Microbiology spectrum最新文献

Clinical studies of vaccines generally require collections of microbial isolates obtained from various body sites over multiple years. Further, large microbe collections are needed for research due to increasing appreciation for the phenotypic and genotypic diversity among a single microbial species. However, large collections are not generally available due to method limitations. We show a new way to create, recover, and manage microbe collections in 96- or 384-well plates using 50% glycerol at -20°C. Fifty percent glycerol remains liquid at -20°C and permits only the chosen isolates to be reliably sampled without first thawing all other isolates in the plate. Consequently, the glycerol sampling allows integration of microbe collection, labeling, recovery, and storage steps. Creating a microbe collection as an array in microplates reduces physical storage space by 6- or 23-fold with 96-well or 384-well plates, respectively. The array permits direct analysis of the collection with high-throughput assay systems. Further, we show that Streptococcus pneumoniae could be stored for 11 years as a microplate array. Standardized microbe arrays created in microplates with the new method could be easily distributed for studies of microbial structures, genetic diversity, antibiotics, and microbe-host interactions. This integrated method suggests how automated microbe management systems can be created.IMPORTANCEEpidemiologic and microbiology studies require large microbial collections, and the use of microplates could facilitate the creation and management of these collections. However, recovering individual isolates from microplates is manual and tedious. In this study, we demonstrate a simple method for recovering a selected individual isolate from a microplate at -20°C using 50% glycerol. Additionally, we found that Streptococcus pneumoniae could be revived for more than 10 years in microplates. This new method of recovering microbes from frozen microplates could greatly streamline many large-scale epidemiologic studies, particularly those related to pneumococcal vaccine studies. This new method may ultimately automate the collection, management, and storage of microbial isolates.

Nucleic acid amplification tests (NAATs) are the method of choice for Chlamydia trachomatis diagnosis, but these strategies are susceptible to target site mutations. C. trachomatis variants escaping detection with the Aptima Combo 2 (AC2) assay on the Hologic Panther instrument from 23S rRNA mutations have been reported in Nordic countries, England, Japan, and the United States. Given the potential for false negative results, this study investigated whether strains of C. trachomatis with AC2 target site mutations were present in Canada. Surveillance was conducted in Canadian laboratories from 2019 to 2021. Specimens suspected of AC2 target site mutations included those with low-value detections on the AC2 assay, with subsequent high-value detections on the Aptima Chlamydia Trachomatis (ACT) assay used for confirmatory testing. Specimens with AC2/ACT discrepant results were subjected to sequencing of the AC2 target (i.e., 23S rRNA). Sequencing revealed 15 (4.8%) diagnostic escape variants which were carrying either C1514T, G1523A, or G1526A mutations. All specimens with a diagnostic escape mutation were detected with a reformulated version of the AC2 assay. Overall, while the prevalence of C. trachomatis variants was rare, their presence in the Canadian population supports the use of the new AC2 kit formulation and the need for ongoing genetic surveillance for NAAT-based assays.

Importance: Molecular tests are commonly used for the detection of sexually transmitted infections (STIs) like Chlamydia trachomatis. Mutations impacting C. trachomatis molecular target detection on the Hologic Panther AC2 assay have been reported in several countries, raising concerns about potential false negative results. This study showed C. trachomatis target detection failures in specimens submitted for C. trachomatis testing in Canadian laboratories from 2019 to 2021. A reformulated version of the AC2 molecular test is now available that can identify C. trachomatis strains harboring target site mutations that were impacted by the previous test formulation. While target site mutations were rare in Canada, revealing their presence is important to ensure accurate molecular detection of C. trachomatis with existing testing methods. This study supports ongoing genetic monitoring of C. trachomatis molecular test target sites, as well as the use of the reformulated test to avoid false negative results and subsequent transmissions.

Incorporating molecular testing for human papillomavirus (HPV) into the screening of cervical specimens can improve risk stratification and, in turn, patient management. Infection with a high-risk (HR) HPV genotype is associated with greater risk for persistent infection, viral integration, and progression of cervical neoplasia. Current guidelines consider HPV 16 or HPV 18 clinically actionable with referral to colposcopy; however, 12 Other HR HPV genotypes have been associated with cervical cancer risk, suggesting a benefit of extended genotyping. In this multi-center study, we evaluated the performance of the Alinity m HR HPV assay, which reports HPV 16, 18, and 45 individually and aggregates of HPV 31/33/52/58 and HPV 35/39/51/56/59/66, compared with cobas HPV and Aptima HPV assays, across a variety of cytology result categories. A total of 746 de-identified residual cervical specimens, collected as part of routine cervical cancer screening programs, were tested using Alinity m HR HPV and at least one comparator assay. The overall percent agreement was ≥90.7% for results from the Alinity m HR HPV assay and cobas HPV assays and 90.5% for results from the Alinity m HR HPV and Aptima HPV assay. In patients with any abnormal cytology result, Alinity m identified 78 specimens with non-HPV 16/18 results, underscoring the benefit of detecting additional HR HPV genotypes to guide patient management more accurately. Among specimens with normal cytology, Alinity m detected 14 additional specimens with non-HPV 16/18 genotypes. Extended HR HPV testing can provide additional information to triage patients for appropriate testing and follow-up.IMPORTANCEExtended genotyping for high-risk human papillomavirus (HPV) types enhances diagnostic precision by identifying additional oncogenic HPV types beyond 16 and 18 therefore offering a more nuanced risk profile. This more comprehensive detection may aid in identifying persistent infections that are more likely to progress, thereby supporting future risk-based patient management strategies.

Escherichia coli synthesizes aromatic amino acids (AAAs) through the common pathway to produce the precursor, chorismate, and the three terminal pathways to convert chorismate into Phe, Tyr, and Trp. E. coli also imports exogenous AAAs through five transporters. GcvB small RNA post-transcriptionally regulates more than 50 genes involved in amino acid uptake and biosynthesis in E. coli, but the full extent of GcvB regulon is still underestimated. This study examined all genes involved in AAA biosynthesis and transport using translation reporter assay and qRT-PCR analysis. In addition to previously verified targets, aroC, aroP, and trpE, we identified new target genes that were significantly repressed by GcvB primarily via the R1 seed region. Exceptionally, GcvB strongly inhibits the expression of aroG, which encodes the major isozyme of the first reaction in the common pathway, through direct base pairing between the aroG translation initiation region and the GcvB R3 seed sequence. RNase E mediates the degradation of target mRNAs except aroC and aroP via its C-terminal domain. GcvB overexpression prolongs the lag phase and reduces the growth rate in minimal media supplemented with AAAs and confers resistance to an antibiotic compound, azaserine, by repressing AAA transporters.IMPORTANCEE. coli strains have been genetically modified in relevant transcription factors and biosynthetic enzymes for industrial use in the fermentative production of aromatic amino acids (AAAs) and their derivative compounds. This study focuses on GcvB small RNA, a global regulator of amino acid metabolism in E. coli, and identifies new GcvB targets involved in AAA biosynthesis and uptake. GcvB represses the expression of the first and last enzymes of the common pathway and the first enzymes of Trp and Phe terminal pathways. GcvB also limits import of AAAs. This paper documents the impact of RNA-mediated regulation on AAA metabolism in E. coli.

Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that can cause epidemics and pandemics. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined three host factors for their contributions to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated the impact on IAV and other RNA viruses. We confirmed that knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface but has no effect on vesicular stomatitis virus infection. Although ADAR1 overexpression does not significantly inhibit IAV replication, it has a pro-viral effect with coxsackie B virus (CVB) infection. This pro-viral effect is not likely secondary to reduced type I interferon (IFN) production, as the induction of the IFN-stimulated genes ISG15 and CXCL10 is negligible in both parent and ADAR1-overexpressing A549 cells following CVB challenge. In contrast, ISG15 and CXCL10 production is robust and equal for parent and ADAR1-overexpressing A549 cells challenged with IAV. Taken together, these data provide insights into how host factors can be further explored to understand the dynamics of pro- and anti-viral factors.IMPORTANCEInfluenza A virus (IAV) remains a global threat due to its ability to cause pandemics, making the identification of host factors essential for developing new antiviral strategies. In this study, we utilized CRISPR-based techniques to investigate host factors that impact IAV infectivity. Knockout of CMAS, a key enzyme in sialic acid biosynthesis, significantly reduced IAV binding and infection by disrupting sialic acid production on the cell surface. Overexpression of B4GALNT2 had similar effects, conferring resistance to IAV infection through diminished cell-surface binding. Overexpression of ADAR1, known for its role in RNA editing and immune regulation, impacted IAV replication minimally but enhanced coxsackie B virus replication. Such findings reveal the diverse roles of host factors in viral infection, offering insights for targeted therapeutic development against IAV and other pathogens.

The aim of this study was to investigate the prevalence of Tropheryma whipplei, Chlamydia psittaci, Chlamydia pneumoniae, Legionella, Brucella, and Francisella tularensis in valve samples from endocarditis patients using the real-time PCR method at a major referral heart hospital in Iran. In this study, 146 paraffin-embedded tissue samples from the heart valves of patients with clinical and pathological evidence of infective endocarditis (IE), who underwent heart valve replacement surgery between 2016 and 2020 at Tehran Heart Center were collected. After DNA extraction from paraffin-embedded valve tissue samples, they were surveyed for the presence of T. whipplei, C. psittaci, C. pneumoniae, Legionella, Brucella, and F. tularensis using quantitative real-time PCR (qPCR). The 16S rRNA gene sequence analysis was used for accurate species identification. Based on the molecular results, T. whipplei, Chlamydia spp., and Legionella spp. were detected in 10 (6.84%), 6 (4.1%), and 3 (2.05%) valve samples, respectively. In addition, one of six positive samples for Chlamydia spp. was identified as C. psittaci. No positive samples for F. tularensis and Brucella were found. In addition, all control valve samples were negative for all investigated pathogens. The findings suggest that specific bacterial species such as T. whipplei, Chlamydia spp., and Legionella spp. are associated with the development of IE. Considering the life-threatening nature of IE, it is critical for healthcare systems to prioritize the identification of its causative agents and develop targeted treatment strategies.IMPORTANCEInfective endocarditis (IE) is a serious and potentially life-threatening condition, and it is associated with significant morbidity, mortality, and complications, making it a major concern in both global and national healthcare systems. Late diagnosis and failure to receive appropriate treatment for patients with culture-negative endocarditis caused by hard-to-grow bacteria can lead to the death of patients. Unfortunately, in Iran, less attention is paid to the role of organisms that are difficult to cultivate in laboratory settings such as Tropheryma whipplei, Chlamydia psittaci, Chlamydia pneumoniae, Legionella, Brucella, and Francisella tularensis in causing culture-negative endocarditis, and these pathogens are overlooked by infectious, cardiologists, and health officials. This study underscores the need for special attention in the diagnosis of the agents of IE.

Sequence type (ST)11 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) can cause life-threatening infections and is therefore of global concern. Despite its importance, the evolutionary history and mechanism for the emergence of ST11 CR-hvKP are unclear. In recent years, the detection rate of ST11 CR-hvKP has increased in a teaching hospital. Based on its clonal transmission, a conjugation experiment was performed between a hvKP strain AP8555 and a ST11 CRKP strain, resulting in two ST11 CR-hvKP strains. Research had confirmed that the virulence plasmid pAP855 was horizontally transferred to the CRKP strain to form the conjugant S270-Tc, which was recombined by mobile genetic elements to evolve into the conjugant S270-Tc-R. The S270-Tc-R had high virulence, high plasmid stability, and greater adaptability. Interestingly, it had high homology to clinically prevalent ST11 CR-hvKP strains using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). This is the first demonstration that plasmid recombination in vitro has led to the formation of ST11 CR-hvKP strains. The clinical setting is a multi-factorial and multi-selection pressure environment that may stimulate the evolution of conjugant strains in the transition period to local strains in the stable period, and surveillance is urgently needed for infection control.

Importance: The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) heralded the onset of a new and rapidly worsening public health disaster on a global scale. More attention has been paid to its evolutionary history and mechanism, which currently remains unclear. In this study, a conjugation experiment was performed between a hvKP strain AP8555 and a ST11 CRKP strain, resulting in two ST11 CR-hvKP strains. We had confirmed that the virulence plasmid pAP855 was horizontally transferred to the CRKP strain to form the conjugant S270-Tc, which was recombined by mobile genetic elements to evolve into the conjugant S270-Tc-R. The S270-Tc-R had high virulence, high plasmid stability, and greater adaptability. Interestingly, it had high homology to clinically prevalent ST11 CR-hvKP strains using pulsed-field gel electrophoresis and whole-genome sequencing.

Immune checkpoint inhibitor (ICI) therapy has yielded revolutionary outcomes among some individuals with skin cancer, but a large percentage of individuals do not benefit from these treatments. The gut microbiota is hypothesized to impact ICI therapy outcomes. However, data on ICI therapy, gut microbiota, and non-melanoma skin cancers are limited. To examine the association of gut microbiota structure and function with non-melanoma skin cancer ICI outcomes, we performed 16S rRNA V1-V2 gene amplicon sequencing of 68 fecal samples collected longitudinally from individuals with basal cell carcinoma (n = 5), Merkel cell carcinoma (n = 5), or cutaneous squamous cell carcinoma (CSCC, n = 11), followed by tumor-dependent differential analyses of bacterial composition and fecal sample analysis by untargeted metabolomics. Across all tumor types, we identified 10 differential bacterial genera between responders (R) or non-responders (NR) to ICI therapy. Among individuals with CSCC, we identified 10 genera and 20 species that differentiated between R and NR and yielded 8 pathways enriched in NR and 12 pathways enriched in R by predicted functional pathway analyses. Untargeted fecal metabolomics to examine putative gut microbiota metabolites associated with CSCC ICI R/NR identified nine KEGG pathways associated with ICI efficacy. In summary, this exploratory study suggests gut microbiota features that are associated with ICI efficacy in individuals with non-melanoma skin cancers and highlights the need for larger studies to validate the results.IMPORTANCEPrior studies examining associations between ICI efficacy and the gut microbiome have focused primarily on individuals with melanoma, for whom ICI therapy was first approved. Meanwhile, data regarding microbiome features associated with ICI responses in individuals with non-melanoma skin cancers (NMSCs) have remained limited. This initial fecal microbiota examination of individuals with NMSCs suggests that larger-scale studies to extend and validate our findings may yield predictive or prognostic biomarkers for individuals with NMSC receiving ICI with potential to provide insight to complementary, effective therapeutic interventions through microbiota modification.

Although much has been learned about the entry mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many details of the entry mechanisms of seasonal human coronaviruses (HCoVs) remain less well understood. In the present study, we used 293T cell lines stably expressing angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2), which support high-level transduction of lentiviral pseudoviruses bearing spike proteins of seasonal HCoVs, HCoV-NL63, -229E, or -HKU1, respectively, to compare spike processing and virus entry pathways among these viruses. Our results showed that the entry of HCoV-NL63, -229E, and -HKU1 pseudoviruses into cells is sensitive to endosomal acidification inhibitors (chloroquine and NH₄Cl), indicating entry via the endocytosis route. Although TMPRSS2 expression on target cell surface was required for HCoV-HKU1 spike-mediated entry and cell-cell fusion, we found that only the serine protease domain of TMPRSS2 and not the serine protease activity of TMPRSS2 was required for viral entry via endocytic route. However, the serine protease activity of TMPRSS2 and a furin processing site (RKRR) at the S1/S2 junction were essential for efficient HCoV-HKU1 spike-mediated cell-cell fusion. Additionally, we show that dibasic and monobasic arginine residues at the S1/S2 junctions of spike proteins of HCoV-NL63 and -229E are essential for virus entry, but multi-basic furin processing site at the S1/S2 junction was dispensable for HCoV-HKU1 viral entry. Our findings highlight features of the entry mechanisms of seasonal HCoVs that may support the development of novel treatment strategies.IMPORTANCEDetails of the entry mechanisms of seasonal human coronaviruses (HCoVs) remain to be fully explored. To investigate spike-mediated virus entry of HCoV-NL63, -229E, and -HKU1 CoVs, we employed 293T cells that stably express angiotensin converting enzyme (ACE2), aminopeptidase N (APN), or transmembrane serine protease 2 (TMPRSS2) to study entry mechanisms of pseudoviruses bearing spike proteins of HCoV-NL63, -229E, and -HKU1, respectively. We found that HCoV-NL63, -229E, and -HKU1 pseudoviruses entered cells via the endocytic route independently of cellular serine protease activity and therefore likely depended on endosomal cathepsin activity. Furthermore, we showed that arginine amino acids in S1/S2 junctions of HCoV-NL63 and -229E spikes were essential for entry but not essential for HCoV-HKU1 entry. Our results provide new insights into the S1/S2 junctional residues, cellular receptors, and protease requirements for seasonal HCoV pseudovirus entry into cells that may support the development of novel inhibitors.