Machine learning based DNA melt curve profiling enables automated novel genotype detection.

Surveillance for genetic variation of microbial pathogens, both within and among species, plays an important role in informing research, diagnostic, prevention, and treatment activities for disease control. However, large-scale systematic screening for novel genotypes remains challenging in part due to technological limitations. Towards addressing this challenge, we present an advancement in universal microbial high resolution melting (HRM) analysis that is capable of accomplishing both known genotype identification and novel genotype detection. Specifically, this novel surveillance functionality is achieved through time-series modeling of sequence-defined HRM curves, which is uniquely enabled by the large-scale melt curve datasets generated using our high-throughput digital HRM platform. Taking the detection of bacterial genotypes as a model application, we demonstrate that our algorithms accomplish an overall classification accuracy over 99.7% and perform novelty detection with a sensitivity of 0.96, specificity of 0.96 and Youden index of 0.92. Since HRM-based DNA profiling is an inexpensive and rapid technique, our results add support for the feasibility of its use in surveillance applications.

A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

Universal Digital High-Resolution Melt Analysis for the Diagnosis of Bacteremia.

Fast and accurate diagnosis of bloodstream infection is necessary to inform treatment decisions for septic patients, who face hourly increases in mortality risk. Blood culture remains the gold standard test but typically requires approximately 15 hours to detect the presence of a pathogen. We, therefore, assessed the potential for universal digital high-resolution melt (U-dHRM) analysis to accomplish faster broad-based bacterial detection, load quantification, and species-level identification directly from whole blood. Analytical validation studies demonstrated strong agreement between U-dHRM load measurement and quantitative blood culture, indicating that U-dHRM detection is highly specific to intact organisms. In a pilot clinical study of 17 whole blood samples from pediatric patients undergoing simultaneous blood culture testing, U-dHRM achieved 100% concordance when compared with blood culture and 88% concordance when compared with clinical adjudication. Moreover, U-dHRM identified the causative pathogen to the species level in all cases where the organism was represented in the melt curve database. These results were achieved with a 1-mL sample input and sample-to-answer time of 6 hours. Overall, this pilot study suggests that U-dHRM may be a promising method to address the challenges of quickly and accurately diagnosing a bloodstream infection.

Jumbo phages are active against extensively drug-resistant eyedrop-associated Pseudomonas aeruginosa infections.

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

A simple solid media assay for detection of synergy between bacteriophages and antibiotics.

The emergence of antibiotic resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. While phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates, and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat Vancomycin Resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.

Universal digital high resolution melt analysis for the diagnosis of bacteremia.

Fast and accurate diagnosis of bloodstream infection is necessary to inform treatment decisions for septic patients, who face hourly increases in mortality risk. Blood culture remains the gold standard test but typically requires ∼15 hours to detect the presence of a pathogen. Here, we assess the potential for universal digital high-resolution melt (U-dHRM) analysis to accomplish faster broad-based bacterial detection, load quantification, and species-level identification directly from whole blood. Analytical validation studies demonstrated strong agreement between U-dHRM load measurement and quantitative blood culture, indicating that U-dHRM detection is highly specific to intact organisms. In a pilot clinical study of 21 whole blood samples from pediatric patients undergoing simultaneous blood culture testing, U-dHRM achieved 100% concordance when compared with blood culture and 90.5% concordance when compared with clinical adjudication. Moreover, U-dHRM identified the causative pathogen to the species level in all cases where the organism was represented in the melt curve database. These results were achieved with a 1 mL sample input and sample-to-answer time of 6 hrs. Overall, this pilot study suggests that U-dHRM may be a promising method to address the challenges of quickly and accurately diagnosing a bloodstream infection. Universal digital high resolution melt analysis for the diagnosis of bacteremia: April Aralar, Tyler Goshia, Nanda Ramchandar, Shelley M. Lawrence, Aparajita Karmakar, Ankit Sharma, Mridu Sinha, David Pride, Peiting Kuo, Khrissa Lecrone, Megan Chiu, Karen Mestan, Eniko Sajti, Michelle Vanderpool, Sarah Lazar, Melanie Crabtree, Yordanos Tesfai, Stephanie I. Fraley.

Common antibiotics, azithromycin and amoxicillin, affect gut metagenomics within a household.

BACKGROUND: The microbiome of the human gut serves a role in a number of physiological processes, but can be altered through effects of age, diet, and disturbances such as antibiotics. Several studies have demonstrated that commonly used antibiotics can have sustained impacts on the diversity and the composition of the gut microbiome. The impact of the two most overused antibiotics, azithromycin, and amoxicillin, in the human microbiome has not been thoroughly described. In this study, we recruited a group of individuals and unrelated controls to decipher the effects of the commonly used antibiotics amoxicillin and azithromycin on their gut microbiomes. RESULTS: We characterized the gut microbiomes by metagenomic sequencing followed by characterization of the resulting microbial communities. We found that there were clear and sustained effects of the antibiotics on the gut microbial community with significant alterations in the representations of Bifidobacterium species in response to azithromycin (macrolide antibiotic). These results were supported by significant increases identified in putative antibiotic resistance genes associated with macrolide resistance. Importantly, we did not identify these trends in the unrelated control individuals. There were no significant changes observed in other members of the microbial community. CONCLUSIONS: As we continue to focus on the role that the gut microbiome plays and how disturbances induced by antibiotics might affect our overall health, elucidating members of the community most affected by their use is of critical importance to understanding the impacts of common antibiotics on those who take them. Clinical Trial Registration Number NCT05169255. This trial was retrospectively registered on 23-12-2021.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

The gastrointestinal microbiome plays a significant role in modulating numerous host processes, including metabolism. Prior studies show that when mice receive fecal transplants from obese donors on high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes, demonstrating the prominent role that gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, the impact of gut viruses on these phenotypes is understudied. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow over a 4-week study. By characterizing the gut bacterial biota via 16S rRNA amplicon sequencing and measuring mouse weights over time, we demonstrate that transplanted viruses affect the gut bacterial community, as well as weight gain/loss. Notably, mice fed chow but gavaged with HFD-derived viromes gained more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained less weight than their counterparts receiving HFD-derived viromes. Results were replicated in two independent experiments and phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Due to methodological limitations, we were unable to identify the specific bacterial strains responsible for respective phenotypic changes. This study confirms that virome-mediated perturbations can alter the fecal microbiome in vivo and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Characterization of SARS-CoV-2 Distribution and Microbial Succession in a Clinical Microbiology Testing Facility during the SARS-CoV-2 Pandemic.

The exchange of microbes between humans and the built environment is a dynamic process that has significant impact on health. Most studies exploring the microbiome of the built environment have been predicated on improving our understanding of pathogen emergence, persistence, and transmission. Previous studies have demonstrated that SARS-CoV-2 presence significantly correlates with the proportional abundance of specific bacteria on surfaces in the built environment. However, in these studies, SARS-CoV-2 originated from infected patients. Here, we perform a similar assessment for a clinical microbiology lab while staff were handling SARS-CoV-2 infected samples. The goal of this study was to understand the distribution and dynamics of microbial population on various surfaces within different sections of a clinical microbiology lab during a short period of 2020 Coronavirus disease (COVID-19) pandemic. We sampled floors, benches, and sinks in 3 sections (bacteriology, molecular microbiology, and COVID) of an active clinical microbiology lab over a 3-month period. Although floor samples harbored SARS-CoV-2, it was rarely identified on other surfaces, and bacterial diversity was significantly greater on floors than sinks and benches. The floors were primarily colonized by bacteria common to natural environments (e.g., soils), and benchtops harbored a greater proportion of human-associated microbes, including Staphylococcus and Streptococcus. Finally, we show that the microbial composition of these surfaces did not change over time and remained stable. Despite finding viruses on the floors, no lab-acquired infections were reported during the study period, which suggests that lab safety protocols and sanitation practices were sufficient to prevent pathogen exposures. IMPORTANCE For decades, diagnostic clinical laboratories have been an integral part of the health care systems that perform diagnostic tests on patient's specimens in bulk on a regular basis. Understanding their microbiota should assist in designing and implementing disinfection, and cleaning regime in more effective way. To our knowledge, there is a lack of information on the composition and dynamics of microbiota in the clinical laboratory environments, and, through this study, we have tried to fill that gap. This study has wider implications as understanding the makeup of microbes on various surfaces within clinical laboratories could help identify any pathogenic bacterial taxa that could have colonized these surfaces, and might act as a potential source of laboratory-acquired infections. Mapping the microbial community within these built environments may also be critical in assessing the reliability of laboratory safety and sanitation practices to lower any potential risk of exposures to health care workers.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

Background: The gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mice’s gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss. Results: We gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes. Conclusions: This study confirms that virome-mediated perturbations can alter the fecal microbiome in an in vivo model and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Multicenter Evaluation of the Cepheid Xpert GBS LB XC Test.

Early-onset neonatal sepsis due to Streptococcus agalactiae (group B Streptococcus [GBS]) infection is one of the leading causes of newborn mortality and morbidity. The latest guidelines published in 2019 recommended universal screening of GBS colonization among all pregnant women and intrapartum antibiotic prophylaxis for positive GBS. The updated procedures allow rapid molecular-based GBS screening using nutrient broth-enriched rectovaginal samples. Commercially available molecular assays for GBS diagnosis target mainly the cfb gene, which encodes a hemolysin protein responsible for producing the Christie-Atkins-Munch-Petersen (CAMP) factor. cfb is considered a conserved gene in essentially all GBS isolates. However, false-negative GBS results on Cepheid Xpert GBS and GBS LB tests due to deletions in or near the region that encodes cfb were reported recently. Therefore, the new Xpert GBS LB XC test was developed. This study is a multicenter evaluation of the new test for GBS identification from nutrient broth-enriched rectal/vaginal samples from antepartum women. A total of 621 samples were prospectively enrolled. The samples were tested with the Xpert GBS LB XC test, the composite comparator method, which included the Hologic Panther Fusion GBS test combined with bacterial culture, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and bacterial culture alone, followed by MALDI-TOF MS identification. The respective sensitivity and specificity of the Xpert GBS LB XC test were 99.3% and 98.7% compared to the composite comparator method and 99.1% and 91.8% compared to bacterial culture alone with MALDI-TOF MS identification. Overall, the Xpert GBS LB XC test performed comparatively to the composite comparator method and is equivalent to traditional bacterial culture followed by MALDI-TOF MS.

Comparison of the APAS Independence Automated Plate Reader System with the Manual Standard of Care for Processing Urine Culture Specimens.

Urine cultures are among the highest-volume tests in clinical microbiology laboratories and usually require considerable manual labor to perform. We evaluated the APAS Independence automated plate reader system and compared it to our manual standard of care (SOC) for processing urine cultures. The APAS device provides automated image interpretation of urine culture plate growth and sorts those images that require further evaluation. We examined 1,519 specimens over a 4-month period and compared the APAS growth interpretations to our SOC. We found that 72 of the 1,519 total specimens (4.74%) had growth discrepancies, where these specimens were interpreted differently by the APAS and the technologist, which required additional evaluation of plate images on the APAS system. Overall, there were 56 discrepancies in pathogen identification, which were present in 3.69% of the cultures. An additional pathogen was uncovered in a majority of these discrepancies; 12 (21.4%) identified an additional pathogen for the SOC, and 40 (71.4%) identified an additional pathogen for the APAS workflow. We found 214 (2.69%) antimicrobial susceptibility test (AST) discrepancies; 136 (1.71%) minor errors (mEs), 41 (0.52%) major errors (MEs), and 36 (0.45%) very major errors (VMEs). Many of the MEs and VMEs occurred in only a small subset of 13 organisms, suggesting that the specimen may have had different strains of the same pathogens with differing AST results. Given the significant labor required to perform urine cultures, the APAS Independence system has the potential to reduce manual labor while maintaining the identity and AST results of urinary pathogens. IMPORTANCE Urine cultures are among the highest-volume tests performed in clinical microbiology facilities and require considerable manual labor to perform. We compared the results of our manual SOC workflow with that of the APAS Independence system, which provides automated image interpretation and sorting of urine culture plates based on growth. We examined 1,519 urine cultures processed using both workflows and found that only 4.74% had growth pattern discrepancies and 3.69% pathogen identification discrepancies. There was substantial agreement in AST results between workflows, with only 2.69% having discrepancies. Only 1.71% of the ASTs had mEs, 0.52% had MEs, and 0.45% had VMEs, with most of the MEs and VMEs belonging to a small subset of organisms. The APAS system significantly decreased manual urine culture processing, while providing similar results to the SOC. As such, incorporating such automation into laboratory workflows has the potential to significantly improve efficiency.

Analysis of the FAST™ system for expedited identification and antimicrobial susceptibility testing of bloodborne pathogens.

The high morbidity and mortality of sepsis can be impacted by expediting identification (ID) and antibiotic susceptibility testing (AST) of causative bacteria. We evaluated the Qvella FAST™ System which creates a Liquid Colony™ (LC) from blood cultures that can be used to expedite results by 24 to 48 hours. We analyzed 289 LC samples and found that there were 17 (5.9%) that resulted in no ID. One hundred percent of the LC samples that produced an ID were concordant with SOC identification. Gram-positive bacteria showed a categorical agreement (CA) of 99.5%, with 3 minor errors (minE), and no major errors (majE) or very major errors (VME), and essential agreement (EA) of 98.9%. For Gram-negatives, the CA was 97.8% and the EA was 98.5% with 31 minE, 0 majE, and 2 VME. The FAST-System™ can accelerate ID and AST by 24 to 48 hours with potential positive impacts on time to effective therapy for sepsis.

Increased Innate Immune Susceptibility in Hyperpigmented Bacteriophage-Resistant Mutants of Pseudomonas aeruginosa.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.

Phage Cocktails Constrain the Growth of Enterococcus.

Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 Enterococcus phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the Myoviridae phages that we found in southern California wastewater were from the Brockvirinae subfamily (formerly Spounavirinae) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. Enterococcus is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear Enterococcus host cultures and delay the emergence of phage-resistant Enterococcus. We found that some phages were ineffective at clearing Enterococcus cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in Enterococcus exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for Enterococcus, which is an important target for phage therapy applications. IMPORTANCE Due to the rise in antibiotic resistance, Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both in vitro and early clinical studies. Here, we established a collection of 19 Enterococcus phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against Enterococcus isolates and reduce the emergence of resistance.

Antimicrobial-Resistant Shigella spp. in San Diego, California, USA, 2017-2020.

Annually, Shigella spp. cause ≈188 million cases of diarrheal disease globally, including 500,000 cases in the United States; rates of antimicrobial resistance are increasing. To determine antimicrobial resistance and risk factors in San Diego, California, USA, we retrospectively reviewed cases of diarrheal disease caused by Shigella flexneri and S. sonnei diagnosed during 2017-2020. Of 128 evaluable cases, S. flexneri was slightly more common than S. sonnei; most cases were in persons who were gay or bisexual cisgender men, were living with HIV, were unhoused, or used methamphetamines. Overall, rates of resistance to azithromycin, fluoroquinolones, ampicillin, and trimethoprim/sulfamethoxazole (TMP/SMX) were comparable to the most recent national data reported from the Centers for Disease Control and Prevention; 55% of isolates were resistant to azithromycin, 23% to fluoroquinolones, 70% to ampicillin, and 83% to TMP/SMX. The rates that we found for TMP/SMX were slightly higher than those in national data.

Molecular Mirror Technology Facilitates High-Throughput, Accurate SARS-CoV-2 Testing.

Tests to diagnose acute SARS-CoV-2 infection are at the center of controlling the COVID-19 pandemic. Rapid tests benefit from providing quick results but suffer from lower sensitivity, while PCR tests usually take longer to provide more reliable results and can be difficult to scale to meet population needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA) using nucleic acid extraction and a nucleic acid extraction-free method to determine its ability to identify SARS-CoV-2 in nasal specimens from individuals suspected of having SARS-CoV-2. We compared the MMA using nucleic acid extraction to the emergency use authorization (EUA)-approved TaqPath reverse transcriptase PCR (RT-PCR) assay to determine its performance characteristics. From 412 total specimens (including 115 previous positives and 297 previous negatives), we found that the positive percent agreement (PPA) was 99.1% (confidence interval [CI], 97.4% to 100.0%) and the negative percent agreement (NPA) was 99.3% (95% CI, 98.4% to 100.0%) for SARS-CoV-2 detection. Using the extraction-free method, we analyzed 109 specimens (51 previous positives and 58 previous negatives) and found that the PPA for the more rapid version of the assay was 87.8% (95% CI, 78.5% to 96.9%) and the NPA was 100.0% (95% CI, 100.0%) for virus detection. The extraction method has performance comparable to what is observed in many PCR-based assays. The extraction-free method has lower PPA but has the advantage of being more rapid and having a higher throughput. Our data offer a proof of concept that nuclear magnetic resonance (NMR) detection can be used in SARS-CoV-2 diagnostic testing and may allow for alternative supply chains to increase testing options. IMPORTANCE Accurate diagnostics for SARS-CoV-2 infections have been critical for responding to the COVID-19 pandemic. Both high-sensitivity/specificity PCR-based tests and lower-sensitivity/specificity rapid antigen assays have been the subject of worldwide supply chain limitations as individual facilities and countries have struggled to meet their population testing needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA), which uses nuclear magnetic resonance to detect the presence of SARS-CoV-2 nucleic acids both with and without full nucleic acid extractions. We found that compared to a U.S. emergency use authorization (EUA) approved assay (TaqPath) that uses reverse transcriptase PCR (RT-PCR), the MMA had high PPA and NPA with full nucleic acid extractions, and acceptable positive percent agreement (PPA) and negative percent agreement (NPA) with an extraction-free protocol. In a landscape marred by supply chain shortages across the world, altered SARS-CoV-2 detection methods such as the MMA can add to testing supplies while providing quality SARS-CoV-2 testing results.

Compositional Differences in the Oral Microbiome of E-cigarette Users.

Electronic (e)-cigarettes have been advocated as a safer alternative to conventional tobacco cigarettes. However, there is a paucity of data regarding the impact of e-cigarette aerosol deposition on the human oral microbiome, a key component in human health and disease. We aimed to fill this knowledge gap through a comparative analysis of the microbial community profiles from e-cigarette users and healthy controls [non-smokers/non-vapers (NSNV)]. Moreover, we sought to determine whether e-cigarette aerosol exposure from vaping induces persistent changes in the oral microbiome. To accomplish this, salivary and buccal mucosa samples were collected from e-cigarette users and NSNV controls, with additional oral samples collected from e-cigarette users after 2 weeks of decreased use. Total DNA was extracted from all samples and subjected to PCR amplification and sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Our analysis revealed several prominent differences associated with vaping, specific to the sample type (i.e., saliva and buccal). In the saliva, e-cigarette users had a significantly higher alpha diversity, observed operational taxonomic units (OTUs) and Faith's phylogenetic diversity (PD) compared to NSNV controls, which declined with decreased vaping. The buccal mucosa swab samples were marked by a significant shift in beta diversity between e-cigarette users and NSNV controls. There were also significant differences in the relative abundance of several bacterial taxa, with a significant increase in Veillonella and Haemophilus in e-cigarette users. In addition, nasal swabs demonstrated a trend toward higher colonization rates with Staphylococcus aureus in e-cigarette users relative to controls (19 vs. 7.1%; p = n.s.). Overall, these data reveal several notable differences in the oral bacterial community composition and diversity in e-cigarette users as compared to NSNV controls.

Animal Models of Phage Therapy.

Amidst the rising tide of antibiotic resistance, phage therapy holds promise as an alternative to antibiotics. Most well-designed studies on phage therapy exist in animal models. In order to progress to human clinical trials, it is important to understand what these models have accomplished and determine how to improve upon them. Here we provide a review of the animal models of phage therapy in Western literature and outline what can be learned from them in order to bring phage therapy closer to becoming a feasible alternative to antibiotics in clinical practice.

Comparison of two nucleic acid amplification tests (NAATs) and two antigen tests for detection of SARS-CoV-2 from upper respiratory specimens.

There are numerous tests available for acute diagnosis of SARS-CoV-2, the virus that causes the disease COVID-19. These tests fall into two main groups: nucleic acid amplification tests (NAATs) and antigen-based assays. We evaluated the clinical performance of two rapid antigen assays (BD Veritor System for Rapid Detection of SARS CoV-2 and Abbott BinaxNOW COVID-19 Ag Card) and one NAAT (Hologic Aptima SARS CoV-2 Assay) by comparing them with the initial test of record, the Roche cobas SARS-CoV-2 assay; the antigen tests were also compared to Aptima. We tested remnant frozen specimens from patients suspected of SARS-CoV-2 infections (either due to symptoms or exposure) on the comparator platforms to evaluate assay performance across a wide range of positive results, including cobas cycle threshold (Ct) values ranging between 12 and 35. We tested 250 previous positive and 50 previous negative specimens and found 95.6% positive percent agreement (PPA) with the Aptima assay. The few discrepancies between the NAATs occurred only when Ct values were >32. Agreement was much lower for the rapid antigen tests, with 45.2%/47.3% PPA for the Veritor and 47.0%/47.0% PPA for the Binax compared to cobas/Aptima. Discrepancies occurred when cobas Ct values were >20 for Veritor and >25 for Binax. The negative percent agreement (NPA) was 100% for all assay comparisons. These data indicate similar performance between the cobas and Aptima NAATs but demonstrate that antigen-based assays may be insufficient to diagnose SARS-CoV-2 infection when lower levels of the virus are shed.