Transmission barrier of the blaKPC plasmid mediated by type I restriction-modification systems in Escherichia coli.

BACKGROUND: Transportation of carbapenem-resistant plasmids contributes to carbapenem resistance in Gram-negative bacteria. KPC enzymes are the most clinically important enzymes among carbapenem-resistant Klebsiella pneumoniae, whereas the rate of blaKPC in Escherichia coli is low. The CRISPR-Cas system and restriction-modification system (R-M system) in bacteria defend against invading genomes. Currently, the role of the immune systems in the low rate of KPC-producing E. coli remains unclear. OBJECTIVES: We investigated the relationship between immune systems and the low detection rate of blaKPC in E. coli. METHODS: We searched for blaKPC among 1039 E. coli whole genomes available in GenBank using nucleotide BLAST. CRISPR-Cas systems and the R-M system were detected in all strains having the ST as blaKPC-positive strains. Nucleotide BLAST was used to search for protospacers on blaKPC plasmids. A conjugation assay was performed to determine whether the R-M system influences the acquisition of blaKPC plasmids by E. coli. RESULTS: ST131 was the dominant ST of KPC-producing E. coli and IncN was the main plasmid type (12/32). CRISPR-Cas systems were frequently present in E. coli carrying blaKPC. Furthermore, CRISPR-Cas systems in E. coli didn't target plasmids with blaKPC. Type I R-M systems were rare in KPC-producing E. coli, but significantly over-represented in KPC-negative strains. E. coli DH5α with hsdR deletion accepted blaKPC-carrying plasmids, whereas those with hsdR complementation impeded blaKPC-carrying plasmid conjugation. CONCLUSIONS: Horizontal transmission of blaKPC occurs among E. coli. The type I R-M system is associated with the defence against blaKPC plasmid transport into E. coli.

In vitro Combined Inhibitory Activities of ß-Lactam Antibiotics and Clavulanic Acid Against bla KPC-2-Positive Klebsiella pneumoniae.

BACKGROUND: The spread of KPC-producing Enterobacteriaceae has triggered a global public health concern, with KPC-2-positive strains being the most prevalent in China. We hereby studied the in vitro combined inhibitory activities of three kinds of ß-lactam antibiotics and clavulanic acid at different concentrations against bla KPC-2-positive Klebsiella pneumoniae to explore the antimicrobial characteristics of these combinations and alternative therapeutic regimens for infections caused by bla KPC-2-positive K. pneumoniae strains. MATERIALS AND METHODS: In this study, 153 clinically isolated bla KPC-2-positive K. pneumoniae strains from 19 provinces in China were collected from 2016 to 2018. Antimicrobial susceptibility testing of imipenem/clavulanic acid, meropenem/clavulanic acid, ceftazidime/clavulanic acid, and each antimicrobial agent alone was performed by broth microdilution technique according to the CLSI guidelines. The concentration ratios of ß-lactam antibiotics to clavulanic acid were as follows: 1:1, 1:2, 1:4, 1:8, 1:16, 1:32. The antimicrobial susceptibility of the combinations was determined according to the breakpoints of Imipenem, meropenem, and ceftazidime established by the CLSI directives for Enterobacteriaceae. RESULTS: The MICs of all three combinations gradually declined with increments in the proportion of clavulanic acid in the regimens, and the most significant decline in the MIC50 and MIC90 was seen in combinations at the concentration ratio of 1:1 (also 1:2 for meropenem/clavulanic acid). When the concentration of clavulanic acid was restricted to 4 mg/L, the susceptibility of more than 70% of the isolates to the regimens could be restored with imipenem MIC 2-4 mg/L, meropenem MIC 2-8 mg/L or ceftazidime MIC 8mg/L. However, the percentage decreased to 30 to 40% when the initial MIC level was higher. CONCLUSION: The highest combined inhibitory activity of ß-lactam antibiotics/clavulanic acid at low concentration ratios against bla KPC-2-positive K. pneumoniae may offer a new way to optimize the effects of these antimicrobial regimens.

Dissemination of Carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) Among Carbapenem-Resistant Enterobacteriaceae Isolated From Adult and Children Patients in China.

This study aimed to investigate the dissemination and characteristics of blaKPC, blaNDM, blaOXA-48-like , blaIMP, and blaVIM among the carbapenem-resistant Enterobacteriaceae (CRE) strains isolated from adult and children patients. A total of 935 non-duplicate CRE strains were collected from 36 hospitals in 24 provinces or cities across China from 2016 to 2018. Antimicrobial susceptibility testing was performed by broth microdilution method and carbapenemase genes blaKPC, blaNDM, blaOXA-48-like , blaIMP, and blaVIM were screened by PCR and confirmed by DNA sequencing. Overall, carbapenemases were produced in 97.4% (911/935) of CRE strains, including KPC-2 (51.6%, 482/935), NDM (35.7%, 334/935), and OXA-48-like carbapenemases (7.3%, 68/935). Overall, the most prevalent carbapenemase gene was blaKPC-2 among Klebsiella pneumoniae (64.6%, 457/709) and the CRE strains isolated from adult patients (70.3%, 307/437), and blaNDM among Escherichia coli (96.0%, 143/149) and the CRE strains from children (49.0%, 247/498). The blaOXA-232-positive carbapenem-resistant K. pneumoniae (9.3%, 66/709) were all isolated from children. Sixteen strains were positive for blaIMP and 9 strains produced multiple carbapenemases. No strain was positive for blaVIM. Most of the CRE strains (>90%) were resistant to cephalosporins and carbapenems, more than half (>50%) were resistant to aminoglycosides and fluoroquinolones, but the majority (95.8 and 98.4%) were susceptible to polymyxin B and tigecycline. Ceftazidime-avibactam showed excellent in vitro activity against blaKPC-2 and blaOXA-48-like positive strains (100% susceptible). In China, KPC-2, NDM, and OXA-48-like carbapenemases were predominant among CRE clinical isolates. The most prevalent carbapenemase gene was blaKPC-2 among K. pneumoniae isolates from adult patients, and blaNDM among E. coli isolates from children.

Evaluation of the Immunochromatographic NG-Test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT for Rapid Detection of KPC-, NDM-, IMP-, VIM-type, and OXA-48-like Carbapenemase Among Enterobacterales.

BACKGROUND: Enterobacterales are the most common pathogens for nosocomial infections. The emergence and spread of KPC, NDM, and OXA-48-like carbapenemase-producing Enterobacterales with their extensively drug-resistant characteristics have posed great threats to public health. This study aimed to evaluate the performance of NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT for rapid detection of five carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like) among Enterobacterales. METHODS: A total of 186 carbapenem-resistant Enterobacterales clinical isolates and 29 reference strains were used in this study. Carbapenemase genes were confirmed by PCR and DNA sequencing. The sensitivities and specificities of these assays were calculated utilizing the VassarStats software. RESULTS: For clinical isolates, the NG-test Carba 5 detected KPC, NDM, OXA-48-like, IMP, and VIM in less than 15 min with the sensitivity and specificity of 100% and 100%, respectively. The RESIST-5 O.O.K.N.V. detected KPC, NDM, OXA-48-like, and VIM with the sensitivity and specificity of 99.4 and 100%. The IMP K-SeT detected all of the IMP producers (6/6). For reference strains, the sensitivity and specificity of NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT were all 100 and 100%, respectively. CONCLUSION: As efficient, rapid, and convenient diagnostic methods, NG-test Carba 5, RESIST-5 O.O.K.N.V., and IMP K-SeT could help to simplify the complex routine workflow for detecting carbapenemases. Rapid and accurate identification of carbapenemase is of significance for both epidemiological and infection control purposes.