The biological function of the type II toxin-antitoxin system ccdAB in recurrent urinary tract infections.

Urinary tract infections (UTIs) represent a significant challenge in clinical practice, with recurrent forms (rUTIs) posing a continual threat to patient health. Escherichia coli (E. coli) is the primary culprit in a vast majority of UTIs, both community-acquired and hospital-acquired, underscoring its clinical importance. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. The type II TA system, prevalent in prokaryotes, emerges as a critical player in stress response, biofilm formation, and cell dormancy. ccdAB, the first identified type II TA module, is renowned for maintaining plasmid stability. This paper aims to unravel the physiological role of the ccdAB in rUTIs caused by E. coli, delving into bacterial characteristics crucial for understanding and managing this disease. We investigated UPEC-induced rUTIs, examining changes in type II TA distribution and number, phylogenetic distribution, and Multi-Locus Sequence Typing (MLST) using polymerase chain reaction (PCR). Furthermore, our findings revealed that the induction of ccdB expression in E. coli BL21 (DE3) inhibited bacterial growth, observed that the expression of both ccdAB and ccdB in E. coli BL21 (DE3) led to an increase in biofilm formation, and confirmed that ccdAB plays a role in the development of persistent bacteria in urinary tract infections. Our findings could pave the way for novel therapeutic approaches targeting these systems, potentially reducing the prevalence of rUTIs. Through this investigation, we hope to contribute significantly to the global effort to combat the persistent challenge of rUTIs.

Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system.

OBJECTIVE: The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system. METHODS: Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance. RESULTS: In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA. CONCLUSION: In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

Elimination of blaKPC-2-mediated carbapenem resistance in Escherichia coli by CRISPR-Cas9 system.

OBJECTIVE: The purpose of this study is to re-sensitive bacteria to carbapenemases and reduce the transmission of the blaKPC-2 gene by curing the blaKPC-2-harboring plasmid of carbapenem-resistant using the CRISPR-Cas9 system. METHODS: The single guide RNA (sgRNA) specifically targeted to the blaKPC-2 gene was designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-sgRNA(blaKPC-2) was transformed into Escherichia coli (E.coli) carrying pET24-blaKPC-2. The elimination efficiency in strains was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by E-test strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance. RESULTS: In the present study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the blaKPC-2 gene. PCR and qPCR results indicated that prokaryotic CRISPR-Cas9 plasmid transforming drug-resistant bacteria can efficiently clear blaKPC-2-harboring plasmids. In addition, the drug susceptibility test results showed that the bacterial resistance to imipenem was significantly reduced and allowed the resistant model bacteria to restore susceptibility to antibiotics after the blaKPC-2-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. CONCLUSION: In conclusion, our study demonstrated that the one plasmid-mediated CRISPR-Cas9 system can be used as a novel tool to remove resistance plasmids and re-sensitize the recipient bacteria to antibiotics. This strategy provided a great potential to counteract the ever-worsening spread of the blaKPC-2 gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

ESBL-Producing and Non-ESBL-Producing Escherichia coli Isolates from Urinary Tract Differ in Clonal Distribution, Virulence Gene Content and Phylogenetic Group.

Purpose: The objectives of this study are to determine the differences in clonality, virulence gene (VG) content and phylogenetic group between non extended-spectrum beta-lactamase-producing E. coli (non-ESBL-EC) and ESBL-EC isolates from urine. Patients and Methods: This study characterized a total of 100 clinical E. coli isolates consecutively obtained from the inpatients hospitalized in The First Affiliated Hospital of Ningbo University in China by polymerase-chain reaction (PCR). Results: Phylogenetic group B2 was found to be the most prevalent in both ESBL-EC and non-ESBL-EC group. Among 100 clinical isolates, the count of acquired virulence genes in group B2 was found to be significantly higher than that in group A, B1, and D (p <0.001). Additionally, the presence of content within virulence genes (the total number of virulence genes detected per isolate) in B2 of non-ESBL-EC and ESBL-EC showed a significant difference (p<0.001). ST131 was detected exclusively in ESBL-EC, while ST95 and ST73 were the main sequence types in non-ESBL-EC. Conclusion: Our study demonstrated the different distribution of MLST, phylogenetic group in ESBL-EC and non-ESBL-EC group. The inverse association between beta-lactamase resistance and VG content performed in this study should get a lot more attention. At the same time, we should also be wary of the appearance of non-ESBL-EC isolates of group B2 harboring more virulence genes which will lead to high pathogenicity.

Analysis of genetic structure and function of clustered regularly interspaced short palindromic repeats loci in 110 Enterococcus strains.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune system involved in specific defenses against the invasion of foreign mobile genetic elements, such as plasmids and phages. This study aims to analyze the gene structure and to explore the function of the CRISPR system in the Enterococcus genome, especially with regard to drug resistance. The whole genome information of 110 enterococci was downloaded from the NCBI database to analyze the distribution and the structure of the CRISPR-Cas system including the Cas gene, repeat sequences, and spacer sequence of the CRISPR-Cas system by bioinformatics methods, and to find drug resistance-related genes and analyze the relationship between them and the CRISPR-Cas system. Multilocus sequence typing (MLST) of enterococci was performed against the reference MLST database. Information on the drug resistance of Enterococcus was retrieved from the CARD database, and its relationship to the presence or absence of CRISPR was statistically analyzed. Among the 110 Enterococcus strains, 39 strains (35.45%) contained a complete CRISPR-Cas system, 87 CRISPR arrays were identified, and 62 strains contained Cas gene clusters. The CRISPR system in the Enterococcus genome was mainly type II-A (59.68%), followed by type II-C (33.87%). The phylogenetic analysis of the cas1 gene sequence was basically consistent with the typing of the CRISPR-Cas system. Of the 74 strains included in the study for MLST typing, only 19 (25.68%) were related to CRISPR-Cas typing, while the majority of the strains (74.32%) of MLST typing were associated with the untyped CRISPR system. Additionally, the CRISPR-Cas system may only be related to the carrying rate of some drug-resistant genes and the drug-resistant phenotype. In conclusion, the distribution of the enterococcus CRISPR-Cas system varies greatly among different species and the presence of CRISPR loci reduces the horizontal transfer of some drug resistance genes.

Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus.

Purpose: This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes. Materials and Methods: Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests. Results: A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene. Conclusion: In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.

Detection rate of SARS-CoV-2 RNA in relation to isolation time and environmental surface type.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) causes environmental contamination via respiratory droplets and persists on contaminants and environmental surfaces for anywhere from a few hours to 6 days. Therefore, it is particularly important to understand the transmission and containment of SARS-CoV-2 on the surface of objects within isolated environments. In this study, 356 environmental surface samples were collected and 79 tested positive, with the highest contamination rate (56.96%) in the wood category (bedside tables, wood floors, and walls). This study revealed differences in the detection rates of environmental surfaces in hospitalized and discharged rooms of patients with confirmed COVID-19 in 2 isolated settings (A: p = 0.001; B: p = 0.505) and suggested that environmental contamination may be an important route of virus transmission, providing a reference to guide the enhancement of ventilation, the use of hotel isolation model, the advocacy of cotton masks, and the effective suppression of virus transmission.

NeuroPP: A Tool for the Prediction of Neuropeptide Precursors Based on Optimal Sequence Composition.

Neuropeptides (NPs) are short secreted peptides produced mainly in the nervous system and digestive system. They activate signaling cascades to control a wide range of biological functions, such as metabolism, sensation, and behavior. NPs are typically produced from a larger NP precursor (NPP) which includes a signal peptide sequence, one or more NP sequences, and other sequences. With the drastic growth of unknown protein sequences generated in the post-genomic age, it is highly desired to develop computational methods for identifying NPP rapidly and efficiently. In this article, we developed a predictor for NPPs based on optimized sequence composition of single amino acid, dipeptide, and tripeptide. Evaluated with independent data set, the predictor showed excellent performance that achieved an accuracy of 88.65% with AUC of 0.95. The corresponding web server was developed, which is freely available at http://i.uestc.edu.cn/neuropeptide/neuropp/home.html . It can help relevant researchers to screen candidate NP precursor, shorten experimental cycle, and reduce costs.

Biopanning data bank 2018: hugging next generation phage display.

Database URL: The BDB database is available at http://immunet.cn/bdb.