Evaluation of PCR-Reverse Blot Hybridization Assay, REBA Sepsis-ID Test, for Simultaneous Identification of Bacterial Pathogens and mecA and van Genes from Blood Culture Bottles

BACKGROUND: The aim of this study was to evaluate a newly developed PCR-based reverse blot hybridization assay (PCR-REBA), REBA Sepsis-ID (M&D, Wonju, Korea), to rapidly detect the presence of bacteremia and antimicrobial resistance gene in blood culture samples. METHODS: One thousand four hundred consecutive blood culture samples from patients with a delta neutrophil index greater than 2.7% were selected from March to July in 2013. Three hundred positive and 1,100 negative for bacterial growth in blood culture bottles samples were tested by conventional and real-time PCR-REBA, respectively. RESULTS: The overall agreement between the conventional identification test and the REBA Sepsis-ID test was 95.3% (286/300). Agreement for gram-positive bacteria, gram-negative bacteria, fungi, and polymicrobials was 94.5% (190/201), 97.3% (71/73), 100% (14/14), and 91.7% (11/12), respectively. The detection rate of the mecA gene from methicillin-resistant Staphylococcus isolates was 97.8% (90/92). The vanA gene was detected in one blood culture sample from which vancomycin-resistant Enterococcus was isolated. When the cycle threshold for real-time PCR was defined as 30.0, 2.4% (26/1,100) of negative blood culture samples tested positive by real-time PCR. CONCLUSIONS: The REBA Sepsis-ID test is capable of simultaneously and quickly detecting both causative agents and antimicrobial resistance genes, such as mecA and van, in blood culture positive samples.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Analysis of Gene Mutations Associated with Antibiotic Resistance in Helicobacter pylori Strains Isolated from Korean Patients

BACKGROUND/AIMS: This study aims to identify the gene mutation pattern associated with antibiotic resistance for mainly used antibiotics in Helicobacter pylori strains isolated from Koreans. MATERIALS AND METHODS: Seventy-one H. pylori strains were isolated from gastric mucosal biopsy specimens. The specimens were cultivated and the resistance to 5 antibiotics were assessed by using agar gel dilution method. DNA sequencing was carried out to detect the resistance-related gene mutations. RESULTS: A point mutation at A2143G of 23S rRNA was observed in all of the clarithromycin resistant strains, but tetracycline resistant strains were not found. Substitution N562Y in penicillin binding protein 1 were observed in an amoxicillin resistant strain (minimum inhibitory concentration [MIC] 2.0microg/mL). Eleven (57.8%) out of 19 levofloxacin resistant strains showed amino acid substitution at N87K (8 strains), N87I, A88V and D91N in GyrA. The truncation in rdxA was detected in 8 (25.0%) out of 32 metronidazole resistant strains. Two out of the 7 patients who failed in first-line treatment of clarithromycin and amoxicillin showed A2143G mutation. CONCLUSIONS: 23S rRNA mutation is closely related to the failure of eradication, however, the fact that five people who have no gene mutation failed eradication implies that other factors are related. As MIC levels in clarithromycin and levofloxacin resistance strains are getting higher, their appropriate gene mutation is more correlated.

Antibiotic Resistance in Helicobacter pylori Strains and its Effect on H. pylori Eradication Rates in a Single Center in Korea

BACKGROUND: Clarithromycin, amoxicillin, metronidazole, tetracycline, and levofloxacin have been commonly used for the eradication of Helicobacter pylori. We compared the change in antibiotic resistance of H. pylori strains during two separate periods and investigated the effect of antibiotic resistance on H. pylori eradication. METHODS: H. pylori strains were isolated from 71 patients between 2009 and 2010 and from 94 patients between 2011 and 2012. The distribution of minimal inhibitory concentration (MIC) of 5 antibiotics was assessed using the agar dilution method, and H. pylori eradication based on the antimicrobial susceptibility of the isolates was investigated retrospectively. RESULTS: Antibiotic resistance rate against clarithromycin, amoxicillin, tetracycline, metronidazole, and levofloxacin for the 2009-2010 isolates were 7.0% (5/71), 2.8% (2/71), 0% (0/71), 45.1% (32/71), and 26.8% (19/71), respectively, and for the 2011-2012 isolates were 16.0% (15/94), 2.1% (2/94), 0% (0/94), 56.3% (53/94), and 22.3% (21/94), respectively. Multi-drug resistance for 2 or more antibiotics increased slightly from 16.9% (12/71) in the 2009-2010 isolates to 23.4% (22/94) in the 2011-2012 isolates. In follow-up testing of 66 patients, first-line treatment successfully eradicated H. pylori in 50 patients (75.8%) and failed in 4 of 7 patients (57.1%) in a clarithromycin-resistant and amoxicillin-susceptible group. CONCLUSIONS: We observed an increase in resistance to clarithromycin and an overall increase in multi-drug resistance during the 2 study periods. The effectiveness of the eradication regimen was low with combinations of clarithromycin and amoxicillin, particularly in the clarithromycin-resistant group. Thus, eradication of H. pylori depends upon periodic monitoring of antimicrobial susceptibility.