Evaluation of Multiplex PCR Techniques for Klebsiella Producing Ampc-Β Lactamases in Clinically Significant Klebsilla Isolates (original) (raw)
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Iranian Red Crescent Medical Journal, 2016
Background: Extended-spectrum β-lactamases (ESBLs) is one of the most important mechanisms of resistance to β-lactams especially among Enterobacteriaceae family including Klebsiella spp. Different types of extended-spectrum β-lactamases including CTX-M-type and PER enzymes are identified among gram negative bacteria. Objectives: The current study aimed to determine the prevalence of CTX-M-type and PER extended-spectrum β-lactamases among Klebsiella spp. isolated from clinical specimens in the teaching hospital of Kashan, Iran. Patients and Methods: One hundred Klebsiella spp. were isolated from clinical specimens of hospitalized patients at Shahid-Beheshti hospital from December 2012 to November 2013. Disk diffusion method was used to determine the susceptibility of these isolates to 14 different antimicrobial agents; disks were purchased from MAST company (United Kingdom). The phenotypic double disk synergy confirmatory test was used to screen the isolates to produce extended-spectrum β-lactamase. DNAs of isolates were extracted using boiling method and PCR assay was used to characterize the bla CTX-M type and bla PER genes. The purified PCR products were sent to Macrogen research company (Korea) for sequencing. Results: Of the total 100 Klebsiella isolates, %93 was susceptible to imipenem. Resistance to ampicillin, ceftazidime, ceftriaxone, aztreonam and cefotaxime was (92%), (67%), (65%), (64%) and (59%), respectively. The phenotypic confirmatory test (PCT) confirmed that 35% (n = 35) of the isolates were ESBL-producing Klebsiella strains. The prevalence of bla CTX-M type and bla RER genes among Klebsiella isolates were 28% (n = 28) and 9% (n = 9), respectively. Conclusions: The prevalence of ESBL-producing Klebsiella strains in Shahid-Beheshti hospital in Kashan has increased. The study concluded that there was a high prevalence of the bla CTX-M type gene among ESBL positive isolates.
Journal of clinical and diagnostic research : JCDR, 2014
AmpC β lactamases are one of the important causes of drug resistance in gram negative bacteria. Failure to detect these enzymes in the laboratory has contributed to therapeutic failures but there are till date no standard guideline available. This study was therefore undertaken to evaluate three phenotypic laboratory tests and the inhibitors used in two of the tests to detect AmpC β lactamases produced by E. coli and Klebsiella species as they are most commonly isolated organisms. E. coli and Klebsiella isolates from different clinical samples were tested for ESBLs production as per CLSI guidelines and excluded from the study. The non-ESBLs isolates were then screened for AmpC β lactamases production, by cefoxitin and then confirmed by three different methods, i.e., Disc Potentiation Test (DPT) , Double Disc Synergy Test (DDST) and Modified Three Dimensional Test (M3DT) which in the absence of molecular methods, was taken as the gold standard. Boronic acid and cloxacillin were used ...
2004
Objective This study was aimed to determine prevalence and resistance pattern like multidrug resistant (MDR) or ESBL nature of E. coli and Klebsiella spp from various sewage drain samples with an idea to deliver baseline information that could be utilized for defining guidelines for the treatment of hospital sewages. Results Of 10 sewage samples analyzed, 7 (70%) contained E. coli while 6 (60%) contained Klebsiella. Except one sample, all positive samples contained both E. coli and Klebsiella spp. E. coli isolates were resistant to ampicillin, amoxicillin, cefoxitin, cefuroxime, and cefpodoxime; while 85.7% were resistant to amoxicillin/clavulanate, ceftazidime, cefotaxime and ceftriaxone. 71.4%, 57.1%, 42.9%, and 28.6% were resistant to aztreonam, trimethoprim/sulfamethoxazole, nitrofurantoin, and gentamicin. Most were sensitive to chloramphenicol, ofloxacin, ciprofloxacin, and azithromycin. 85.7% and 57.1% of E. coli were MDR and ESBL isolates respectively. Klebsiella were resistant to ampicillin, amoxicillin, and amoxicillin/clavulanate. 83.4% of Klebsiella were resistant to cefoxitin. 66.7% of strains were resistant to cefuroxime, ceftazidime, cefotaxime, ceftriaxone, and cefpodoxime. Klebsiella showed 50% resistant to aztreonam and trimethoprim/sulfamethoxazole, while 33.3% were resistant to chloramphenicol, nitrofurantoin, ofloxacin, and ciprofloxacin. Klebsiella were sensitive to azithromycin and gentamicin. 66.7% and 33.3% of Klebsiella were MDR and ESBL isolates respectively.
The Indian journal of medical research, 2007
Extended-spectrum beta-lactamases (ESBLs) are rapidly evolving group of beta-lactamase enzymes produced by the Gram negative bacteria. These enzymes have been derived from TEM and SHV genes by mutations and have been well described in Klebsiella pneumoniae. Information on molecular types of ESBL positive Klebsiella sp. is lacking from India. We therefore undertook this study to look for the TEM and SHV genes in ESBL positive Klebsiella sp. isolated from the patients admitted to a tertiary care hospital in north India. A total of 204 multidrug-resistant isolates of Klebsiellae obtained from clinical samples; blood (n=108), urine (n=15), pus (n=2) and sputum (n=79) were obtained and screened for resistance to 3rd generation cephalosporins (3GC). The ESBL status was determined by double disk diffusion test (DDDT) and further by ESBL E-test. Multiplex PCR specific for TEM and SHV genes was performed to distinguish four different genotypes: TEM-positive, SHV-positive, TEM- and SHV-positi...
Diagnostic Microbiology and Infectious Disease, 2000
Escherichia coli and Klebsiella spp. were screened for ESBL based on routine susceptibility testing results. Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporins or aztreonam were reported as probable ESBL producers. By using the NCCLS proposed ESBL confirmatory method, we tested 61 screen-positive isolates from 42 patients, 30 randomly selected susceptible isolates, and 12 isolates with previously characterized -lactamases. Ceftazidime contributed to 97% of screen-positive isolates, whereas aztreonam added a single patient isolate. An ESBL was confirmed in 86% of K. pneumoniae, 100% of K. oxytoca, and 20% of E. coli screen-positive single patient isolates. None of the susceptible isolates were shown to produce ESBL. Based on these findings a comment regarding the presence of ESBL seems sufficient for Klebsiella spp. but confirmatory testing is indicated for E. coli. There was 85% agreement between the type of -lactamase and the result of the ESBL confirmatory test. When a cefotaxime MIC Ͼ 0.25 g/mL was used to indicate the presence of ESBL, the specificity of the assay increased to 100%. The NCCLS ESBL phenotypic confirmatory method was reproducible and accurate enough to be used in the clinical laboratory.
RADS Journal of Biological Research & Applied Sciences
Multi drug resistance has now become a worldwide therapeutic challenge due to the widespread use of broad spectrum antibiotics. Klebsiella species have significant importance in clinical field as they cause various infections in human and are considered as potential pathogens that express antibiotic resistance through their strong enzymatic activity. Extended spectrum beta lactamases (ESBLs) are plasmid mediated enzymes produced mostly because of mutation and few other factors. These enzymes confer resistance against various β-lactam drugs including cephalosporins and monobactams. Among the genus Klebsiella, ESBLs are highly prevalent in K. pneumoniae followed by K. oxytoca. This study was conducted in Pakistan to assess the distribution of ESBL producers among Klebsiella spp., an important member of the family Enterobacteriaceae. From January 2010 to January 2012, a total of 236 gram-negative isolates were collected from different renowned microbiological laboratories. Out of the...
Acta Tropica, 2005
Extended Spectrum -Lactamases (ESBLs) producer and multidrug resistant Klebsiella spp. are becoming a major nosocomial pathogen globally. There are no documented reports yet on the occurrence of ESBL enzymes in Klebsiella spp. species from Ethiopia. This study was undertaken to isolate and determine the occurrence of ESBLs and multi-drug resistant Klebsiella spp. in different clinical samples obtained from patients. A cross-sectional survey was conducted in four different hospitals of Harar region (Hiwot Fana, Misrak-Arbegnoch, Police and Army) from December 2003 to February 2004. Three hundred and eighty four clinical specimens (202 sputum, 164 urine and 18 pus) were collected from patients admitted in different wards. Antimicrobial susceptibilities were performed on 57 clinical isolates by standard disk diffusion procedures against eight antimicrobial agents. The ESBLs detection was made by using cefotaxime and ceftazidime alone and in combination with clavulanate. A total of 57 (15%) Klebsiella spp. were isolated from 384 patients. Of the 57 isolates, 33 (58%) were from sputum, 18 (31.5%) from urine and 6 (10.5%) from pus. Of the 57 Klebsiella spp., 54 (94.7%) were identified as K. pneumoniae and 3 (5.3%) as K. oxytoca. Resistance was found against cephalosporins [cefotaxime (39.0%), cefoxitin (39.0%), ceftazidime (40.0%), ceftriaxone (40.0%), cephalothin (42.0%)], chloramphenicol (70.0%), gentamicin (61.0%) and trimethoprim-sulphamethoxazole (65.0%). Analyzed Klebsiella isolates were characterized also by a high degree of multi-resistance (67.0%). In 19/57 (33.3%) of the Klebsiella isolates, ESBL production was detected. Rates of detection of ESBL producers were 42.1, 26.3, 26.3 and 5.3% in Hiwot-Fana, Misrak-Arbegnoch, Police and Army hospitals, respectively. Multi-drug resistant isolates were more prevalent among the ESBLs producers (95.0%) than non-producers (53.0%) (p = 0.24). In conclusion, our results show that awareness of ESBL production by Klebsiella spp. is clinically important. In the absence of infection control measures, ESBL producing organisms readily pass horizontally from patient to patient. These strains also transiently colonize the hands of hospital staff members, thereby facilitating patient-to-patient transmission of the organism.
Background: The resistance to antimicrobials has become a serious global health concern with negative consequences on treatment strategies and increasing health-care costs. The extended spectrum beta lactamases producing bacteria stands outs as bacteria of great concern among Gram negative bacilli. Lack of capacity to effectively diagnose these organisms in developing countries result in sub-optimal treatment. We compared two phenotypic methods for the confirmatory testing of ESBL in Klebsiella pneumoniae to identify an easy and efficient method for their laboratory diagnosis Materials and Methods: We screened all patients admitted into various units of University of Maiduguri Teaching Hospital between from the 01/01/2014 to 31/06/2014 to isolate Klebsiella pneumoniae. All confirmed isolates were screened for ESBL enzyme using CLSI breakpoints. Suspected ESBLs producers were subjected to confirmation using two phenotypic methods. The double disk synergy method (with ceftazidime; 30 µg, cefotaxime;30 µg, and amoxicillin;20 µg, plus clavulanate;10µg,:[augmentin;30 µg].) and Etest method for MIC determination (using cefotaxime and cefotaxime + amoxicillin-clavulanic acid). Multiplex polymerase chain reaction (PCR) method was considered as a gold standard for confirmation. We compared the two methods with the gold standard to determine their sensitivity, specificity and predictive value positive. Results: We detected 178 isolates of Klebsiella pneumoniae among of hospitalized patients. The DDST method revealed 59 out of the 178 isolates resistant with a sensitivity of 100%, specificity of 97%, positive predictive value of 93% and negative predictive value of 100%. Using the Etest MIC, 56 resistant isolates were identified with a sensitivity of 100%, specificity of 99%, positive predictive value of 98% and negative predictive value of 100%. Only 55resistant organisms were found based on the multiplex Polymerase chain reaction method.