: The frequencies of the Epsilon variant among outpatients, hospitalized patients, and ICU patients were indifferent. CONCLUSIONS: Our study suggests that the Epsilon variant is not associated with increased hospitalization and ICU admission.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , California/epidemiology , Genomics , Hospitalization , Humans , Intensive Care Units , SARS-CoV-2/geneticsABSTRACT
Gardnerella is a frequent member of the urogenital microbiota. Given the association between Gardnerella vaginalis and bacterial vaginosis (BV), significant efforts have been focused on characterizing this species in the vaginal microbiota. However, Gardnerella also is a frequent member of the urinary microbiota. In an effort to characterize the bacterial species of the urinary microbiota, we present here 10 genomes of urinary Gardnerella isolates from women with and without lower urinary tract symptoms. These genomes complement those of 22 urinary Gardnerella strains previously isolated and sequenced by our team. We included these genomes in a comparative genome analysis of all publicly available Gardnerella genomes, which include 33 urinary isolates, 78 vaginal isolates, and 2 other isolates. While once this genus was thought to consist of a single species, recent comparative genome analyses have revealed 3 new species and an additional 9 groups within Gardnerella Based upon our analysis, we suggest a new group for the species. We also find that distinction between these Gardnerella species/groups is possible only when considering the core or whole-genome sequence, as neither the sialidase nor vaginolysin genes are sufficient for distinguishing between species/groups despite their clinical importance. In contrast to the vaginal microbiota, we found that only five Gardnerella species/groups have been detected within the lower urinary tract. Although we found no association between a particular Gardnerella species/group(s) and urinary symptoms, further sequencing of urinary Gardnerella isolates is needed for both comprehensive taxonomic characterization and etiological classification of Gardnerella in the urinary tract.IMPORTANCE Prior research into the bacterium Gardnerella vaginalis has largely focused on its association with bacterial vaginosis (BV). However, G. vaginalis is also frequently found within the urinary microbiota of women with and without lower urinary tract symptoms as well as individuals with chronic kidney disease, interstitial cystitis, and BV. This prompted our investigation into Gardnerella from the urinary microbiota and all publicly available Gardnerella genomes from the urogenital tract. Our work suggests that while some Gardnerella species can survive in both the urinary tract and vagina, others likely cannot. This study provides the foundation for future studies of Gardnerella within the urinary tract and its possible contribution to lower urinary tract symptoms.
Subject(s)
Gardnerella/classification , Gardnerella/genetics , Genome, Bacterial , Gram-Positive Bacterial Infections/urine , Microbiota/genetics , Vagina/microbiology , Vaginosis, Bacterial/urine , Female , Gardnerella/pathogenicity , Genotype , Gram-Positive Bacterial Infections/microbiology , Humans , Microbiota/physiology , Phylogeny , RNA, Ribosomal, 16S/genetics , Urinary Tract Infections/microbiology , Vaginosis, Bacterial/microbiology , Whole Genome SequencingABSTRACT
Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.
Subject(s)
RNA, Viral/genetics , SARS-CoV-2/pathogenicity , Saliva/virology , High-Throughput Nucleotide Sequencing , Humans , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission1,2. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals. We have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. After setting up SwabSeq in a high-complexity CLIA laboratory, we performed more than 80,000 tests for COVID-19 in less than two months, confirming in a real world setting that SwabSeq inexpensively delivers highly sensitive and specific results at scale, with a turn-around of less than 24 hours. Our clinical laboratory uses SwabSeq to test both nasal and saliva samples without RNA extraction, while maintaining analytical sensitivity comparable to or better than traditional RT-qPCR tests. Moving forward, SwabSeq can rapidly scale up testing to mitigate devastating spread of novel pathogens.
ABSTRACT
Aeromonas hydrophila resides in a variety of aquatic environments. Infections with A. hydrophila mainly occur after contact with fresh or brackish water. Nosocomial infections with A. hydrophila can also occur. A. hydrophila infections can be difficult to treat due to both intrinsic and acquired antimicrobial resistance (AMR) mechanisms. In 2018-19, we isolated multi-drug resistant (MDR) A. hyrodphila from two solid organ transplant patients with intra-abdominal infections. We aimed to characterize their AMR mechanisms and to determine their genetic relatedness to aid epidemiological investigation. We performed whole genome sequencing (WGS) using Illumina MiSeq and Nanopore MinIon on 3 A. hydrophila isolates, with one isolate from Patient A (blood) and two isolates from Patient B (abdominal and T-tube fluid, isolated 2 weeks apart). Phenotypic assays included: Broth Microdilution (BMD), Modified Hodge Test (MHT), Modified Carbapenem Inactivation Method (mCIM), and EDTA Carbapenem Inactivation Method (eCIM). Data analyses were performed using CLCbio and Geneious. AMR genomic analysis revealed that all three isolates possess chromosomally encoded genes including blaOXA-12(oxacillinase), blacepS (AmpC), and blacphA7(metallo-beta-lactamase). All isolates tested strongly positive by MHT and mCIM, but only Patient B's second isolate (after 2 weeks of meropenem treatment) tested positive by eCIM. More intriguingly, Patient B's first isolate (before meropenem treatment) tested falsely susceptible to carbapenems by BMD, suggesting blacphA7 gene was not expressed constitutively. Phylogenetic analysis showed the two isolates from Patient B were highly similar with only 1 SNP difference. The isolate from Patient A only differed from Patient B's isolates by 35 and 36 SNPs, respectively, suggesting close genetic relatedness. Further epidemiological investigation is undergoing. We report the first cases of CphA-mediated carbapenem resistant A. hydrophila in the U.S. It is concerning that 1 out of 3 isolates tested falsely susceptible to carbapenems by BMD despite clear carbapenemase production shown by strongly positive MHT and mCIM. In both cases, meropenem was initially used to treat the patients. Clinicians and microbiologists in the US should be aware of the emerging MDR Aeromonas nosocomial infections and the potential false carbapenem susceptible results due to CphA-type carbapenemase, which may be induced during treatment.
Subject(s)
Aeromonas hydrophila , Organ Transplantation , Aeromonas hydrophila/genetics , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Humans , Microbial Sensitivity Tests , Phylogeny , beta-Lactamases/geneticsABSTRACT
A novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. In this study, we performed a comprehensive epidemiological and genomic analysis of SARS-CoV-2 genomes from 10 patients in Shaoxing (Zhejiang Province), a mid-sized city outside of the epicenter Hubei province, China, during the early stage of the outbreak (late January to early February, 2020). We obtained viral genomes with >99% coverage and a mean depth of 296X demonstrating that viral genomic analysis is feasible via metagenomics sequencing directly on nasopharyngeal samples with SARS-CoV-2 Real-time PCR Ct values <28. We found that a cluster of four patients with travel history to Hubei shared the exact same virus with patients from Wuhan, Taiwan, Belgium, and Australia, highlighting how quickly this virus spread to the globe. The virus from another cluster of two family members living together without travel history but with a sick contact of a confirmed case from another city outside of Hubei accumulated significantly more mutations (9 SNPs vs. average 4 SNPs), suggesting a complex and dynamic nature of this outbreak. Our findings add to the growing knowledge of the epidemiological and genomic characteristics of SARS-CoV-2 and offers a glimpse into the early phase of this viral infection outside of Hubei, China.