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.

Integrated Genomic and Social Network Analyses of SARS-CoV-2 Transmission in the Healthcare Setting.

BACKGROUND: Infection prevention (IP) measures are designed to mitigate the transmission of pathogens in healthcare. Using large-scale viral genomic and social network analyses, we determined if IP measures used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic were adequate in protecting healthcare workers (HCWs) and patients from acquiring SARS-CoV-2. METHODS: We performed retrospective cross-sectional analyses of viral genomics from all available SARS-CoV-2 viral samples collected at UC San Diego Health and social network analysis using the electronic medical record to derive temporospatial overlap of infections among related viromes and supplemented with contact tracing data. The outcome measure was any instance of healthcare transmission, defined as cases with closely related viral genomes and epidemiological connection within the healthcare setting during the infection window. Between November 2020 through January 2022, 12 933 viral genomes were obtained from 35 666 patients and HCWs. RESULTS: Among 5112 SARS-CoV-2 viral samples sequenced from the second and third waves of SARS-CoV-2 (pre-Omicron), 291 pairs were derived from persons with a plausible healthcare overlap. Of these, 34 pairs (12%) were phylogenetically linked: 19 attributable to household and 14 to healthcare transmission. During the Omicron wave, 2106 contact pairs among 7821 sequences resulted in 120 (6%) related pairs among 32 clusters, of which 10 were consistent with healthcare transmission. Transmission was more likely to occur in shared spaces in the older hospital compared with the newer hospital (2.54 vs 0.63 transmission events per 1000 admissions, P < .001). CONCLUSIONS: IP strategies were effective at identifying and preventing healthcare SARS-CoV-2 transmission.

Genomic surveillance reveals dynamic shifts in the connectivity of COVID-19 epidemics.

The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of "local" when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

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.

Validating Tools to Detect and Inactivate Monkeypox Virus in Human Milk.

Objectives: Breastfeeding and human milk (HM) improve maternal and infant morbidities and mortality. Therefore, monitoring the safety of breastfeeding and access to HM is of critical importance. In this study, we assessed tools to monitor the presence of monkeypox virus (MPXV) in HM and whether standard Holder pasteurization inactivates MPXV. Materials and Methods: Heat-inactivated MPXV was added to HM or viral transport media (VTM) and analyzed using both research and clinical MPXV quantitative polymerase chain reaction (qPCR) tests. Infectious MPXV was added to HM and was exposed to 1 cycle of freeze-thaw, incubation for 1 hour at room temperature, or conditions of Holder pasteurization (62.5°C for 30 minutes) followed by infectious unit quantification by plaque assay. Results: Research and clinical nucleic acid tests detect MPXV that was added to HM but with reduced sensitivity compared with equivalent samples in VTM at low virus inoculum. MPXV added to HM to achieve a starting concentration of 225,000 plaque forming units (pfu)/mL remains infectious after freeze-thaw or 1 hour storage at room temperature. However, Holder pasteurization reduced infectious virus below the limit of detection, >2,000-fold reduction in viral titer. Conclusion: MPXV can be detected when added to HM using a clinical laboratory-developed qPCR test without modification, but the detection limit is reduced compared with equivalent samples in VTM. MPXV remains viable in HM should the virus ever gain access to HM, but Holder pasteurization reduces infectious MPXV to below detection limits and can be used to reduce the risk of MPXV transmission to infants who receive pasteurized (donor) HM.

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.

CORE: A Global Aggregation Service for Open Access Papers.

This paper introduces CORE, a widely used scholarly service, which provides access to the world's largest collection of open access research publications, acquired from a global network of repositories and journals. CORE was created with the goal of enabling text and data mining of scientific literature and thus supporting scientific discovery, but it is now used in a wide range of use cases within higher education, industry, not-for-profit organisations, as well as by the general public. Through the provided services, CORE powers innovative use cases, such as plagiarism detection, in market-leading third-party organisations. CORE has played a pivotal role in the global move towards universal open access by making scientific knowledge more easily and freely discoverable. In this paper, we describe CORE's continuously growing dataset and the motivation behind its creation, present the challenges associated with systematically gathering research papers from thousands of data providers worldwide at scale, and introduce the novel solutions that were developed to overcome these challenges. The paper then provides an in-depth discussion of the services and tools built on top of the aggregated data and finally examines several use cases that have leveraged the CORE dataset and services.

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.

Pangenome comparison of Bacteroides fragilis genomospecies unveil genetic diversity and ecological insights.

Bacteroides fragilis is a Gram-negative commensal bacterium commonly found in the human colon that differentiates into two genomospecies termed division I and II. We leverage a comprehensive collection of 694 B. fragilis whole genome sequences and report differential gene abundance to further support the recent proposal that divisions I and II represent separate species. In division I strains, we identify an increased abundance of genes related to complex carbohydrate degradation, colonization, and host niche occupancy, confirming the role of division I strains as gut commensals. In contrast, division II strains display an increased prevalence of plant cell wall degradation genes and exhibit a distinct geographic distribution, primarily originating from Asian countries, suggesting dietary influences. Notably, division II strains have an increased abundance of genes linked to virulence, survival in toxic conditions, and antimicrobial resistance, consistent with a higher incidence of these strains in bloodstream infections. This study provides new evidence supporting a recent proposal for classifying divisions I and II B. fragilis strains as distinct species, and our comparative genomic analysis reveals their niche-specific roles.

Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Bacteriophages (phages) are a promising anti-infective option for human disease. Major gaps remain in understanding their potential utility. METHODS: This is a randomized, placebo-controlled, double-blind study of a single dose of intravenous phage in approximately 72 clinically stable adult cystic fibrosis volunteers recruited from up to 20 US sites with Pseudomonas aeruginosa airway colonization. The single dose of phage consists of a mixture of four anti-pseudomonal phages. Six sentinel participants will be sequentially enrolled with dose escalation of the phage mixture by one log10 beginning with 4 × 107 plaque-forming units in an unblinded stage 1. If no serious adverse events related to the study product are identified, the trial will proceed to a double-blinded stage 2. In stage 2a, 32 participants will be randomly assigned to one of three phage dosages or placebo in a 1:1:1:1 allocation. An interim analysis will be performed to determine the phage dosage with the most favorable safety and microbiological activity profile to inform phage dosing in stage 2b. During stage 2b, up to 32 additional volunteers will be randomized 1:1 to the phage or placebo arm. Primary outcomes include (1) the number of grade 2 or higher treatment-emergent adverse events, (2) change in log10 P. aeruginosa total colony counts in sputum, and (3) the probability of a randomly selected subject having a more favorable outcome ranking if assigned to receive phage therapy versus placebo. Exploratory outcomes include (1) sputum and serum phage pharmacokinetics, (2) the impact of phage on lung function, (3) the proportion of P. aeruginosa isolates susceptible to the phage mixture before and after study product administration, and (4) changes in quality of life. DISCUSSION: This trial will investigate the activity of phages in reducing P. aeruginosa colony counts and provide insights into the safety profile of phage therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT05453578. Registered on 12 July 2022.

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.

Isolation of a Peptide That Binds to Pseudomonas aeruginosa Lytic Bacteriophage.

Antimicrobial resistance is a global health threat that is exacerbated by the overuse and misuse of antibiotics in medicine and agriculture. As an alternative to conventional antimicrobial drugs, phage therapy involves the treatment of infected patients with a bacteriophage that naturally destroys bacterial pathogens. With the re-emergence of phage therapy, novel tools are needed to study phages. In this work we set out to screen and isolate peptide candidates that bind to phages and act as affinity tags. Such peptides functionalized with an imaging agent could serves as versatile tools for tracking and imaging of phages. Specifically, we screened a phage display library for peptides that bind to the Good Vibes phage (GV), which lyses the bacterial pathogen Pseudomonas aeruginosa. Isolated monoclonal library phages featured a highly conserved consensus motif, LPPIXRX. The corresponding peptide WDLPPIGRLSGN was synthesized with a GGGSK linker and conjugated to cyanine 5 or biotin. The specific binding of the LPPIXRX motif to GV in vitro was confirmed using an enzyme-linked immunosorbent assay. We demonstrated imaging and tracking of GV in bacterial populations using the fluorescent targeting peptide and flow cytometry. In conclusion, we developed fluorescent labeled peptides that can bind to bacteriophage GV specifically, which may enable real-time analysis of phage in vivo and monitor the efficacy of phage therapy.

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.