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Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this study, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS 1 , IS 609 , and enzymes of DNA metabolism were closely related to CRISPR-Cas. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS 5 were observed near the CRISPR loci in plasmid, which is also confirmed by the results of PCA that they may be important factors affecting the plasmid with CRISPR. [ABSTRACT FROM AUTHOR]

This research aimed to investigate the presence and transferability of the extended-spectrum β-lactamase resistance genes to identify the genetic context of multi-drug resistant (MDR) loci in two Escherichia coli plasmids from livestock and poultry breeding environment. MICs were determined by broth microdilution. A total of 137 E. coli resistant to extended-spectrum β-lactam antibiotics were screened for the presence of the ESBL genes by PCR. Only two E. coli out of 206 strains produced carbapenemases, including strain 11011 that produced enzyme A, and strain 417957 that produced enzyme B. The genes were bla KPC and bla NDM , respectively. The plasmids containing bla CTX–M were conjugatable, and the plasmids containing carbapenem resistance gene were not conjugatable. Six extended-spectrum β-lactamase resistance genes were detected in this research, including bla TEM, bla CTX–M, bla SHV, bla OAX–1, bla KPC, and bla NDM , and the detection rates were 94.89% (130/137), 92.7% (127/137), 24.81% (34/137), 20.43% (28/137), 0.72% (1/137), and 0.72% (1/137), respectively. Two conjugative lncFII multi-resistance plasmids carrying bla CTX–M, p11011-fosA and p417957-CTXM, were sequenced and analyzed. Both conjugative plasmids were larger than 100 kb and contained three accessory modules, including MDR region. The MDR region of the two plasmids contained many antibiotic resistance genes, including bla CTX–M, mph (A) , dfrA17 , aadA5 , sul1 , etc. After transfer, both the transconjugants displayed elevated MICs of the respective antimicrobial agents. A large number of resistance genes clusters in specific regions may contribute to the MDR profile of the strains. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterobacteriaceae through inter-replicon gene transfer. Our study provides beta-lactam resistance profile of bacteria, reveals the prevalence of β-lactamase resistance genes in livestock and poultry breeding environment in Zhejiang Province, and enriches the research on IncFII plasmids containing bla CTX–M. [ABSTRACT FROM AUTHOR]

Background: IncR, IncFII, IncpA1763-KPC, and IncN1 plasmids have been increasingly found among Enterobacteriaceae species, but plasmids with hybrid structures derived from the above-mentioned incompatibility groups have not yet been described. Methods: Plasmids p721005-KPC, p504051-KPC, and pA3295-KPC were fully sequenced and compared with previously sequenced related plasmids pHN84KPC (IncR), pKPHS2 (IncFIIK), pKOX_NDM1 (IncFIIY), pHN7A8 (IncFIIpHN7A8), and R46 (IncN1). Results: The backbone of p721005-KPC/p504051-KPC was a hybrid of the entire 10-kb IncR-type backbone from pHN84KPC, the entire 64.3-kb IncFIIK-type maintenance, and conjugal transfer regions from pKPHS2, a 15.5-kb IncFIIY-type maintenance region from pKOX_NDM1 and a 5.6-kb IncpA1763-KPC-type backbone region from pA1763-KPC, and it contained a primary IncR replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. The backbone of pA3295-KPC was a hybrid of a 7.2-kb IncFIIpHN7A8-type backbone region from pHN7A8, the almost entire 33.3-kb IncN1-type maintenance and conjugal transfer regions highly similar to R46, a 26.2-kb IncFIIK-type maintenance regions from pKPHS2, the above 15.5-kb IncFIIY-type maintenance region, and the above 5.6-kb IncpA1763-KPC-type backbone region, and it contained a primary IncFIIpHN7A8 replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. Each of p721005-KPC, p504051-KPC, and pA3295-KPC acquired a wealth of accessory modules, carrying a range of intact and residue mobile elements (such as insertion sequences, unit transposons, and integrons) and resistance markers (such as blaKPC, tetA, dfrA, and qnr). Conclusion: In each of p721005-KPC, p504051-KPC, and pA3295-KPC, multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number. [ABSTRACT FROM AUTHOR]

The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system. CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system. [ABSTRACT FROM AUTHOR]