Dataset Information

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

ABSTRACT: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE).116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes.Among the carbapenem-resistant isolates the genes bla KPC, bla VIM, bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC, bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials.The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

SUBMITTER: Smiljanic M

PROVIDER: S-EPMC5504714 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Plazomicin and comparator agents were tested by using the CLSI reference broth microdilution method against 4,825 clinical isolates collected during 2014 and 2015 in 70 U.S. hospitals as part of the ALERT (Antimicrobial Longitudinal Evaluation and Resistance Trends) program. Plazomicin (MIC50/MIC90, 0.5/2 ?g/ml) inhibited 99.2% of 4,362 Enterobacteriaceae at ?4 ?g/ml. Amikacin, gentamicin, and tobramycin inhibited 98.9%, 90.3%, and 90.3% of these isolates, respectively, by applying CLSI breakpoints. The activities of plazomicin were similar among Enterobacteriaceae species, with MIC50 values ranging from 0.25 to 1 ?g/ml, with the exception of Proteus mirabilis and indole-positive Proteeae that displayed MIC50 values of 2 ?g/ml. For 97 carbapenem-resistant Enterobacteriaceae (CRE), which included 87 isolates carrying blaKPC, plazomicin inhibited all but 1 isolate at ?2 ?g/ml (99.0% and 98.9%, respectively). Amikacin and gentamicin inhibited 64.9% and 56.7% of the CRE isolates at the respective CLSI breakpoints. Plazomicin inhibited 96.5 and 95.5% of the gentamicin-resistant isolates, 96.9 and 96.5% of the tobramycin-resistant isolates, and 64.3 and 90.0% of the amikacin-resistant isolates according to CLSI and EUCAST breakpoints, respectively. The activities of plazomicin against Pseudomonas aeruginosa (MIC50/MIC90, 4/16 ?g/ml) and Acinetobacter species (MIC50/MIC90, 2/16 ?g/ml) isolates were similar. Plazomicin was active against coagulase-negative staphylococci (MIC50/MIC90, 0.12/0.5 ?g/ml) and Staphylococcus aureus (MIC50/MIC90, 0.5/0.5 ?g/ml) but had limited activity against Enterococcus spp. (MIC50/MIC90, 16/64 ?g/ml) and Streptococcus pneumoniae (MIC50/MIC90, 32/64 ?g/ml). Plazomicin activity against the Enterobacteriaceae tested, including CRE and isolates carrying blaKPC from U.S. hospitals, supports the development plan for plazomicin to treat serious infections caused by resistant Enterobacteriaceae in patients with limited treatment options.

Project description:The development of antibiotic resistance is a major public health issue, as infections are increasingly unresponsive to antibiotics. Emerging antimicrobial resistance has raised researchers' interest in the development of alternative strategies using natural compounds with antibacterial activity, like honey, which has emerged as an agent to treat several infections and wound injuries. Nevertheless, the antibacterial effect of honey was mostly evaluated against Gram-positive bacteria. Hence, the objective of our study was to evaluate the antibacterial activity, as well as the physicochemical parameters, of genuine Greek honeys against multidrug-resistant Gram-negative pathogenic bacteria. In this vein, we aimed to study the in vitro antibacterial potential of rare Greek honeys against Verona integron-encoded metallo-β-lactamase (VIM)- or Klebsiella pneumoniae carbapenemase-producing multidrug-resistant Gram-negative pathogens. Physicochemical parameters such as pH, hydrogen peroxide, free acidity, lactonic acid, total phenols total flavonoids, free radical scavenging activities, tyrosinase enzyme inhibitory activity and kojic acid were examined. Moreover, the antimicrobial activity of 10 different honey types was evaluated in five consecutive dilutions (75%, 50%, 25%, 12.5% and 6.25%) against the clinical isolates by the well diffusion method, as well as by the determination of the minimum inhibition concentration after the addition of catalase and protease. Almost all the physicochemical parameters varied significantly among the different honeys. Fir and manuka honey showed the highest values in pH and H2O2, while the free acidity and lactonic acid levels were higher in chestnut honey. Total phenols, total flavonoids and free radical scavenging activities were found higher in cotton, arbutus and manuka honey, and finally, manuka and oregano honeys showed higher tyrosinase inhibition activity and kojic acid levels. The antimicrobial susceptibility depended on the type of honey, on its dilution, on the treatment methodology and on the microorganism. Arbutus honey was the most potent against VIM-producing Enterobacter cloacae subsp. dissolvens in 75% concentration, while fir honey was more lethal for the same microorganism in the 25% concentration. Many honeys outperformed manuka honey in their antibacterial potency. It is of interest that, for any given concentration in the well diffusion method and for any given type of honey, significant differences were not detected among the four multidrug-resistant pathogens, which explains that the damaging effect to the bacterial cells was the same regardless of the bacterial species or strain. Although the antimicrobial potency of different honey varieties dependents on their geographical origin and on their compositional differences, the exact underlying mechanism remains yet unclear.

Dataset Information

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets