Identification And Molecular Resistance Mechanisms Of A New Type Of Metallo-?-lactamase SIM

Objectives:1.To investigate the distribution of metallo-?-lactamase SIM in the carbapenemresistant Gram-negative bacteria in Fuzhou and Ningde, Fujian Province, and to study the epidemiological characteristics and drug resistance mechanisms.2.Compare the ability of hydrolyzing imipenem among MBLs(SIM-1,SIM-2, ND M-1 and VIM-2).Methods:1. Imipenem-resistant Gram-negative bacilli were collected from August 2013 to August 2014 in Fuzhou and Ningde, which were identified by VITEK-2 compact system. 441 strains were collected, including Acinetobacter spp(325), Klebsiella pneumonia(73), Enterobacter(3), Pseudomonas aeruginosa(19), Pseudomonas putida(1), Escherichia coli(14) and meninges sepsis Flavobacterium(6).2. Metallo-?-lactamase SIM was screened by PCR method with SIM-LIKE primers within carbapenem antibiotic-resistant gram-negative bacilli. The positive PCR products were sequenced, and the sequence was compared with the nucleotide sequence database by BLAST tool.3. The full-length sequence of SIM was amplified by using the DNA of SIM-positive strains as template. The positive PCR products were sequenced, and the sequence was compared with the nucleotide sequence database by BLAST tool.4. Whether the SIM-positive strains were resistant to carbapenem and whether they carried metallo-?-lactamase were conformed by IPM E-test and MBL E-test.5. SIM-positive strains were conformed as Pseudomonas aeruginosa and Pseudomonas putida by 16 s r RNA sequence analysis.6. Type A, B and D of carbapenem gene, outer membrane porin gene, efflux pump gene and part of ESBL gene were determined by PCR method to further study their resistance mechanisms.7. The SIM gene was positioned by plasmid conjugation test, which used SIM-positive strain as the donor strain and E.coli J53 Azr as the recipient strain.8. Genotype of SIM-positive strain was performed by MLST method.9. Expression Vector of SIM-1, SIM-2 and NDM-1 were constructed and then transformed into TOP10 competent cells to compare the ability of hydrolyzing imipenem.Results:1. SIM gene was detected in Pseudomonas aeruginosa B99 and Pseudomonas putida C285.2. A “G” to “A” mutation was identified at position 587 base of SIM gene, which resulted in Glycine changed into Aspartic acid. This type of mutation is not reported and named after SIM-2.3. The IPM E-test result of B99 and C285 exceeded the ceiling limit; MBL E-test results were positive.4. 16 S rRNA sequencing confirmed that B99 is a Pseudomonas aeruginosa; C285 is a Pseudomonas putida.5. VIM-2 gene was found both in B99 and C285 by PCR method; No other related genes but mex A, mex C and mex E efflux pump genes were found in B99; no other related genes except SIM-2 and VIM-2 were found in B99 and C285 strains.6. The result of conjugation experiments of B99 and C285 was negative.7. The Pseudomonas aeruginosa B99 MLST genotyping is ST1756.8. The imipenem MIC values of SIM-1, SIM-2, NDM-1 and VIM-2 were respectively8?g / ml, 64?g / ml, 32?g / ml and 2 ?g / ml.Conclusions:1. A new SIM-2 gene were found in a Pseudomonas aeruginosa and a Pseudomonas putida. The SIM-2 neucleotide of Pseudomonas aeruginosa B99 has been submitted to NCBI, the accession number is KT013203.2. Not only SIM-2 gene but also VIM-2 gene were found in Pseudomonas aeruginosa B99 and Pseudomonas putida C285. The resistance of carbapenem antibiotics of Pseudomonas aeruginosa B99 and Pseudomonas putida C285 is related to VIM-2 as well as to SIM-2.3. Efflux pump gene mex A, mex C and mex E were detected in Pseudomonas aeruginosa B99, while the outer membrane porin gene was absent, which indicated that the drug resistance was not only related to VIM-2 and SIM-2 but also the lack of outer membrane porin and efflux pump.4.Pseudomonas aeruginosa C285 and Pseudomonas aeruginosa B99 plasmid conjugation experiment negative suggested that the SIM-2 gene may be located on the chromosome.5. If ranked according to ability of hodrolyzing imipenem, the order should be SIM-2,NDM-1,SIM-1 and VIM-2 from strong to weak, indicating that the mutation of base587 leads to the increase of resistance to imipenem.