Evaluation of Six Phenotypic Methods for the Detection of Carbapenemases in Gram-Negative Bacteria With Characterized Resistance Mechanisms

BACKGROUND: We compared the performance of the modified Hodge test (MHT), Triton Hodge test (THT), Carba NP test (CNPt), simplified Carba NP test (CNPt-direct), blue-Carba NP test (BCT), and carbapenem inactivation method (CIM) for rapid and accurate carbapenemase detection. METHODS: The methods were evaluated by using 256 gram-negative isolates, including 197 Enterobacteriaceae (79 Enterobacter spp., 74 Klebsiella spp., 33 Escherichia coli, 10 Citrobacter spp., and 1 Serratia marcescens), 51 Acinetobacter baumannii, and 8 Pseudomonas aeruginosa strains. The collection included 117 non-carbapenemase, 18 Klebsiella pneumoniae carbapenemases (KPC) producers, 46 New Delhi metallo-β-lactamases (NDM) producers, 11 imipenemases (IMP) producers, and 51 oxacillinases (OXA) producers, and 13 strains harboring two different carbapenemase genes. RESULTS: The specificity of the THT (91.5%) was significantly lower than other methods, each of which had 100% specificity (P0.999). Because of improved detection of NDM carriers, THT showed significantly higher sensitivity than the MHT (84.9% vs 75.5%, P<0.001). However, poor performances in detecting OXA still influenced the sensitivities of the CNPt (66.2%) and BCT (82.0%), as well as the MHT and THT. CONCLUSIONS: CNPt-direct and CIM demonstrated the best performance for the efficient detection of carbapenemase among the six evaluated methods. Except the MHT and THT, the detection of carbapenemase-producing Enterobacteriaceae by all the other methods was acceptable, when the OXA-type carbapenemase was not prevalent.

Multidrug Resistance Mechanisms of Carbapenem Resistant Klebsiella pneumoniae Strains Isolated in Chongqing, China

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered a serious global threat. However, little is known regarding the multidrug resistance (MDR) mechanisms of CRKP. This study investigated the phenotypes and MDR mechanisms of CRKP and identified their clonal characteristics. METHODS: PCR and sequencing were utilized to identify antibiotic resistance determinants. Integron gene cassette arrays were determined by restriction fragment length polymorphism (RFLP) analysis. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. Plasmids were typed by using a PCR-based replicon typing and analyzed by conjugation and transformation assays. RESULTS: Seventy-eight strains were identified as resistant to at least one carbapenem; these CRKP strains had a high prevalence rate (38.5%, 30/78) of carbapenemase producers. Additionally, most isolates harbored MDR genes, including Extended spectrum β-lactamases (ESBLs), AmpC, and quinolone and aminoglycoside resistance genes. Loss of porin genes was observed, and Class 1 integron was detected in 66.7% of the investigated isolates. PFGE and MLST results excluded the occurrence of clonal dissemination among these isolates. CONCLUSIONS: A high prevalence of NDM-1 genes encoding carbapenem resistance determinants was demonstrated among the K. pneumoniae isolates. Importantly, this is the first report of bla(NDM-1) carriage in a K. pneumoniae ST1383 clone in China and of a MDR CRKP isolate co-harboring bla(NDM-1), bla(KPC-2), bla(CTX-M), bla(SHV), acc(6′)-Ib, rmtB, qnrB, and acc(6′)-Ib-cr.

The molecular epidemiology and infections risk factors of clinical linezolid-resistant Enterococci isolates / 中华检验医学杂志

Objective To investigate the molecular epidemiology and infectious risk factors of linezolid-resistant Enterococci (LRE) isolates in the First Affiliated Hospital of Chongqing Medical University.Methods Thirteen LRE isolates were collected from 2011 to 2013 and confirmed by broth dilution susceptibility testing.The minimum inhibitory concentrations (MIC) of twelve antimicrobial agents were analyzed using Vitek 2 compact.The molecular epidemiology of LRE isolates was determined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and the Diversilab.A casecontrol study was conducted for the analysis of risk factors, and Logistic regression was performed to evaluate the independent risk factors.Results All thirteen LRE isolates showed low-level resistance to linezolid, and most of these isolates were resistant to tetracycline, erythromycin and ciprofloxacin.However, they had high sensitivity to penicillin, ampicillin and tigecycline.Sequence type 480 was predominant in the hospital, and three isolates (isolates 3, 4, and 5) from July to September 2013 were found to have the same ST, PFGE pattern and rep-PCR group, indicating the same resistance clone.Admission to intensive care unit (ICU), peripheral vascular disease, males, hypoalbuminaemia, enema and linezolid therapy were identified as significant risk factors for LRE infections.Among these factors, admission to ICU, enema and linezolid therapy were independent risk factors for the acquisition of LRE.Conclusions Thirteen LRE isolates collected in the hospital showed a multidrug-resistant phenotype, and a small-scale prevalence was detected from 2011 to 2013.Therefore, attention should be paid to monitor the LRE in the hospital to decrease the prevalence of LRE infections.

Study on the culture and the function of dendritic cells of patients with laryngeal squamous cell carcinoma in vitro / 中国耳鼻咽喉头颈外科

OBJECTIVE To culture dendritic cells from the peripheral blood ofpatients with laryngeal squamous cell carcinoma. METHODS Peripheral blood was collected from patients under sterile conditions. The mononuclear cells were separated by centrifugation through a density gradient (Ficoll-Hypaque). The adhesion cells, collected from the mononuclear cells, were cultured with interleukin-4 (IL-4, 100ng),granulocyte/macrophage colony stimulating factor (GM-CSF, 100ng) and tumor necrosis factor ? (TNF-?, 100ng) in RPMI 1640 medium for 7 days. The cell morphology was observed under electronic microscope, and the characteristic surface marker CD80 of the cells was analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the mixed lymphocytes. The mixed lymphocytes were stimulated by DC loaded with laryngeal carcinoma antigen. RESULTS A large quantity of cells characteristic of dendritic cell was obtained. Cytometry analysis showed that 33.76 % of mononuclear cells expressed the characteristic marker CD80. After being cultured for 7 days, the expression level of CD80 was significantly increased (69.15 %). Few DC with laryngeal carcinoma antigen could play a role in stimulating lymphocyte proliferation, and the proliferation effect was optimized when the ratio between DC and T cells was 1:5. CONCLUSION A large number of DCs with high purityand sufficient treatment dose can be cultured in vitro from the peripheral blood of patients with laryngeal carcinoma. This technique may play an important role in experimental studies for clinical applications.