Objective To evaluate the performance of VITEK 2-Compact GN13 methods for testing imipenem susceptibility of Klebsiella pneumoniae and assess the reliability of its Advanced Expert System (AES).Methods A retrospective study was conducted with a total of 157K. pneumoniae strains, which were isolated from blood and intra-abdominal infections in the First Affiliated Hospital of Nanchang University from 2014 to 2015. Thein vitro minimum inhibitory concentration (MIC) values of imipenem were determined by disc diffusion, VITEK 2-Compact GN13 and broth microdilution methods, respectively. Categorical agreement (CA) rates of disc diffusion and VITEK 2-Compact GN13 methods were determined using broth microdilution as reference method. The genes encoding ESBLs and carbapenemase were screened by PCR and sequencing analysis. The phenotypic confirmatory tests such as modified Hodge test, PCR and DNA sequencing were used to confirm the resistance mechanism and evaluate the reliability of AES in interpreting the imipenem susceptibility of K. pneumoniae.Results Among the 157 isolates, 64 and 8 were identified as resistant and intermediate strains by broth microdilution method, respectively; 52 and 10 were tested as resistant and intermediate strains by disc diffusion method, respectively; 54 and 13 were determined as resistant and intermediate strains by VITEK 2-Compact GN13 method, respectively, while 70 and 3 were judged as resistant and intermediate strains by VITEK 2-Compact GN13 method plus AES validation. The CA of disc diffusion and VITEK 2-Compact GN13 methods compared with broth microdilution method were all higher than 90 %. However, the major error (ME) rate was 3.8 % and very major error (VME) rates were all 0.6 % in imipenem susceptibility testing by VITEK 2-Compact GN13 and disc diffusion. The imipenem susceptibility of 16 strains were modified by the AES, which eliminated 0.6 % VME, but increased major error by 1.3 % and minor error by 1.9 %. Phenotypic confirmatory tests showed that 75 % (12/16) of these strains were validated as producers of both ESBLs and carbapenemase, which was consistent with the result of AES validation. PCR and DNA sequencing analysis proved that 62.5 % (10/16) of these strains produce IMP-4/KPC-2 /NDM-1 and ESBLs.Conclusions Both disc diffusion and VITEK 2-Compact GN13 methods can be used for testing the imipenem susceptibility of K. pneumoniae isolates with reliable and accurate results. Attention should be paid to the possibility of ME and VME when testing imipenem susceptibility. The VME can be avoided by the AES mechanism. However, AES intervention will increase ME and minor error, which may be associated with decreased expression of carbapenemase.

Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.

Objective To investigate the molecule phenotype, epidemiology, and resistance genes of the New Delhi metallo- β-lactamase-1 ( NDM-1 ) producing Klebsiella pneumoniae ( K. pneumoniae ) . Methods Retrospective study was made on one hundred and ten non-repetitive carbepenem-resistant K. pneumoniae clinical isolated strains, which were collected from January 2011 to December 2012 in our hospital. The minimal inhibitory concentrations ( MICs ) of antibiotics were tested by the GN13 cards of BioMerieux Company. Modified Hodge test were used for the detection of carbapenemases. The blaNDM-1 encoding gene and linkage of ISAba125-NDM were detected by PCR method. The purified PCR products were cloned and sequenced. The homology of the K. pneumoniae were analyzed by the multilocus sequence typing ( MLST ) . Plasmid conjugation experiment and curing method were used to study the transfer of bacterial resistance. The Fisher′s exact probability test was used to compare the data. Results 13% NDM-1-producing K. pneumoniae were detected and confirmed as blaNDM-1 by sequencing (14/110). The resistance rates of the 14 NDM-1-producing K. pneumoniae strains to imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, amikacin, and aztreonam were 14/14, 14/14, 13/14, 10/14, 9/14, 5/14, and 11/14. Meanwhile, the positive rate of ISAba125-NDM linkage of those 14 NDM-1-producing K. pneumoniae strains was 14/14. The E. coli J53 transconjugants, whose MICs of imipenem, meropenem, and ertapenem were increased by 4 to 64 times, were blaNDM-1 gene and ISAba125-NDM linkage positive. In addition, it was showed that the blaNDM-1 gene and ISAba125-NDM linkage were located on a plasmid with a size of approximately 65 000 bp. Conclusions The NDM-1 producing K. pneumoniae strains in this study were resistant to many commonly used antibiotics, however, the resistance rate to aminoglycoside and aztreonam were relatively low. The carbapenemase-resistant genotype spread by blaNDM-1 carried plasmid. Attention should be paid to its easily transmissible feature among the strains in clinic. The insertion sequence ISAba125 may be involved in the blaNDM-1 gene mediated carbapenemase-resistant genotype.

Objective To investigate the capsular polysaccharide (CPS) serotypes and molecular characteristics of carbapenem resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP) and study the possible mechanism of carbapenem resistance. Methods A retrospective study was conducted on 18 nonduplicate CR-HMKP strains which were collected from the First Affiliated Hospital of Nanchang University from 2012 to 2016. The clinical data were retrieved from medical records. The capsular serotypes, resistance genes and virulence factors were detected by polymerase chain reaction and DNA sequencing. Antimicrobial susceptibility testing was determined on VITEK 2 compact system. The CR-HMKP strains were characterized molecularly by using PCR, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). Modified carbapenem inactivation method was used to screen carbapenemase-producing strains. Plasmid conjugation transfer experiments were carried out to study transmission of carbapenem resistance. Results Eighteen (2.7％) CR-HMKP isolates were identified, which belonged to 4 serotypes, including wzi128-K1 (n=1), wzi206-K57 (n=1), wzi2-K2 (n=2), and wzi64-K14.64 (n=14). PCR and sequencing analysis identified blaNDM-1 gene in 2 CR-HMKP strains, blaKPC-2 gene in 17 strains, qnrS1 gene in 18 strains, blaCTX-M-3 gene in 3 strains, blaCTX-M-14 gene in 18 strains, blaTEM-1 gene in 16 strains, blaSHV-12 gene in 17 strains, and rmtB in 5 strains. All the 18 CR-HMKP strains carried virulence-associated genes, including rmpA (88.9％, 16/18), magA (5.6％, 1/18), iroN (83.3％, 15/18), aerobactin (27.8％, 5/18), rmpA2 (66.7％, 12/18) and mrkD (100％, 18/18). Three sequence types (STs) were identified by MLST, including ST11 (15 strains), ST86 (2 strains), and ST412 (1 strain). PFGE resulted in three major PFGE clusters, of which cluster A corresponds to ST1 isolates, and cluster B corresponds to ST86 isolates, and cluster C corresponds to ST412 isolates. All the blaKPC-2- positive strains belonged to ST11. Plasmid conjugation was successful in 5 (27.8％) of the 18 CR-HMKP isolates. Conclusions wzi64-K14.64 is the predominant capsule serotype of the CR-HMKP strains in this hospital. KPC-2 gene conjugationmay contribute to the emergence of CR-HMKP isolates. In addition, CRHMKP strain may be the highly prevalent ST11, and highly virulent CPS serotypes harboring K1/K2.

Objective To investigate the antimicrobial resistant mechanisms of high level carbapenem resistant Klebsiella pneumoniae infection of burn patients .Methods A retrospective study was conducted on totally 18 non-repetitive high level CR-KP which were isolated from burn patients hospitalized between July 2014 and June 2015.MIC of antibiotics were determined by using the GN 13 cards and agar dilution method.The specific PCR and DNA sequence analysis were performed to confirm the β-lactamase type.Plasmid conjugation transfer experiments and southem hybridization were applied to study the mode of carbapenem resistance transmission .Outer membrane proteins ( Omps) were isolated and examined by PCR and ( sodium dodecyl sulfate polyacrylamide gel electrophores ) SDS-PAGE.Pulsed-field gel electrophoresis( PFGE ) and Multilocus sequence typing ( MLST ) was used to determine the genotypes . Results Susceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance . The resistance rate to piperacillin/tazobactam, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, tobramycin, cefotetan, ceftazidime, cefepime, aztreonam, imipenem, and meropenem was 100％ (18/18).Moreover, the resistance rate of CR-KP isolates to amikacin was 72.2％ ( 13/18 ) , compound sulfamethoxazole was 61.1％(11/18), tigecycline was 0％(0/18).Conjugation study with Escherictda coli J53 resulted in the transfer of significant reduced carbapenem susceptibility from donors (MICs increased at least 8-fold).By PCR, eighteen strains of Klebsiella pneumoniae carried NDM-1 gene, 5 strains carried KPC-2 gene.The blaNDM-1 was transferable by plasmids.Southern blot hybridization indicated that the blaNDM-1 gene was located on plasmid in size of 46 kb.The plasmid belonged to incompatibility group IncX 3.Seven types of CR-KP were detected by PFGE.In addition, MLST assigned them to sequence type ( ST)11, ST395, ST17, ST37, ST263, ST14 and ST76 types.SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that there was absence of outer membrane proteins OmpK 36 in ST11, ST395, ST37 strains.However, the other STs strains expressed lower quantities of OmpK 36.Conclusions High level carbapenem resistance in K.pneumoniae causing infection in burn patients is attributable to production of plasmid-mediated metallo· β-laetamase NDM-1 combined with porin OmpK36 deficiency or low expression .The K.pneumoniae with NDM-1 and KPC-2 carbapenemase were detected .

OBJECTIVETo study the resistance mechanism and homology of carbapenems-resistant Pseudomonas aeruginosa (PA).

METHODSA total of 812 strains of PA (identified) were isolated from sputum, urine, blood, pus, and drainage of patients with burn, severe pneumonia, diabetes, chronic obstructive pneumonia, myocarditis, liver transplantation, or brainstem hemorrhage hospitalized from January to September 2012. Drug resistance of the 812 strains of PA to 15 antibiotics commonly used in clinic, including piperacillin, imipenem, etc., was tested using the automatic microorganism identifying and drug sensitivity analyzer. Among the carbapenems-resistant PA isolates, synergism test with imipenem-ethylene diamine tetraacetic acid (EDTA) and enhancement test with imipenem-EDTA and ceftazidime-EDTA were used to screen metallo-β-lactamase (MBL)-producing strains; modified Hodge test was used to screen strains producing Klebsiella pneumoniae carbapenemases (KPC); the carbapenemase gene, plasmid mediated quinolone resistant (PMQR) gene, and mobile genetic elements (MGE) were detected by polymerase chain reaction (PCR). In addition, a comparative analysis of the PMQR gene carrying level between the carbapenemase gene positive strains and carbapenemase gene negative strains was carried out. The repetitive consensus sequence of Enterobacteriaceae genome PCR (ERIC-PCR) was carried out for gene typing. Moreover, the source and resistance genes of strains with the same genotype were analyzed. Data were processed with Fisher's exact probability test.

RESULTSThe sensitive rates of the 812 strains of PA to ceftriaxone and trimethoprim-sulfamethoxazole were high, respectively 83.07% and 88.19%, and those of the other antibiotics ranged from 17.30% to 55.18%. Twenty-four carbapenems-resistant PA strains were screened, including 11 MBL-producing strains and 2 KPC-producing strains. Eleven carbapenems-resistant PA strains were found to harbor the blaVIM-2 gene, accounting for 45.83%; 2 carbapenems-resistant PA strains carried the blaKPC-2 gene, accounting for 8.33%. Fourteen carbapenems-resistant PA strains only harbored the PMQR gene acc (6')-Ib-cr, accounting for 58.33%; 3 carbapenems-resistant PA strains (12.50%) harbored the PMQR genes acc (6')-Ib-cr and qnr, including 1 strain with qnr A1 and 2 strains with qnr B4. Ten carbapenems-resistant PA strains carried the MGE gene ISCR1, accounting for 41.67%; 6 carbapenems-resistant PA strains carried the MGE gene ISEcp1, accounting for 25.00%. In addition, 3 carbapenems-resistant PA strains co-harbored the MGE genes ISCR1 and ISEcp1 (accounting for 12.50%), while only 1 carbapenems-resistant PA strain co-harbored the MGE genes class 1 integron and ISEcp1, accounting for 4.17%. Twelve out of the 13 carbapenemase gene positive strains carried one or two PMQR gene (s), which was significantly higher than that of the carbapenemase gene negative strains (with only five strains harboring one PMQR gene, P = 0.023). The 24 carbapenems-resistant PA strains were classified into 6 genotypes by the ERIC-PCR. Thirteen strains (accounting for 54.17%), mainly isolated from pus and blood samples, which were collected from burn department, were in genotype A. Eight out of the 13 strains harbored genes blaVIM-2, acc (6')-Ib-cr, and ISCR1. Five strains (accounting for 20.83%), mainly isolated from sputum samples which were collected from ICU, were in genotype B. Only 2 out of the 5 strains co-harbored the carbapenemase gene, PMQR gene, and MGE gene. There were respectively 2 strains in genotypes C and D, both accounting for 8.33%; the strains in different pattern were isolated from different wards, and they harbored diverse resistance genes. There were respectively 1 strain in genotypes E and F, both accounting for 4.17%.

CONCLUSIONSThe resistance mechanism of PA to carbapenems is mainly mediated by the VIM-2 type MBL in our hospital during 2012, followed by KPC-2 type carbapenemase, and the prevalent genotype is type A. The carbapenemase genes and PMQR genes co-carrying phenomenon exists among these strains of PA, which disseminated by clones.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Carbapenems ; pharmacology ; DNA, Bacterial ; Drug Resistance, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification ; beta-Lactamases ; genetics