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.

Anti-restriction protein ArdA promotes clinical Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae spread and its molecular mechanism.

BACKGROUND: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) has spread worldwide and has become a major threat to public health. The restriction modification system provides an innate defence of bacteria against plasmids or transposons, while many different types of plasmid encoding the anti-restriction protein ArdA can specifically affect the restriction activity in bacteria. OBJECTIVES: To detect the codistribution of ArdA and blaKPC-2 plasmids in KPC-KP and explore the molecular mechanism of ArdA promoting KPC-KP spread. METHODS: We collected 65 clinical CRKP isolates from Ningbo, China, and 68 cases of plasmid complete sequences in GenBank to determine the prevalence of ArdA gene on the K. pneumoniae blaKPC-2 plasmid. The anti-restriction function of ArdA in promoting horizontal gene transfer (HGT) was verified by transformation, conjugation and transduction methods, and the pull-down experiment was used to investigate the molecular mechanism of ArdA protein in vitro. RESULTS: We found that ArdA was widely distributed in KPC-KP in 100% of cases, which was detected in 0% of drug susceptible K. pneumoniae, and the plasmids containing the ArdA gene in 90% of the 30 cases randomly retrieved from the database. We also verified that ArdA has a good anti-restriction function (P < 0.05) through two aspects of HGT (transformation, transduction), and explored the non-occurrence interaction of ArdA and the hsdM subunit protein of EcoKI enzyme from the perspective of protein molecules. CONCLUSIONS: These findings suggest that the coexistence advantage of ArdA with the blaKPC-2 plasmids may provide KPC-producing K. pneumoniae with a very efficient evasion of the restriction of type I systems, which not only favours ArdA-containing mobile genetic elements in the same species HGT between bacteria also facilitates HGT between other bacterial species.

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.

Functional comparison of anti-restriction and anti-methylation activities of ArdA, KlcA, and KlcAHS from Klebsiella pneumoniae.

Anti-restriction proteins are typically encoded by plasmids, conjugative transposons, or phages to improve their chances of entering a new bacterial host with a type I DNA restriction and modification (RM) system. The invading DNA is normally destroyed by the RM system. The anti-restriction proteins ArdA, KlcA, and their homologues are usually encoded on plasmid of carbapenemase-resistant Klebsiella pneumoniae. We found that the plasmid sequence and restriction proteins affected horizontal gene transfer, and confirmed the anti-restriction and anti-methylation activities of ArdA and KlcA during transformation and transduction. Among the three anti-restriction proteins, ArdA shows stronger anti-restriction and anti-methylation effects, and KlcAHS was weaker. KlcA shows anti-methylation only during transformation. Understanding the molecular mechanism underlying the clinical dissemination of K. pneumoniae and other clinically resistant strains from the perspective of restrictive and anti-restrictive systems will provide basic theoretical support for the prevention and control of multidrug-resistant bacteria, and new strategies for delaying or even controlling the clinical dissemination of resistant strains in the future.

The Application of the CRISPR-Cas System in Antibiotic Resistance.

The emergence and global epidemic of antimicrobial resistance (AMR) poses a serious threat to global public health in recent years. AMR genes are shared between bacterial pathogens mainly via horizontal gene transfer (HGT) on mobile genetic elements (MGEs), thereby accelerating the spread of antimicrobial resistance (AMR) and increasing the burden of drug resistance. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are an RNA-guided adaptive immune system in prokaryotes that recognizes and defends against invasive genetic elements such as phages and plasmids. Because of its specifically target and cleave DNA sequences encoding antibiotic resistance genes, CRISPR/Cas system has been developed into a new gene-editing tool for the prevention and control of bacterial drug resistance. CRISPR-Cas plays a potentially important role in controlling horizontal gene transfer and limiting the spread of antibiotic resistance. In this review, we will introduce the structure and working mechanism of CRISPR-Cas systems, followed by delivery strategies, and then focus on the relationship between antimicrobial resistance and CRISPR-Cas. Moreover, the challenges and prospects of this research field are discussed, thereby providing a reference for the prevention and control of the spread of antibiotic resistance.

The Value of Combined Detection of Megakaryocyte and Platelet Parameters for the Diagnosis of Primary Immune Thrombocytopenia.

Objective:To investigate the application value of bone marrow megakaryocyte count, the proportion of megakaryocytes at each stage, and the platelet parameter in the clinical diagnosis of primary immune thrombocytopenia (ITP). Methods: The megakaryocyte and platelet parameter level in 62 ITP and 40 control group patients were compared and analyzed. Linear correlation analysis, Pearson correlation analysis, and ROC curves were performed for the correlation between megakaryocytes and platelet parameters. Results: Compared to the control group, the total number of megakaryocytes, the promegakaryocytes the granular megakaryocytes (GMeg), and naked megakaryocytes (NMeg), MPV, and P-LCR% in the ITP group increased. All differences were statistically significant (P<0.05). While the proportion of platelet-producing megakaryocytes (PMeg), PLT, and PCT decreased in the ITP group. These differences were statistically significant (P < 0.05). PLT was strongly positively correlated with PCT (r = 0.921, p<0.01). PCT was weakly positively with MPV (r = 0.309, p<0.05). MPV was positively correlated with P-LCR (r = 0.856, p<0.01). PDW was weakly positively correlated with P-LCR (r = 0.296, p<0.05) and Meg (r = 0.301, p<0.05), and negatively correlated with PMeg (r = -0.336, p<0.05). ROC curve analysis showed that PLT, PCT MPV and P-LCR% gave a high sensitivity（100.0%，81.0%，74.6%，90.5%，respectively.) and specificity (100.0%, 92.5%, 80.0%, 77.5%, respectively.) in diagnosis of ITP. Conclusion: The combined analysis of bone marrow megakaryocyte count, the proportion of megakaryocyte classification at each stage, and platelet parameters have an important reference value for auxiliary diagnosis of ITP.

The Spread of Antibiotic Resistance Genes In Vivo Model.

Infections caused by antibiotic-resistant bacteria are a major public health threat. The emergence and spread of antibiotic resistance genes (ARGs) in the environment or clinical setting pose a serious threat to human and animal health worldwide. Horizontal gene transfer (HGT) of ARGs is one of the main reasons for the dissemination of antibiotic resistance in vitro and in vivo environments. There is a consensus on the role of mobile genetic elements (MGEs) in the spread of bacterial resistance. Most drug resistance genes are located on plasmids, and the spread of drug resistance genes among microorganisms through plasmid-mediated conjugation transfer is the most common and effective way for the spread of multidrug resistance. Experimental studies of the processes driving the spread of antibiotic resistance have focused on simple in vitro model systems, but the current in vitro protocols might not correctly reflect the HGT of antibiotic resistance genes in realistic conditions. This calls for better models of how resistance genes transfer and disseminate in vivo. The in vivo model can better mimic the situation that occurs in patients, helping study the situation in more detail. This is crucial to develop innovative strategies to curtail the spread of antibiotic resistance genes in the future. This review aims to give an overview of the mechanisms of the spread of antibiotic resistance genes and then demonstrate the spread of antibiotic resistance genes in the in vivo model. Finally, we discuss the challenges in controlling the spread of antibiotic resistance genes and their potential solutions.

Rapid Detection of Klebsiella pneumoniae Carrying Virulence Gene rmpA2 by Recombinase Polymerase Amplification Combined With Lateral Flow Strips.

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10-1 ng/µL and 10 copies/µL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2.