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Klebsiella variicola (K. variicola) is an emerging human pathogen, which poses a threat to public health. The horizontal gene transfer (HGT) of plasmids is an important driver for the emergence of multiple antibiotic-resistant K. variicola. The clustered regularly interspersed short palindromic repeats coupled with the CRISPR-associated genes (CRISPR/Cas) constitute an adaptive immune system in bacteria, which provide acquired immunity against HGT. However, the information about CRISPR/Cas system in K. variicola is still limited. In this study, a total of 487 genomes from NCBI database were used to analyze the characterization of CRISPR/Cas systems. 105 of the 487 genomes harbored at least one confirmed CRISPR array. Three types of CRISPR/Cas system, including types I-E, I-E*, and Ⅳ-A systems, were identified among 105 strains. The distribution of type I system was strongly associated with MLST, whereas type IV system was randomly distributed. Approximately one-third of spacer origins were homologous with plasmids or phages, indicating the role of CRISPR/Cas systems in controlling HGT. Moreover, spacers in K. variicola tended to target mobile genetic elements (MGEs) from Klebsiella pneumoniae, which provides new evidence for their interaction during evolution. Collectively, our results provide valuable insights into the role of CRISPR/Cas systems in K. variicola.

Staphylococcus aureus is a serious threat to public health. S. aureus infection can cause acute or long-term persistent infections that are often resistant to antibiotics and are associated with high morbidity and death. Understanding the defensive systems of S. aureus can help clinicians make the best use of antimicrobial drugs and can also help with antimicrobial stewardship. The mechanisms and clinical implications of S. aureus defense systems, as well as potential response systems, were discussed in this study. Because resistance to all currently available antibiotics is unavoidable, new medicines are always being developed. Alternative techniques, such as anti-virulence and bacteriophage therapies, are being researched and may become major tools in the fight against staphylococcal infections in the future, in addition to the development of new small compounds that affect cell viability.

CRISPR systems are often encoded by many prokaryotes as adaptive defense against mobile genetic elements (MGEs), but several MGEs also recruit CRISPR components to perform additional biological functions. Type IV-A systems are identified in Klebsiella plasmids, yet the distribution, characterization, and role of these plasmids carrying CRISPR systems in the whole Klebsiella genus remain unclear. Here, we performed large-scale comparative analysis of these plasmids using publicly available plasmid genomes.

The coronavirus disease 2019 (COVID-19) pandemic is a public health emergency of international concern. However, its stress on the mental health of young to middle-aged adults is largely unexplored. This study aimed to evaluate the mental health difficulties during the resurgent phase of COVID-19 among young to middle-aged adults in China. There were 1,478 participants with a median age of 26 years (IQR, 23 - 30), including 535 males (36.2%). The prevalence of anxiety, depression, and insomnia were 8.6%, 11.4%, and 13.7%, respectively. Participants aged 29 - 59 years (OR, 95% CI: 2.46, 1.23 - 4.91) and females (2.49, 1.55 - 4.01) had a higher risk of anxiety. Education status, worried level about the current COVID-19, and the level of COVID-19's impact on life were significantly associated with the prevalence of anxiety. Besides, the level of COVID-19's impact on life was positively related to the prevalence of depression and insomnia. Our study provided novel evidence of psychological difficulties among young to middle-aged adults during the resurgent stage of the COVID-19 epidemic. Psychological intervention should be continuously implemented to prevent long-term psychological comorbidities during the COVID-19 epidemic.

The CRISPR-Cas system is widely distributed in prokaryotes and plays an important role in the adaptive immunity of bacteria and archaea. Bifidobacterium is an important component of the intestinal flora of humans and animals, and some species of this bacterium can be employed as food additives. However, the Bifidobacterium CRISPR-Cas system has not been fully elucidated to date. In this study, the genomes of 110 strains of Bifidobacterium were employed to research the diversity of the type I-U system. The 110 strains were divided into five groups according to the genes adjacent to the CRISPR locus, including group A, B, C, D and E. Strains in the intergroup had unique species classifications and MLST types. An evolutionary tree was constructed based on the conserved cas4/cas1 fusion gene. The results showed that group A had a different evolutionary branch compared with the other groups and had a relatively low spacer number. Notably, group B, C and E had exhibited ABC transporter regulators in the genes adjacent to the CRISPR locus. ABC transporters play important roles in the exocytosis of many antibiotics and are involved in horizontal gene transfer. This mechanism may have promoted the evolution of Bifidobacterium and the horizontal gene transfer of the type I-U system, which may have promoted the generation of system diversity. In summary, our results help to elucidate the role of the type I-U system in the evolution of Bifidobacterium.

The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml–1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.

The hypervariable nature of clustered regularly interspaced short palindromic repeats (CRISPRs) makes them valuable biomarkers for subtyping and epidemiological investigation of Escherichia coli. Shiga toxin-producing E. coli (STEC) serogroup O80 is one hybrid pathotype that is emerging recently in Europe and is involved in hemolytic uremic syndrome with bacteremia. However, whether STEC O80 strains can be genotyped using CRISPR has not been evaluated. In this study, we aimed to characterize the genetic diversity of 81 E. coli serogroup O80 isolates deposited in the National Center for Biotechnology Information databases using CRISPR typing and to explore the association between virulence potential and CRISPR types (CTs). A total of 21 CTs were identified in 80 O80 strains. CRISRP typing provided discrimination with variants of a single serotype, which suggested a stronger discriminatory power. Based on CRISPR spacer profiles, 70 O80:H2 isolates were further divided into four lineages (lineage LI, LII, LIII, and LIV), which correlated well with whole-genome single nucleotide polymorphisms typing and virulence gene profiles. Moreover, the association between CRISPR lineages and virulence gene profiles hinted that STEC O80:H2 strains may originate from O80:H19 or O80:H26 and that lineage LI may have been evolved from lineage LII. CT2 and CT13 were shared by human and cattle isolates, suggesting that there might be the potential transmission between cattle and human. Collectively, CRISPR typing is one technology that can be used to monitor the transmission of STEC O80 strains and provide new insights into microevolution of serogroup O80.

E. coli of phylogenetic group B2 is responsible for many extraintestinal infections, posing a great threat to health. The relatively polymorphic nature of CRISPR in phylogenetically related E. coli strains makes them potential markers for bacterial typing and evolutionary studies. In the current work, we investigated the occurrence and diversity of CRISPR/Cas system and explored its potential for genotyping. Type I-F CRISPR/Cas systems were found in 413 of 1190 strains of E. coli and exhibited the clustering within certain CCs and STs. And CRISPR spacer contents correlated well with MLST types. The divergence analysis of CRISPR showed stronger discriminatory power than MLST, and CRISPR polymorphism was instrumental for differentiating highly closely related strains. The timeline of spacer acquisition and deletion provided important information for inferring the evolution model between distinct serotypes. Identical spacer sequences were shared by strains with the same H-antigen type but not strains with the same O-antigen type. The homology between spacers and antibiotic-resistant plasmids demonstrated the role of Type I-F system in limiting the acquisition of antimicrobial resistance. Collectively, our data presents the dynamic nature of Type I-F CRISPR in E. coli of phylogenetic group B2 and provides new insights into the application of CRISPR-based typing in the species.

Clostridium perfringens is an important pathogen of human and livestock infections, posing a threat to health. The horizontal gene transfer (HGT) of plasmids that carry toxin-related genes is involved in C. perfringens pathogenicity. The CRISPR/Cas system, which has been identified in a wide range of prokaryotes, provides acquired immunity against HGT. However, information about the CRISPR/Cas system in Clostridium perfringens is still limited. In this study, 111 C. perfringens strains with publicly available genomes were used to analyze the occurrence and diversity of CRISPR/Cas system and evaluate the potential of CRISPR-based genotyping in this multi-host pathogen. A total of 59 out of the 111 genomes harbored at least one confirmed CRISPR array. Four CRISPR/Cas system subtypes, including subtypes IB, IIA, IIC, and IIID systems, were identified in 32 strains. Subtype IB system was the most prevalent in this species, which was subdivided into four subgroups displaying subgroup specificity in terms of cas gene content, repeat sequence content, and PAM. We showed that the CRISPR spacer polymorphism can be used for evolutionary studies, and that it can provide discriminatory power for typing strains. Nevertheless, the application of this approach was largely limited to strains that contain the CRISPR/Cas system. Spacer origin analysis revealed that approximately one-fifth of spacers showed significant matches to plasmids and phages, thereby suggesting the implication of CRISPR/Cas systems in controlling HGT. Collectively, our results provide new insights into the diversity and evolution of CRISPR/Cas system in C. perfringens.

Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.