Use of viability PCR for detection of live Chlamydia trachomatis in clinical specimens

BackgroundThe current testing approach to diagnose Chlamydia trachomatis (CT) infection relies on nucleic acid amplification tests (NAATs). These tests are highly sensitive, but do not distinguish between active infection and residual bacterial nucleic acid which may remain after resolution of infection, or via cross-contamination. Better methods to assess the viability of CT detected in clinical samples would be useful in determining the relevance of CT detection in a variety of clinical settings. The goal of this study was to test viability PCR (vPCR) as a method to distinguish viable bacteria from non-viable CT.MethodsThe vPCR relies on a propidium monoazide dye (PMAxx), which intercalates into accessible DNA from dead organisms and prevents their detection in a PCR assay for the CT ompA gene. We used digital PCR to quantify absolute genome copy numbers from samples. We validated the vPCR approach using laboratory stocks of CT with known viability. Then, we tested total DNA, viable CT DNA, and culture results from 18 clinical vaginal specimens and 25 rectal clinical specimens, all of which had tested positive by NAAT.ResultsIn laboratory stocks of CT, vPCR using defined ratios of heat-killed to live bacteria tracked closely with expected results. In vaginal clinical specimens, vPCR and total DNA results were correlated, though total DNA genomes outnumbered viable genomes by 2.2–52.6-fold more copies. As expected, vPCR detected more total genomes than culture results. Both vPCR and total DNA correlated with culture results (Spearman correlation R = 0.8425 for total DNA and 0.8056 for vPCR). Ten rectal NAAT positive specimens were negative by total DNA PCR, vPCR, and were negative or inconclusive by culture. Of the 6 rectal specimens that were culture positive, all were total DNA and vPCR positive. vPCR additionally detected viable bacterial DNA in 8 specimens which were NAAT + and culture negative, though levels were very low (mean 1,357 copies/ml)ConclusionsvPCR is a fast and easy method to assess viability in clinical specimens and is more correlated with culture results than total DNA PCR. Inconsistent ratios between total DNA and vPCR results suggest that the amount of dead bacteria varies considerably in clinical specimens. Results from rectal specimens suggest that many NAAT positive specimens do not in fact represent live replicating bacteria, and likely result in significant overuse of unnecessary antibiotics

Utility of EC 3MTM PetrifilmTM and sanitary surveys for source water assessment in Nyabushozi County, south-western Uganda

The majority of people in developing nations rely on untreated or minimally treated surface and shallow groundwater sources which are prone to faecal contamination. This study evaluated the utility of EC 3M™ Petrifilm™ and sanitary inspection forms (SIFs) as tools to assess 47 water sources and identify hazards of contamination in two rural Ugandan villages (90% were surface sources). Water samples were cultured on EC 3MTM PetrifilmTM, which are intended for the enumeration of E. coli and total coliforms following 24 h incubation at 37ºC. Isolated bacteria were cultured on MacConkey agar and identified using standard biochemical tests, while selected isolates were verified by sequencing 16S rRNA genes. From 105 Petrifilms, 110 presumptive E. coli were isolated and identified to genus level. However, only 33 presumptive E. coli isolates from 14 water sources (representing 27 distinct strains as determined by PFGE) were confirmed E. coli. The other presumptive E. coli isolates were identified as Citrobacter, Enterobacter, Proteus, Salmonella and Yersinia species. SIFs used an adapted survey designed for urban water sources of Uganda. The form yielded an SIF score based on binary data and characterized potential sources of contamination. SIF scores alone offered little information to distinguish between contamination levels of surface water sources, but the information collected in the surveys could be used to identify ways to improve sources. The results of this study suggest that the use of sanitary surveys may assist in identifying potential pollution sources that may be targeted to protect water sources. Bacterial monitoring using EC 3MTM PetrifilmsTM may be effective for the screening of relative levels of contamination of source waters, including surface sources.Keywords: drinking water, developing countries, sanitary survey, EC 3MTM PetrifilmT

Surfactant-enhanced DNA accessibility to nuclease accelerates phenotypic β-lactam antibiotic susceptibility testing of Neisseria gonorrhoeae

Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for β-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen’s slow doubling time and the lack of methods to quickly quantify the pathogen’s response to β-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the β-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to β-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to β-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15–30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats

Mycoplasma genitalium in the US (MyGeniUS): Surveillance data from sexual health clinics in 4 US regions

BACKGROUND: Mycoplasma genitalium (MG) is on the CDC Watch List of Antimicrobial Resistance Threats, yet there is no systematic surveillance to monitor change. METHODS: We initiated surveillance in sexual health clinics in 6 cities, selecting a quota sample of urogenital specimens tested for gonorrhea and/or chlamydia. We abstracted patient data from medical records and detected MG and macrolide-resistance mutations (MRMs) by nucleic acid amplification testing. We used Poisson regression to estimate adjusted prevalence ratios (aPRs) and 95% CIs, adjusting for sampling criteria (site, birth sex, symptom status). RESULTS: From October-December 2020 we tested 1743 urogenital specimens: 57.0% from males, 46.1% from non-Hispanic Black persons, and 43.8% from symptomatic patients. MG prevalence was 16.6% (95% CI: 14.9-18.5%; site-specific range: 9.9-23.5%) and higher in St Louis (aPR: 1.9; 1.27-2.85), Greensboro (aPR: 1.8; 1.18-2.79), and Denver (aPR: 1.7; 1.12-2.44) than Seattle. Prevalence was highest in persons \u3c18 years (30.4%) and declined 3% per each additional year of age (aPR: .97; .955-.982). MG was detected in 26.8%, 21.1%, 11.8%, and 15.4% of urethritis, vaginitis, cervicitis, and pelvic inflammatory disease (PID), respectively. It was present in 9% of asymptomatic males and 15.4% of asymptomatic females, and associated with male urethritis (aPR: 1.7; 1.22-2.50) and chlamydia (aPR: 1.7; 1.13-2.53). MRM prevalence was 59.1% (95% CI: 53.1-64.8%; site-specific range: 51.3-70.6%). MRMs were associated with vaginitis (aPR: 1.8; 1.14-2.85), cervicitis (aPR: 3.5; 1.69-7.30), and PID cervicitis (aPR: 1.8; 1.09-3.08). CONCLUSIONS: MG infection is common in persons at high risk of sexually transmitted infections; testing symptomatic patients would facilitate appropriate therapy. Macrolide resistance is high and azithromycin should not be used without resistance testing

Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform—isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry—to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.This is the publisher’s final pdf. The published article is copyrighted by The American Society for Biochemistry and Molecular Biology and can be found at: http://www.mcponline.org/content/15/7/233

WHO global research priorities for antimicrobial resistance in human health

The WHO research agenda for antimicrobial resistance (AMR) in human health has identified 40 research priorities to be addressed by the year 2030. These priorities focus on bacterial and fungal pathogens of crucial importance in addressing AMR, including drug-resistant pathogens causing tuberculosis. These research priorities encompass the entire people-centred journey, covering prevention, diagnosis, and treatment of antimicrobial-resistant infections, in addition to addressing the overarching knowledge gaps in AMR epidemiology, burden and drivers, policies and regulations, and awareness and education. The research priorities were identified through a multistage process, starting with a comprehensive scoping review of knowledge gaps, with expert inputs gathered through a survey and open call. The priority setting involved a rigorous modified Child Health and Nutrition Research Initiative approach, ensuring global representation and applicability of the findings. The ultimate goal of this research agenda is to encourage research and investment in the generation of evidence to better understand AMR dynamics and facilitate policy translation for reducing the burden and consequences of AMR

Comparison of multi-drug resistant environmental methicillin-resistant Staphylococcus aureus [MRSA] isolated from recreational beaches and high touch surfaces in built environments

Over the last decade community-acquired methicillin-resistant Staphylococcus aureus [MRSA] has emerged as a major cause of disease in the general population with no health care exposure or known classical risk factors for MRSA infections. The potential community reservoirs have not been well defined though certain strains such as ST398 and USA300 have been well studied in some settings. MRSA has been isolated from recreational beaches, high-touch surfaces in homes, universities and other community environmental surfaces. However, in most cases the strains were not characterized to determine if they are related to community-acquired or hospital-acquired clinical strains. We compared 55 environmental MRSA from 805 samples including sand, fresh and marine water samples from local marine and fresh water recreational beaches (n=296), high touch surfaces on the University of Washington campus (n=294), surfaces in UW undergraduate housing (n=85), and the local community (n=130). Eleven USA300, representing 20% of the isolates, were found on the UW campus surfaces, student housing surfaces and on the community surfaces but not in the recreational beach samples from the Northwest USA. Similarly, the predominant animal ST133 was found in the recreational beach samples but not in the high touch surface samples. All USA300 isolates were multi-drug resistant carrying 2-6 different antibiotic resistance genes coding for kanamycin, macrolides and/or macrolides-lincosamides-streptogramin B and tetracycline, with the majority [72%] carrying 4-6 different antibiotic resistance genes. A surprising 98% of the 55 MRSA isolates were resistant to other classes of antibiotics and most likely represent reservoirs for these genes in the environment