Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Capture of armA by a novel ISCR element, ISCR28.

ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5'-GXXT-3' sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase.

Enhancing pollutants removal in hospital wastewater: Comparative analysis of PAC coagulation vs. bio-contact oxidation, highlighting the impact of outdated treatment plants.

While the effectiveness of Poly-Aluminum Chloride (PAC) coagulation for pollutant removal has been documented across various wastewater scenarios, its specific application in hospital wastewater (HWW) treatment to remove conventional pollutants and hazardous genetic pollutants has not been studied. The research compared three hospital wastewater treatment plants (HWTPs) to address a knowledge gap, including the PAC coagulation-sodium hypochlorite disinfection process (PAC-HWTP), the biological contact oxidation-precipitation-sodium hypochlorite process (BCO-HWTP), and a system using outdated equipment with PAC coagulation (ODE-PAC-HWTP). Effluent compliance with national discharge standards is assessed, with BCO-HWTP meeting standards for direct or indirect discharge into natural aquatic environments. ODE-PAC-HWTP exceeds pretreatment standards for COD and BOD5 concentrations. PAC-HWTP effluent largely adheres to national pretreatment standards, enabling release into municipal sewers for further treatment. Metagenomic analysis reveals that PAC-HWTP exhibits higher removal efficiencies for antibiotic resistance genes, metal resistance genes, mobile genetic elements, and pathogens compared to BCO-HWTP and ODE-PAC-HWTP, achieving average removal rates of 45.13%, 57.54%, 80.61%, and 72.17%, respectively. These results suggests that when discharging treated HWW into municipal sewers for further processing, the use of PAC coagulation process is more feasible and cost-effective compared to BCO technologies. The analysis emphasizes the urgent need to upgrade outdated equipment HWTPs.

Profiles of phage in global hospital wastewater: Association with microbial hosts, antibiotic resistance genes, metal resistance genes, and mobile genetic elements.

Hospital wastewater (HWW) is known to host taxonomically diverse microbial communities, yet limited information is available on the phages infecting these microorganisms. To fill this knowledge gap, we conducted an in-depth analysis using 377 publicly available HWW metagenomic datasets from 16 countries across 4 continents in the NCBI SRA database to elucidate phage-host dynamics and phage contributions to resistance gene transmission. We first assembled a metagenomic HWW phage catalog comprising 13,812 phage operational taxonomic units (pOTUs). The majority of these pOTUs belonged to the Caudoviricetes order, representing 75.29 % of this catalog. Based on the lifestyle of phages, we found that potentially virulent phages predominated in HWW. Specifically, 583 pOTUs have been predicted to have the capability to lyse 81 potentially pathogenic bacteria, suggesting the promising role of HWW phages as a viable alternative to antibiotics. Among all pOTUs, 1.56 % of pOTUs carry 108 subtypes of antibiotic resistance genes (ARGs), 0.96 % of pOTUs carry 76 subtypes of metal resistance genes (MRGs), and 0.96 % of pOTUs carry 22 subtypes of non-phage mobile genetic elements (MGEs). Predictions indicate that certain phages carrying ARGs, MRGs, and non-phage MGEs could infect bacteria hosts, even potential pathogens. This suggests that phages in HWW may contribute to the dissemination of resistance-associated genes in the environment. This meta-analysis provides the first global catalog of HWW phages, revealing their correlations with microbial hosts and pahge-associated ARGs, MRG, and non-phage MGEs. The insights gained from this research hold promise for advancing the applications of phages in medical and industrial contexts.

Meta-analysis addressing the characterization of antibiotic resistome in global hospital wastewater.

Hospital wastewater (HWW) is a significant environmental reservoir of antibiotic resistance genes (ARGs). However, currently, no comprehensive understanding exists of the antibiotic resistome in global HWW. In this study, we attempted to address this knowledge gap through an in silico reanalysis of publicly accessible global HWW metagenomic data. We reanalyzed ARGs in 338 HWW samples from 13 countries in Africa, Asia, and Europe. In total, 2420 ARG subtypes belonging to 30 ARG types were detected, dominated by multidrug, beta-lactam, and aminoglycoside resistance genes. ARG composition in Europe differed from that in Asia and Africa. Notably, the ARGs presented co-occurrence with mobile genetic elements (MGEs), metal resistance genes (MRGs), and human bacterial pathogens (HBP), indicating a potential dissemination risk of ARGs in the HWW. Multidrug resistance genes presented co-occurrence with MGEs, MRGs, and HBP, is particularly pronounced. The abundance of contigs that contained ARG, contigs that contained ARG and HBP, contigs that contained ARG and MGE, contigs that contained ARG and MRG were used for health and transmission risk assessment of antibiotic resistome and screened out 40 high risk ARGs in the global HWW. This study first provides a comprehensive characterization and risk of the antibiotic resistome in global HWW.

Genomic Features and Comparative Genomic Analysis of Streptococcus sp. v1. nov., Isolated from an Endophthalmitis Patient.

Endophthalmitis is an acute inflammatory intraocular condition that can cause permanent vision loss. The treatment strategy and visual outcome partly depend on the identification of the agents of pathogens. In this study, metagenomic sequencing was conducted to investigate the microbial and antibiotic resistance genes (ARGs) composition in the vitreous (intraocular body fluid) of an endophthalmitis patient, who progressed rapidly and accompanied by severe pain. Metagenomic sequencing data revealed that the vitreous sample was predominated by Streptococcus, with a low-diversity microbiome in the vitreous. This strain harbor's the ARGs mainly against beta-lactam, macrolide-lincosamide-streptogramin, and multidrug. Additionally, metagenome-assembled genome sequence of Streptococcus sp. v1. nov. was identified. The Tetra Correlation Search (TCS) analysis uncovered that the closest relative of the Streptococcus sp. v1. nov. was Streptococcus mitis SK321. Pan/core genome analysis for Streptococcus sp. v1. nov. and TCS top 25 hits strains revealed that most unique genes of Streptococcus sp. v1. nov. were linked to ATP-binding cassette transport system, which could indicate unique virulence and pathogenic potentials of Streptococcus sp. v1. nov. In addition, a total of 7 virulence factors were identified, and the overwhelming of them were classified into "offensive virulence factors". The high pathogenicity of Streptococcus sp. v1. nov. could be a reason for the patient's rapid disease progression. Our study was first isolated an ocular pathogen with highly virulent based on metagenomic sequencing and bioinformatics analysis, which has important reference value for revealing the composition and genome characteristics of pathogens in endophthalmitis patient in the future.

Metagenomic analysis reveals antibiotic resistance profiles in tissue samples from patients with diabetic foot infections.

OBJECTIVES: Treating patients with diabetic foot infection (DFI) is challenging because of high rates of antibiotic resistance. Therefore, to administer a suitable antibiotic treatment, it is necessary to know the antibiotic resistance patterns in DFIs. METHODS: To explore this question, we selected metagenomic data of 36 tissue samples from patients with DFI in the National Center for Biotechnology Information Sequence Read Archive database. RESULTS: A total of 229 antibiotic-resistant gene (ARG) subtypes belonging to 20 ARG types were detected. The antibiotic resistome of 229 different genes in the tissue samples of patients with DFI comprised 24 core and 205 accessory resistance genes. Among the core antibiotic resistome, multidrug, tetracycline, macrolide-lincosamide-streptogramin, and beta-lactam resistance genes were the dominant categories. Procrustes analysis indicated that both the microbial community composition and mobile genetic elements (MGEs) were determinants of the ARGs. In the network analysis, 29 species were speculated to be potential hosts of 28 ARGs based on the co-occurrence results. Plasmids and transposons were the most common elements that co-occurred with ARGs. CONCLUSIONS: Our study provided detailed information about antibiotic resistance patterns in DFI, which has practical implications for suggesting a more specific antibiotic choice.

Factors affecting hospital discharge outcomes in patients with community-acquired pneumonia: A retrospective epidemiological study (2014-2021).

BACKGROUND: In patients with community-acquired pneumonia (CAP), the risk and protective factors influencing discharge outcomes have not been fully elucidated. Therefore, we aimed to investigate the factors affecting discharge outcomes and provide a theoretical basis for improving the cure rate of patients with CAP. METHODS: We describe a retrospective epidemiological study of patients with CAP conducted from 2014 to 2021. We used age, sex, co-morbidities, multilobar involvement, severe pneumonia, the main abnormal symptoms present on admission, and pathogen-targeted therapy as variables that may affect discharge outcomes. These variables were included in subsequent logistic regression analyses. Discharge outcomes were divided into remission and cure. RESULTS: Of a total of 1008 patients with CAP, 247 patients were discharged as remission. The results of multivariate logistic regression analyses showed that age >65 years, smoking history, co-morbidity of chronic obstructive pulmonary disease, co-morbidity of chronic heart disease, co-morbidity of diabetes, co-morbidity of malignancy, co-morbidity of cerebrovascular disease, pleural effusion, hypoxemia, respiratory failure, electrolyte disturbances, and severe pneumonia were independently associated with poor discharge outcomes (all P < 0.05), while pathogen-targeted therapy (odds ratio: 0.32, 95% confidence interval: 0.16-0.62) was found as a protective factor. CONCLUSIONS: Age > 65 years, the presence of co-morbidities, the presence of admission symptoms such as electrolyte disturbances, and severe pneumonia are associated with a poor discharge outcome, while pathogen-targeted therapy is associated with a good discharge outcome. Patients with CAP with a defined pathogen are more likely to be cured. Our results suggest that accurate and efficient pathogen testing is essential for CAP inpatients.

Application of metagenomic next-generation sequencing in the diagnosis and resistome analysis of community-acquired pneumonia pathogens from bronchoalveolar lavage samples.

AIMS: To perform a prospective diagnostic study exploring the clinical utility of metagenomic next-generation sequencing (mNGS) in diagnosing community-acquired pneumonia (CAP), and revealing resistome differences in bronchoalveolar lavage fluid (BALF) from CAP patients with varying severity of admission base on Pneumonia Patient Outcomes Research Team (PORT) risk classes. METHODS AND RESULTS: We compared the diagnostic performances of mNGS and conventional testing for the detection of pathogens in BALF from 59 CAP patients, and performed resistome differences analysis of metagenomic data from 59 BALF samples, namely, 25 from CAP patients with PORT score I (I group), 14 from CAP patients with PORT score II (II group), 12 from CAP patients with PORT score III (III group), and 8 from CAP patients with PORT score IV (IV group). The diagnostic sensitivities of mNGS and conventional testing for the detection of pathogens in BALF in patients with CAP were 96.6% (57/59) and 30.5% (18/59), respectively. There was a significant difference in the overall relative abundance of resistance genes between the four groups (P = 0.014). The results of principal coordinate analysis based on Bray-Curtis dissimilarities showed that there were significant differences in the composition of resistance genes among the I, II, III, and IV groups (P = 0.007). A large number of antibiotic resistance genes, such as those affiliated with multidrug, tetracycline, aminoglycoside, and fosfomycin resistance, were enriched in the IV group. CONCLUSIONS: In conclusion, mNGS has a high diagnostic value in CAP. There were significant differences present in microbiota resistance to antibiotics in BALF from CAP patients in different PORT risk classes, which should attract enough attention.

Lipid Mediators Metabolic Chaos of Asthmatic Mice Reversed by Rosmarinic Acid.

BACKGROUND AND OBJECTIVE: Asthma is a common chronic inflammatory disease of the airways with no known cure. Lipid mediators (LMs) are a kind of inflammatory signaling molecules which are believed to be involved in the development of asthma. Hyssopus cuspidatus Boriss. is a traditional Uyghur medicine, which is widely used in the treatment of asthma and other respiratory diseases. Extraction of Hyssopus cuspidatus Boriss. was reported to neutralize asthma symptoms. The purpose of the study was to investigate both the anti-inflammatory and immunoregulation properties of the Hyssopus cuspidatus Boriss. extract (SXCF) and its main active constituent, rosmarinic acid (RosA), in vivo. The effect of RosA, a major constituent of SXCF, was evaluated on an asthmatic model, with both anti-inflammatory and immunoregulation properties. MATERIALS AND METHODS: Anti-inflammatory effect of SXCF and RosA was assessed using OVA-induced asthma model mice by UPLC-MS/MS method. RESULTS: Overall, RosA played a critical role in anti-asthma treatment. In total, 90% of LMs species that were significantly regulated by SXCF were covered. On the most important LMs associated with asthma, RosA equivalent induced similar effects as SXCF did. It is believed that some constituents in SXCF could neutralize RosA excessive impacts on LMs.

Nocardia pulmonis sp. nov., an actinomycete isolated from a patient with pulmonary infection.

A novel bacterial strain, CDC141T, was isolated from sputum samples of a patient with pulmonary infection in Hainan Province, PR China. We performed a polyphasic study to assess the taxonomic position of the new species. Based on the results of 16S rRNA gene sequence analyses, strain CDC141T belonged to the genus Nocardia with the highest sequence similarity to Nocardia nova NBRC 15556T (98.84 %) and Nocardia macrotermitis RB20T (98.54 %). The dapb1 gene sequence-based phylogenetic and phylogenomic trees further showed that the novel strain was clustered in a distinct clade adjacent to Nocardia pseudobrasiliensis DSM 44290T. The DNA G+C content of strain CDC141T was 68.57 mol%. The genomic diversity analysis revealed low average nucleotide identity and in silico DNA‒DNA hybridization values (<84.7 and <28.9 %, respectively) with its closest relative. Growth occurred at 20-40 °C, pH 6.0-9.0 and with NaCl concentrations of 0.5-2.5 % (w/v). The main fatty acids of strain CDC141T were C16 : 0, C18 : 0 10-methyl, TBSA, C16 : 1 ω6c/C16 : 1 ω7c, C18 : 1 ω9c, C18 : 0, C17 : 1 iso I/anteiso B and C17 : 0. The polar lipid profile was dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, unidentified glycolipids, unidentified phospholipids and unidentified lipids. MK8 (H4ω-cycl) and MK8 (H4) were the major respiratory quinones. These characteristics were consistent with the typical chemotaxonomic properties of members of the genus Nocardia. Based on the results of phenotypic and genetic analyses, strain CDC141T was identified as representing a new species of the genus Nocardia, with the proposed name Nocardia pulmonis sp. nov. (CDC141T=JCM 34955T=GDMCC 4.207T).

Epidemiological Investigation of Hospital Transmission of Corynebacterium striatum Infection by Core Genome Multilocus Sequence Typing Approach.

Corynebacterium striatum has recently received increasing attention due to its multiple antimicrobial resistances and its role as an invasive infection/outbreak agent. Recently, whole-genome sequencing (WGS)-based core genome multilocus sequence typing (cgMLST) has been used in epidemiological studies of specific human pathogens. However, this method has not been reported in studies of C. striatum. In this work, we aim to propose a cgMLST scheme for C. striatum. All publicly available C. striatum genomes, 30 C. striatum strains isolated from the same hospital, and 1 epidemiologically unrelated outgroup C. striatum strain were used to establish a cgMLST scheme targeting 1,795 genes (hereinafter referred to as 1,795-cgMLST). The genotyping results of cgMLST showed good congruence with core genome-based single-nucleotide polymorphism typing in terms of tree topology. In addition, the cgMLST provided a greater discrimination than the MLST method based on 6 housekeeping genes (gyrA, gyrB, hsp65, rpoB, secA1, and sodA). We established a clonal group (CG) threshold based on 104 allelic differences; a total of 56 CGs were identified from among 263 C. striatum strains. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying closely related isolates that could give clues on hospital transmission. According to the results of analysis of drug-resistant genes and virulence genes, we identified CG4, CG5, CG26, CG28, and CG55 as potentially hypervirulent and multidrug-resistant CGs of C. striatum. This study provides valuable genomic epidemiological data on the diversity, resistance, and virulence profiles of this potentially pathogenic microorganism. IMPORTANCE Recently, WGS of many human and animal pathogens has been successfully used to investigate microbial outbreaks. The cgMLST schema are powerful genotyping tools that can be used to investigate potential epidemics and provide classification of the strains precise and reliable. In this study, we proposed the development of a cgMLST typing scheme for C. striatum, and then we evaluated this scheme for its applicability to hospital transmission investigations. This report describes the first cgMLST schema for C. striatum. The analysis of hospital transmission of C. striatum based on cgMLST methods has important clinical epidemiological significance for improving nosocomial infection monitoring of C. striatum and in-depth understanding of its nosocomial transmission routes.

Whole-genome sequencing-based prediction and analysis of antimicrobial resistance in Yersinia enterocolitica from Ningxia, China.

Focusing on resistance trends and transmission patterns of pathogenic microorganisms is a major priority for national surveillance programs. The use of whole-genome sequencing for antimicrobial susceptibility testing (WGS-AST) is a powerful alternative to traditional microbiology laboratory methods. Yersinia enterocolitica antimicrobial resistance (AMR) in the Ningxia Hui Autonomous Region has yet to be described thoroughly in current studies. We assessed and monitored the development of Y. enterocolitica AMR in the Ningxia Hui Autonomous Region during 2007-2019 based on WGS-AST. Resistance genotypes were predicted based on WGS. Antimicrobial resistance testing using classical microbiology determined resistance to 13 antimicrobial agents in 189 Y. enterocolitica isolates from Ningxia. The highest resistance level was 97.88% for cefazolin, followed by ampicillin (AMP) (44.97%), ciprofloxacin (CIP) (25.40%), streptomycin (STR) (11.11%), and tetracycline (TET) (10.58%). Isolates emerged as chloramphenicol (CHL) and trimethoprim/sulfamethoxazole (SXT) resistant. The primary plasmid types were IncFII(Y) and ColRNAI. The TET, STR, and SXT resistance were mediated by the tetA, aph(6)-Id, aph(3″)-Ib, and sul2 genes located on the IncQ1 plasmid. The resistant strains were predominantly biotype 4/O:3/ST429 and the hosts were pigs and patients. The number of multidrug-resistant (MDR) strains was of concern, at 27.51%. At present, the prediction of antimicrobial resistance based on WGS requires a combination of phenotypes. From 2007 to 2019, Y. enterocolitica isolates from the Ningxia Hui Autonomous Region showed a relatively high rate of resistance to cefazolin (CZO) and some resistance to AMP, CIP, STR, and TET. CIP, SXT, and TET showed a relatively clear trend of increasing resistance. Plasmids carrying multiple drug resistance genes are an important mechanism for the spread of antimicrobial resistance. Isolates with low pathogenicity were more likely to present an AMR phenotype than non-pathogenic isolates.

Diagnostic performance of metagenomic next-generation sequencing for the detection of pathogens in bronchoalveolar lavage fluid in patients with pulmonary infections: Systematic review and meta-analysis.

OBJECTIVES: Identifying pathogens in patients with pulmonary infection (PI) has always been a major challenge in medicine. Compared with sputum and throat swabs, bronchoalveolar lavage fluid (BALF) can better reflect the actual state of the lungs. However, there has not been a meta-analysis of the diagnostic efficacy of metagenomic next-generation sequencing (mNGS) in detecting pathogens in BALF from patients with PIs. METHODS: Data sources were PubMed, Web of Science, Embase, and the China National Knowledge Infrastructure. The pooled sensitivity and specificity were estimated using random-effects or fixed-effect models. Subgroup analysis was performed to reveal the effect of potential explanatory factors on the diagnostic performance measures. RESULTS: The pooled sensitivity was 78% (95% confidence interval [CI]: 67-87%; I2 = 92%) and the pooled specificity was 77% (95% CI: 64-94%; I2 = 74%) for mNGS. Subgroup analyses for the sensitivity of mNGS revealed that patients with PIs who were severely ill or immunocompromised significantly affected heterogeneity (P < 0.001). The positive detection rate of mNGS for pathogens in BALF of severely or immunocompromised pulmonary-infected patients was 92% (95% CI: 78-100%). CONCLUSION: mNGS has high diagnostic performance for BALF pathogens in patients with PIs, especially in critically ill or immunocompromised patients.

Complete genome sequencing of transposon-mediated sulfamethoxazole resistance encoded by the Sul1 gene in multidrug-resistant Nocardia farcinica SZ 1509.

OBJECTIVES: The objective of this study is to explore the molecular basis of trimethoprim-sulfamethoxazole (SXT) resistance in Nocardia, an SXT-resistant N. farcinica strain, named SZ 1509, by whole-genome sequencing. METHODS: Antimicrobial susceptibility testing of SZ 1509 was performed by broth microdilution, Etest, and disk diffusion arrays. Genome sequencing and analysis were performed to discover the SXT resistance determinant and its genetic context. Inverse PCR was conducted to confirm the circular form of the composite transposon. PCR for the sul1 gene was performed among SXT-susceptible isolates. RESULTS: SZ 1509 is resistant to many drugs, especially SXT, with a minimum inhibitory concentration (MIC) of up to 32/608 µg/mL (ratio of 1:19 for trimethoprim: sulfamethoxazole). Its assembled genome consists of one chromosome and four plasmids with a total size of 6 613 629 bp and 71.1% of GC content. The plasmid 2 was found to carry one IS6-composite transposon containing IS6100 carrying the sul1 gene, one tellurite resistance gene TerC, and several transcriptional regulators. Inverse PCR analyses showed its circular form. All 10 SXT-susceptible isolates do not contain sul1. In addition, mutations with strong associations to SXT resistance were not conclusive. CONCLUSION: This is the first study to elucidate the transposon-mediated sulfamethoxazole resistance in N. farcinica. Our results provide insights on acquired drug resistance of N. farcinica and further suggest that the prevalence and correlation of this resistance's determinants in clinical isolates should be continuously monitored to provide effective clinical management of its resultant diseases.

Characterization of the Ocular Surface Microbiome in Keratitis Patients after Repeated Ophthalmic Antibiotic Exposure.

In human medicine, antibiotics have been widely used to treat microbial infections. The extensive use of antibiotics is a leading cause of antibiotic resistance. Currently, the influence of the use of antibiotics on the ocular surface microbiome in the course of keratitis treatment remains to be explored in depth. We performed metagenomic analyses in a cohort of 26 healthy controls (HCs), 28 keratitis patients (KPs) who received antibiotics [KP (abx+) group], and 12 KPs who were antibiotic naive [KP (abx-) group]. We identified that the dissimilarities in microbial community structure (Bray-Curtis and Jaccard analyses) between the KP (abx+) group and the HC group were greater than those between the KP (abx-) group and the HC group. Pseudomonas lactis, P. aeruginosa, Pseudomonas sp. FDAARGOS_380, Pseudomonas sp. J380, Corynebacterium simulans, Streptococcus pyogenes, Finegoldia magna, and Aspergillus oryzae had no statistically significant differences between the KP (abx+) and KP (abx-) groups but did have statistically significant differences between the KP (abx+) and HC groups and between the KP (abx-) and HC groups. Among them, Pseudomonas lactis, P. aeruginosa, Pseudomonas sp. FDAARGOS_380, and Pseudomonas sp. J380 were identified as possible hosts carrying multidrug-resistant genes. The total abundance and number of antibiotic resistance genes (ARGs) were greater in the KP (abx+) group than in the HC and KP (abx-) groups. The functional profile analysis of ocular surface microbiota revealed that pathogenesis-related functional pathways and virulence functions were enriched in KPs. In conclusion, our results show that empirical antibiotic treatment in KPs leads to increases in the antibiotic resistance of ocular surface microbiota. IMPORTANCE Treatment for keratitis is based on appropriate antimicrobial therapy. A direct correlation between antibiotic use and the extent of antibiotic resistance has been reported. Therefore, knowledge of the antibiotic resistance patterns of ocular surface microbial flora in KPs is important for clinical treatment. To the best of our knowledge, this is the first study to use metagenomic approaches to investigate the associations between ophthalmic antibiotic use and the ocular surface microbiome of KPs. Monitoring the microbiota and antibiotic resistome profiles for the ocular surface has huge potential to help ophthalmologists choose the appropriate antibiotics and will thereby improve the efficacy of treatment regimens, which has important implications for reducing the development of antibiotic resistance of the ocular surface to a certain extent.

A CRISPR-Cas12a-Assisted Fluorescence Platform for Rapid and Accurate Detection of Nocardia cyriacigeorgica.

Nocardia cyriacigeorgica has gradually become a common pathogen in clinical microbial infections. Identification of Nocardia at the species level is essential to assess the susceptibility and pathogenicity of antimicrobials. However, there is no suitable method for rapid and accurate laboratory detection of N. cyriacigeorgica. In this study, we combined PCR amplification with the CRISPR-Cas12a system to establish a novel detection platform, named CRISPR-PCR, and applied it to the detection of N. cyriacigeorgica in clinical samples. The Cas12a protein exhibited collateral cleavage activity following CRISPR RNA binding to specific targets, then indiscriminately cleaved nearby single-stranded DNA, and this was evaluated for diagnostic nucleic acid detection by measuring the fluorescence signal using a fluorescence reader. The assay takes only 2 h, including DNA extraction for 20 min, nucleic acid pre-amplification for 70 min, and fluorescence detection for 20 min. The limit of detection for N. cyriacigeorgica was 10-3 ng and the specificity was 100%. Thus, the N. cyriacigeorgica CRISPR-PCR assay is a rapid and specific method for detecting N. cyriacigeorgica, and the CRISPR-PCR fluorescence detection platform has great potential for detection of other pathogens.

Alterations of fecal antibiotic resistome in COVID-19 patients after empirical antibiotic exposure.

As the COVID-19 pandemic spread globally, the consumption of antibiotics increased. However, no studies exist evaluating the effect of antibiotics use on the antibiotic resistance of intestinal flora in COVID-19 patients during the pandemic. To explore this issue, we collected 15 metagenomic data of fecal samples from healthy controls (HCs) with no use history of antibiotics, 23 metagenomic data of fecal samples from COVID-19 patients who received empirical antibiotics [COVID-19 (abx+)], 18 metagenomic data of fecal samples from antibiotics-naïve COVID-19 patients [COVID-19 (abx-)], and six metagenomic data of fecal samples from patients with community-acquired pneumonia [PC (abx+)] from the Sequence Read Archive database. A total of 513 antibiotic-resistant gene (ARG) subtypes of 18 ARG types were found. Antibiotic treatment resulted in a significant increase in the abundance of ARGs in intestinal flora of COVID-19 patients and markedly altered the composition of ARG profiles. Grouped comparisons of pairs of Bray-Curtis dissimilarity values demonstrated that the dissimilarity of the HC versus the COVID-19 (abx+) group was significantly higher than the dissimilarity of the HC versus the COVID-19 (abx-) group. The mexF, mexD, OXA_209, major facilitator superfamily transporter, and EmrB_QacA family major facilitator transporter genes were the discriminative ARG subtypes for the COVID-19 (abx+) group. IS621, qacEdelta, transposase, and ISCR were significantly increased in COVID-19 (abx+) group; they greatly contributed toward explaining variation in the relative abundance of ARG types. Overall, our data provide important insights into the effect of antibiotics use on the antibiotic resistance of COVID-19 patients during the COVID-19 epidemic.

A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica.

AIMS: To establish a CRISPR-based nucleic acid detection platform and apply it to the detection of Nocardia farcinica. METHODS AND RESULTS: A CRISPR-based nucleic acid detection platform, termed CRISPR-CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre-amplification of target sequences and CRISPR-Cas12a-based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR-CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR-CPA. The whole detection process of N. farcinica CRISPR-CPA assay, including sample pre-treatment and DNA extraction (~20 min), PCR pre-amplification (60 min), CRISPR-based detection (10 min), can be completed within 90 min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR-CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR-CPA assay is 1 pg DNA per reaction in pure cultures and 105  CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving. CONCLUSIONS: The N. farcinica CRISPR-CPA assay is an economic and specific method to detect N. farcinica and provides a high-efficiency tool for screening of pathogens especially of some hard-to-culture and slow-growth infectious agents. SIGNIFICANCE AND IMPACT OF THE STUDY: In CRISPR-CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5' end of the engineered PCR primer for protecting PAM site, thus the CRISPR-CPA can detect any sequence. Also, we applied CRISPR-CPA to rapidly detect N. farcinica, which is slow-growing bacteria and is firstly detected by a CRISPR-based method.

Dental Plaque Microbial Resistomes of Periodontal Health and Disease and Their Changes after Scaling and Root Planing Therapy.

The human oral microbial community has been considered a reservoir of antibiotic resistance. Currently, the effects of periodontitis and the scaling and root planing (SRP) treatment on the performance of antibiotic-resistant genes (ARGs) and metal-resistant genes (MRGs) in the dental plaque microbiota are not well characterized. To explore this issue, we selected 48 healthy-state (HS), 40 periodontitis-state (PS; before treatment), and 24 resolved-state (RS; after SRP treatment) metagenomic data of dental plaque samples from the Sequence Read Archive (SRA) database. NetShift analysis identified Fretibacterium fastidiosum, Tannerella forsythia, and Campylobacter rectus as key drivers during dental plaque microbiota alteration in the progression of periodontitis. Periodontitis and SRP treatment resulted in an increase in the number of ARGs and MRGs in dental plaque and significantly altered the composition of ARG and MRG profiles. Bacitracin, beta-lactam, macrolide-lincosamide-streptogramin (MLS), tetracycline, and multidrug resistance genes were the main classes of ARGs with high relative abundance, whereas multimetal, iron, chromium, and copper resistance genes were the primary types of MRGs in dental plaque microbiota. The cooccurrence of ARGs, MRGs, and mobile genetic elements (MGEs) indicated that a coselection phenomenon exists in the resistomes of dental plaque microbiota. Overall, our data provide new insights into the standing of the distribution of ARGs and MRGs in oral microbiota of periodontitis patients, and it was possible to contribute to the understanding of the complicated correlations among microorganisms, resistomes, and MGEs. IMPORTANCE The emergence and development of resistance to antibiotics in periodontal pathogens have affected the success rate of treatment for periodontitis. The development of new antibacterial strategies is urgently needed to help control and treat periodontal disease, and dental plaque microbiome studies offer a promising new angle of attack. In this study, we investigated the dental plaque microbiota and resistomes in periodontal health and disease states and their changes after SRP therapy. This is the first analysis of the profile of the microbial community and antibiotic and metal resistance genes in dental plaque by the metagenomic approach, to the best of our knowledge. Monitoring the profile of these resistomes has huge potential to provide reference levels for proper antibiotics use and the development of new antimicrobial strategies in periodontitis therapy and thereby improve actual efficacy of the treatment regimens.