ABSTRACT
ObjectiveThe nucleic acid technology for detecting drug-resistant genes has become one of the powerful tools for monitoring and controlling the spreading of drug-resistant bacteria. This study was to establish a method for rapid detection of the drug-resistant genes KPC and NDM and provide some guidance in clinical drug use and monitoring the prevalence of drug-resistant bacteria in the hospital.MethodsAccording to the conserved regions of Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM), we designed the primers of duplex PCR, optimized the amplification system and established a method for simultaneous detection of the drug-resistant genes KPC and NDM. Then, we analyzed the sensitivity and specificity of the method and applied it to the detection of Pseudomonas aeruginosa and Klebsiella pneumoniae.ResultsThe sequences of KPC and NDM exhibited a 100% consistency with those of the original ones. Target fragments of the desired size of 151 bp were detected in the KPC-2 positive standard and Klebsiella pneumoniae ATCC BAA 1705 standard strains, and those of the desired size of 261 bp were observed in the NDM-2 positive standard strain and NDM-positive pneumococcal bacteria, neither with non-specific amplification. Sequencing of the PCR products showed a 100% consistency between the sequences of the products and those of the drug-resistant genes KPC-2 and NDM-1. The detectable limits of KPC and NDM for duplex PCR were 7×102 and 5×102 copies per reaction respectively. Drug-resistant genes were detected in 12 (92.3%) of the 13 carbapenems-resistant strains, including 10 KPC-positive (83.3%) and 2 NDM positive ones (16.7%), but neither KPC nor NDM in the other 10 carbapenems-sensitive strains. In the 13 strains of Pseudomonas aeruginosa, KPC was detected in 2 (33.3%) of the 6 carbapenems-resistant ones, but neither KPC nor NDM in the other 7.ConclusionThe duplex PCR method can be used for rapid and effective detection of the drug-resistance genes KPC and NDM, with the advantages of high sensitivity and specificity, and is therefore of great significance for guiding clinical drug use and monitoring the spreading of carbapenems-resistant bacteria in the hospital.
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Objective To observe the T cells immune response to Enolase (Eno),an immunodominant antigen of Candida albicans.Methods Determined the frequencies of positive spot-forming cells (SFCs) of Eno antigen-specific T cells secreting IFN-γ,IL-4 and IL-17A in the PBMCs of 25 healthy individuals by ELISPOT assay.Results After Eno stimulation,the SFCs of IFN-γ,IL-4 and IL 17A in 25 healthy persons were 14.00(8.50,39.00),0(0,0) and 2(1,4.50),respectively.Either the SFCs of IFN-γ or those of IL-17A were significantly higher than those of IL-4 (P＜0.05).The difference between SFCs of IFN-γ and those of IL-17A was also significant (P=0).The response rates of IFN-γ,IL-4 and IL-17A were 100％ (25/25),4.00％ (1/25) and 88.00％ (22/25),respectively.The difference between either IFN-γor IL 17A and IL-4 was significant (values all P＜0.05).Eno induced strong response (SCFs≥20) for IFN-γ in 10 healthy individuals (40.00％,10/25),but failed to induce strong response for IL-17A and IL-4 in all the volunteers.Major healthy individuals (84.00％,21/25)showed both Th1 and Th17 cells response against Eno,12.00％ (3/25) showing Th1 cells response in isolation,and none showed Th2 or Th17 cells response individually.Conclusion Eno of Candida albicans could induce immunodominant responses of Th1 and Th17 cells,which was considered to provide protection to IC.Eno might be a potential protective vaccine against IC.
ABSTRACT
<p><b>OBJECTIVE</b>To study the genotype distribution of extended-spectrum p-lactamases (ESBLs) and AmpC p-lactamases produced in E. coli isolated from men with urinary infection in Nanjing.</p><p><b>METHODS</b>Organisms of clinical infection were identified by automatic microbial system (Vitek-32). ESBLs were detected by disk diffusion confirmatory test, and ESBLs and AmpC p-lactamases by three-dimensional extract test (TDET) , the presence of plasmid-mediated ESBLs and ampC genes determined by PCR, and conjugal transfer assays of the ampC resistance determinants carried out by a broth mating procedure.</p><p><b>RESULTS</b>ESBLs were produced in 24. 6% (46/187) of the E. coli and the 46 E. coli isolates showed p-lactamase activity in TDET, 3 positive for both ESBLs and AmpC beta-lactamases and 43 for ESBLs only. Forty-four of the 46 isolates were shown by PCR to contain at least one of the genes blaTEM, blaOXA, bla(CTX-M), but no blaSHA. AmpC specific amplication products were observed in 3 of the 46 isolates, of which 2 were of CIT type, and 1 of DHA type. All of the 3 transconjugants transferred the plasmids harbouring ampC genes to recipients.</p><p><b>CONCLUSION</b>CTX-M is the most common genotype in plasmid-mediated ESBLs produced by E. coli isolated from men with urinary infection in Nanjing. Present findings indicate that AmpC-producing E. coli are present in this hospital, and ampC-encoding plasmids are transferable.</p>