Direct detection of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles by multiplex-touchdown PCR assay. - Related Documents




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148901.0000Direct detection of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles by multiplex-touchdown PCR assay. Methicillin-resistant staphylococci (MRS) and ESBL(Extended-Spectrum β-Lactamase)-producing bacteria are the most important resistant pathogens in sepsis. In this study, a new multiplex-touchdown PCR method (MT-PCR) was developed to detect rapidly and simultaneously the presence of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles. The technique showed a sensitivity of 10(3 ) CFU ml(-1) for mecA detection and of 10(2)  CFU ml(-1) for other genes, and 100% specificity in the detection of all genes. All genes were detected in the spiked blood culture bottles artificially contaminated with reference strains. Three methicillin-resistant S. aureus (MRSA), two methicillin-resistant S. epidermidis (MRSE) and 32 ESBL-producing bacteria, were isolated from the clinical blood culture specimens in 48 h by standard microbiological procedures. The corresponding genes were detected directly in the three MRSA, two MRSE and 29 ESBL-producing bacteria from the clinical blood culture specimens in 4 h by MT-PCR assay. None of the bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes were detected in three other bottles with ESBL-producing bacteria because of other ESBL genotypes in the pathogens. Likewise, all bottles proven negative by culture remained negative by PCR. The proposed method was rapid, sensitive and specific, and was able to directly detect the genes of MRS and ESBL-producing bacteria from the blood culture bottles. SIGNIFICANCE AND IMPACT OF THE STUDY: Many studies on the development of PCR for the detection of resistance genes have already been published, including multiplex PCR methods. However, cross-amplification reactions can be a major concern in multiplex PCR methods. In this study, we developed a highly sensitive and specific multiplex-touchdown PCR assay for simultaneous detection of mecA, bla(SHV) , bla(CTX)(-M) , bla(TEM) and bla(OXA) genes from positive blood culture bottles, cross-amplification was absent and false-positive results were not obtained.201727699804
222010.9998Rapid detection and molecular survey of blaVIM, blaIMP and blaNDM genes among clinical isolates of Acinetobacter baumannii using new multiplex real-time PCR and melting curve analysis. BACKGROUND: Acinetobacter baumannii is a cosmopolitan bacterium that is frequently reported from hospitalized patients, especially those patients who admitted in the intensive care unit. Recently, multiplex real-time PCR has been introduced for rapid detection of the resistance genes in clinical isolates of bacteria. The current study aimed to develop and evaluate multiplex real-time PCR to detect common resistance genes among clinical isolates of A. baumannii. RESULTS: Multiplex real-time PCR based on melting curve analysis showed different T(m) corresponding to the amplified fragment consisted of 83.5 °C, 93.3 °C and 89.3 °C for blaIMP, blaVIM and blaNDM, respectively. Results of multiplex real-time PCR showed that the prevalence of blaIMP, blaVIM and blaNDM among the clinical isolates of A. baumannii were 5/128(3.9%), 9/128(7.03%) and 0/128(0%), respectively. Multiplex real-time PCR was able to simultaneously identify the resistance genes, while showed 100% concordance with the results of conventional PCR. CONCLUSIONS: The current study showed that blaVIM, was the most prevalent MBL gene among the clinical isolates of A. baumannii while no amplification of blaNDM was seen. Multiplex real-time PCR can be sensitive and reliable technique for rapid detection of resistance genes in clinical isolates.201931182026
222320.9998Evaluation of a new real-time PCR assay (Check-Direct CPE) for rapid detection of KPC, OXA-48, VIM, and NDM carbapenemases using spiked rectal swabs. To prevent the spread of carbapenemase-producing bacteria, a fast and accurate detection of patients carrying these bacteria is extremely important. The Check-Direct CPE assay (Check-Points, Wageningen, The Netherlands) is a new multiplex real-time PCR assay, which has been developed to detect and differentiate between the most prevalent carbapenemase genes encountered in Enterobacteriaceae (blaKPC, blaOXA-48, blaVIM, and blaNDM) directly from rectal swabs. Evaluation of this assay using 83 non-duplicate isolates demonstrated 100% sensitivity and specificity and the correct identification of the carbapenemase gene(s) present in all carbapenemase-producing isolates. Moreover, the limit of detection (LoD) of the real-time PCR assay in spiked rectal swabs was determined and showed comparable LoDs with the ChromID CARBA agar. With an excellent performance on clinical isolates and spiked rectal swabs, this assay appeared to be an accurate and rapid method to detect blaKPC, blaOXA-48, blaVIM, and blaNDM genes directly from a rectal screening swab.201324135412
144530.9998Rapid Detection of Beta-Lactamases Genes among Enterobacterales in Urine Samples by Using Real-Time PCR. The objective of this study was to develop and evaluate newly improved, rapid, and reliable strategies based on real-time PCR to detect the most frequent beta-lactamase genes recorded in clinical Enterobacterales strains, particularly in Tunisia (bla(SHV12) , bla(TEM) , bla(CTX-M-15) , bla(CTX-M-9) , bla(CMY-2) , bla(OXA-48) , bla(NDM-1) , and bla(IMP) ) directly from the urine. Following the design of primers for a specific gene pool and their validation, a series of real-time PCR reactions were performed to detect these genes in 78 urine samples showing high antibiotic resistance after culture and susceptibility testing. Assays were applied to DNA extracted from cultured bacteria and collected urine. qPCR results were compared for phenotypic sensitivity. qPCR results were similar regardless of whether cultures or urine were collected, with 100% sensitivity and specificity. Out of 78 multiresistant uropathogenic, strains of Enterobacterales (44 E. coli and 34 K. pneumoniae strains) show the presence of the genes of the bla group. In all, 44% E. coli and 36 of K. pneumoniae clinical strains harbored the bla group genes with 36.4%, 52.3%, 70.5%, 68.2%, 18.2%, and 4.5% of E. coli having bla(SHV-12) , bla(TEM) , bla(CTX-M 15) , bla(CTX-M-9) , bla(CMY-2) , and bla(OXA-48) group genes, respectively, whereas 52.9%, 67.6%, 76.5%, 35.5%, 61.8, 14.7, and 1.28% of K. pneumoniae had bla(SHV-12) , bla(TEM) , bla(CTX-M 15) , bla(CTX-M-9) , bla(CMY-2) , bla(OXA-48) , and bla(NDM-1) group genes, respectively. The time required to have a result was 3 hours by real-time PCR and 2 to 3 days by the conventional method. Resistance genes of Gram-negative bacteria in urine, as well as cultured bacteria, were rapidly detected using qPCR techniques. These techniques will be used as rapid and cost-effective methods in the laboratory. Therefore, this test could be a good candidate to create real-time PCR kits for the detection of resistance genes directly from urine in clinical or epidemiological settings.202235978630
149040.9998Rapid detection of Gram-negative bacteria and their drug resistance genes from positive blood cultures using an automated microarray assay. We evaluated the performance of the Verigene Gram-negative blood culture (BC-GN) assay (CE-IVD version) for identification of Gram-negative (GN) bacteria and detection of resistance genes. A total of 163 GN organisms (72 characterized strains and 91 clinical isolates from 86 patients) were tested; among the clinical isolates, 86 (94.5%) isolates were included in the BC-GN panel. For identification, the agreement was 98.6% (146/148, 95% confidence interval [CI], 92.1-100) and 70% (7/10, 95% CI, 53.5-100) for monomicrobial and polymicrobial cultures, respectively. Of the 48 resistance genes harbored by 43 characterized strains, all were correctly detected. Of the 19 clinical isolates harboring resistance genes, 1 CTX-M-producing Escherichia coli isolated in polymicrobial culture was not detected. Overall, BC-GN assay provides acceptable accuracy for rapid identification of Gram-negative bacteria and detection of resistance genes, compared with routine laboratory methods despite that it has limitations in the number of genus/species and resistance gene included in the panel and it shows lower sensitivity in polymicrobial cultures.201525591999
221950.9998Development and validation of a multiplex TaqMan real-time PCR for rapid detection of genes encoding four types of class D carbapenemase in Acinetobacter baumannii. A multiplex TaqMan real-time PCR to detect carbapenem-hydrolysing class D β-lactamases (bla(OXA-23)-like, bla(OXA-24/40)-like, bla(OXA-51)-like and bla(OXA-58)-like genes) was developed and evaluated for early detection of imipenem (IMP) resistance in clinically significant Acinetobacter baumannii isolates. Well-characterized strains of A. baumannii were used as positive controls and non-Acinetobacter strains were used to assess specificity. Analytical sensitivity was quantified by comparison with the number of bacterial c.f.u. Forty of 46 (87 %) clinically significant and IMP-resistant A. baumannii isolates were positive for the bla(OXA-23)-like gene, and one isolate (2 %) was positive for the bla(OXA-58)-like gene. The bla(OXA-24/40)-like gene was not detected in any of the 46 IMP-resistant strains and the bla(OXA-51)-like gene was identified in both IMP-resistant and non-resistant A. baumannii. All 11 non-Acinetobacter bacteria produced a negative result for each of the four bla(OXA) genes. This assay was able to detect as few as 10 c.f.u. per assay. This real-time PCR method demonstrated rapid detection of OXA-like carbapenem resistance in A. baumannii in comparison with phenotypic susceptibility testing methodology. This method could be adapted to a multiplexed single reaction for rapid detection of genes associated with carbapenem resistance in A. baumannii and potentially other clinically significant multidrug-resistant Gram-negative bacteria.201222878252
222160.9998Rapid detection of blaKPC carbapenemase genes by real-time PCR. Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla(KPC)) enzymes are among the most common beta-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of bla(KPC) genes using TaqMan chemistry. The q-PCR amplification of bla(KPC) DNA was linear over 7 log dilutions (r(2) = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for bla(KPC) genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla(KPC) genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla(KPC) genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla(KPC) q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla(KPC) detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.200818614657
149170.9997Evaluation of an expanded antibiotic resistance gene panel on prediction of antimicrobial susceptibility results for Gram-negative bacteria in blood cultures. The QIAstat-Dx BCID Panels (RUO) ("QIAstat," QIAGEN, Hilden, Germany) for identification of 13 Gram-negative bacteria and 18 antimicrobial resistance (AMR) gene groups was evaluated. The study was conducted in two phases; in phase 1, analytical performance was evaluated against 154 challenge isolates against whole genome sequencing data. In this phase, sensitivity and specificity of organism identification calls were 153/154 (99.3%) and 1,748/1,749 (99.8%), respectively. For AMR genes, sensitivity was 434/435 (99.8%) and specificity was 2,334/2,337 (99.9%). One false-negative bla(IMP), one false-positive bla(CTX-M), and two false-positive aac-6'-lb detections were noted in this challenge set of organisms. In phase 2, 101 clinical blood culture isolates of Gram-negative rods were evaluated by the multiplexed PCR versus reference broth microdilution, for the ability of identification combined with AMR genes to predict final susceptibility results. Negative predictive values were 92.8% for ampicillin resistance (100% for Escherichia coli), 93.4% for ceftriaxone, 97.4% for ceftazidime, and 98.7% for cefepime. In constrast, negative predictive values for current standard of care (identification plus detection of bla(CTX-M)) ranged from 56.5% to 88.8%. This study demonstrated additive value of additional beta-lactamase genes for bacteria isolated from blood cultures. IMPORTANCE: Prediction of Gram-negative bacteria resistance through detection of resistance genes is complex. This study evaluated a novel, direct-from-blood or bacterial isolate multiplexed PCR for the detection of 17 resistance genes, and evaluated the prediction of antimicrobial susceptibility.202439297627
221880.9997Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates. BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.201728693493
222290.9997Multiplex real-time PCR assay for the detection of extended-spectrum β-lactamase and carbapenemase genes using melting curve analysis. Real-time PCR melt curve assays for the detection of β-lactamase, extended-spectrum β-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common β-lactamase genes: blaKPC-like, blaOXA-48-like, blaNDM-like, blaVIM-like, blaIMP-like, blaCTX-M-1+2-group, blaCMY-like, blaACC-like, blaSHV-like and blaTEM-like. The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n=56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent β-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens.201627021662
1467100.9997Detection of bla (CTX-M15) and bla (OXA-48) genes in Gram-negative isolates from neonatal sepsis in central of Iran. BACKGROUND AND OBJECTIVES: The aim of this study was to determine the prevalence of neonatal sepsis with a focus on antibiotic resistance and the frequency of the bla (CTX-M-15) and bla (OXA-48) genes in Gram-negative isolates. MATERIALS AND METHODS: A total of 108 Umbilical Cord Blood (UCB) and 153 peripheral blood samples were cultured via BACTEC from May 2017 to June 2018. The bacterial isolates were identified using phenotypic and genotypic analyses. The antibiotic susceptibility profile of the isolates was determined by disk diffusion. PCR was used to determine the frequency of β-lactamase genes. RESULTS: Among the 153 infants, 21 (13.7%) proved positive for sepsis. Escherichia coli, Staphylococcus epidermidis and Klebsiella pneumoniae were the most frequent isolates in the peripheral blood cultures. E. coli and Stenotrophomonas maltophilia were isolated from two UCB cultures. The highest resistance among the Gram-positive strains was to cefixime, ceftriaxone, cefotaxime and clindamycin. In the Gram-negative bacteria the highest rates of resistance were to ampicillin (91.7%). The frequency of bla (OXA-48) and bla (CTX-M-15) genes was 25% and 50%, respectively. CONCLUSION: The high antibiotic resistance among the isolates reveals the importance of monitoring antibiotic consumption and improving control standards in the health care system, especially in neonatal wards.201931719958
1488110.9997Evaluation of an automated rapid diagnostic assay for detection of Gram-negative bacteria and their drug-resistance genes in positive blood cultures. We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla(CTX-M), 119 bla(IMP), 8 bla(KPC), 16 bla(NDM), 24 bla(OXA-23), 1 bla(OXA-24/40), 1 bla(OXA-48), 4 bla(OXA-58), and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.201424705449
1437120.9997Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany. Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants.Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available.Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany.Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla (VIM), bla (OXA-48), bla (OXA-23), bla (KPC), bla (NDM), bla (OXA-40), bla (OXA-58), bla (IMP), bla (GIM), bla (GES), ISAba1-bla (OXA-51), bla (IMI), bla (FIM) and bla (DIM). We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla (VIM), bla (OXA-48), bla (OXA-23), bla (KPC), bla (NDM) and bla (OXA-40), while multiplex-2 included bla (OXA-58), bla (IMP), bla (GIM), bla (GES), ISAba1-bla (OXA-51) and bla (IMI).Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected.Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.202133448924
924130.9997Screening of Antimicrobial Resistance Genes and Epidemiological Features in Hospital and Community-Associated Carbapenem-Resistant Pseudomonas aeruginosa Infections. INTRODUCTION: Researching carbapenem-resistant isolates enables the identification of carbapenemase-producing bacteria and prevents their spread. METHODS: P. aeruginosa isolates were recovered from Medicine Faculty of Recep Tayyip Erdoğan University and identified by conventional methods and the automated Vitek 2 Compact system. Antimicrobial susceptibility experiments were performed in accordance with CLSI criteria and the automated Vitek 2 Compact system. The PCR method was investigated for the presence of β-lactamase resistance genes. PFGE typing was performed to show clonal relation among samples. RESULTS: Seventy P. aeruginosa isolates were isolated from seventy patients. Of the patients, 67.1% had contact with the health service in the last 90 days and 75.7% of the patients had received antimicrobial therapy in the previous 90 days. Twenty-four isolates were carbapenem resistant, 2 isolates were multidrug-resistant except colistin, and none of the samples had colistin resistance. The gene encoding β-lactamase or metallo-β-lactamase was found in a total of 36 isolates. The bla (VEB) and bla (PER) genes were identified in 1 and 5 isolates alone or 17 and 13 isolates in combination with other resistance genes, respectively. The bla (NDM) was the most detected metallo-β-lactamase encoding gene (n=18), followed by bla (KPC) (n=12). bla (IMP) and bla (VIM) were detected in 5 and 1 isolates, respectively. Also, the association of bla (VEB)-bla (PER) and bla (VEB)-bla (KPC)-bla (NDM) was found to be very high. Much more resistance genes and co-occurrence were detected in hospital-acquired samples than community-acquired samples. No difference was found between the community and hospital-associated isolates according to PFGE results. Simultaneously from 6 patients, other microorganisms were also isolated and 5 of them died. CONCLUSION: The average length of stay (days) was found to be significantly higher in HAI group than CAI group. The death of 5 patients with fewer or no resistance genes showed that the co-existence of other microorganisms in addition to resistance genes was important on death.202133907430
997140.9997Prevalence and antibacterial resistance patterns of extended-spectrum beta-lactamase producing Gram-negative bacteria isolated from ocular infections. PURPOSE: Extended-spectrum beta-lactamases (ESBLs) mediated resistance is more prevalent worldwide, especially among Gram-negative bacterial isolates, conferring resistance to the expanded spectrum cephalosporins. As limited data were available on the prevalence of ESBLs in this area, the current study was undertaken to determine the prevalence, antibacterial resistance patterns, and molecular detection and characterization of ESBL encoding resistance genes among ocular Gram-negative bacterial isolates from ocular infections. MATERIALS AND METHODS: A prospective study was done on 252 ocular Gram-negative bacterial isolates recovered from ocular infections during a study period from February 2011 to January 2014. All isolates were subjected to detection of ESBLs by cephalosporin/clavulanate combination disc test and their antibacterial resistance pattern was studied. Molecular detection and characterization of ESBL encoding blaTEM -, blaSHV , blaOXA -, and blaCTX-M (phylogenetic groups 1, 2, 9, and 8/25) resistance genes by multiplex polymerase chain reaction and DNA sequence analysis. RESULTS: Of all Gram-negative bacteria, Pseudomonas aeruginosa (44%) was the most common strain, followed by Enterobacter agglomerans and Klebsiella pneumoniae each (10%). Among the 252, 42 (17%) were ESBL producers. The major source of ESBL producers were corneal scraping specimens, highest ESBL production was observed in P. aeruginosa 16 (38%) and Escherichia coli 7 (16.6%). Among ESBL-producing genes, the prevalence of blaTEM -gene was the highest (83%) followed by blaOXA -gene (35%), blaSHV -gene (18.5%), and blaCTX-M-1 -gene (18.5%) alone or together. CONCLUSION: The higher rate of prevalence of ESBLs-encoding genes among ocular Gram-negative bacteria is of great concern, as it causes limitation to therapeutic options. This regional knowledge will help in guiding appropriate antibiotic use which is highly warranted.201627221683
1128150.9997Molecular detection of ESBLs production and antibiotic resistance patterns in Gram negative bacilli isolated from urinary tract infections. BACKGROUND: β-lactam resistance is more prevalent in Gram negative bacterial isolates worldwide, particularly in developing countries. In order to provide data relating to antibiotic therapy and resistance control, routine monitoring of corresponding antibiotic resistance genes is necessary. AIMS: The aim of this study was the characterization of β-lactam resistance genes and its plasmid profile in bacteria isolated from urinary tract infection samples. MATERIALS AND METHODS: In this study, 298 Gram negative bacteria isolated from 6739 urine specimens were identified by biochemical standard tests. Antimicrobial susceptibility testing was performed by the disk diffusion method. Extended-spectrum β-lactamase (ESBL)-producing strains were also detected by the double-disk synergy test. The presence of blaTEM and blaSHV genes in the strains studied was ascertained by polymerase chain reaction. RESULTS: Of all Gram negative bacteria, Escherichia coli (69.1%) was the most common strain, followed by Klebsiella sp. (12.1%), Enterobacter sp. (8.4%), Proteus sp. (4.4%), Citrobacter (4%) and Pseudomonas sp. (2%). The most antibiotic resistance was shown to tetracycline (95.16%), nalidixic acid (89.78%) and gentamycin (73.20%) antibiotics. Among all the strains tested, 35 isolates (11.75%) expressed ESBL activity. The prevalence of TEM and SHV positivity among these isolates was 34.29%, followed by TEM (31.43%), TEM and SHV negativity (20.0%) and SHV (14.29%), respectively. CONCLUSIONS: Regular monitoring of antimicrobial drug resistance seems necessary to improve our guidelines in the use of the empirical antibiotic therapy.201424943757
2124160.9997Evaluation of Phenotypic and Genotypic Characteristics of Carbapnemases-producing Enterobacteriaceae and Its Prevalence in a Referral Hospital in Tehran City. BACKGROUND & OBJECTIVE: Carbapenem-resistant Enterobacteriaceae is a growing concern worldwide including Iran. The emergence of this pathogen is worrying as carbapenem is one of the 'last-line' antibiotics for treatment of infections caused by multi drug resistant gram- negative bacteria. The main objective of this study was to determine the prevalence of carbapenem-resistant Enterobacteriaceae in a referral hospital in Tehran, Iran. METHODS: In this study, all positive isolates of Enterobacteriaceae recorded in blood, urine, and other body fluids were studied during April 2017 to April 2018 in a referral hospital in Tehran. All cases of resistance to carbapenems were first tested by modified Hodge test. All cases with positive or negative test, after gene extraction, were examined genotypically based on the primers designed for the three Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), and OXA-48 genes by conventional PCR method. RESULTS: 108 isolates (13.6%) were resistant to all cephalosporins as well as to imipenem and meropenem. In a genotypic study, including 45 isolates, 13 isolates were positive for OXA-48 gene, 11 isolates for OXA-48 and NDM genes, 11 isolates for OXA-48, NDM and KPC genes, 4 isolates for OXA-48 genes and KPC, 3 isolates for NDM, one isolate for KPC. On the other hand, two isolates were negative for all three genes examined. CONCLUSION: OXA-48 gene was one of the most common genes resistant to carbapenems in Iran. According to studies, the prevalence of antibiotic resistance in Iran is rising dramatically, which reduces the choice of antibiotics to treat severe infections in the future.202032215024
1127170.9997Extended spectrum beta-lactamase and aminoglycoside modifying enzyme genes in multi drug resistant Gram-negative bacteria: A snapshot from a tertiary care centre. BACKGROUND: This study aims to enhance the existing knowledge of the prevalence of genes responsible for beta-lactam resistance and aminoglycoside resistance in gram negative organisms by molecular detection of extended spectrum beta-lactamase and aminoglycoside modifying enzymes in multidrug-resistant gram-negative bacteria. METHODS: Out of 864 gram-negative isolates, 710 were phenotypically identified as multidrug-resistant by antibiotic susceptibility testing. From the above isolates, 102 representative isolates as per sample size calculated were selected for further molecular studies. The presence of blaTEM, blaCTX-M blaSHV, and five AmpC genes was detected by real-time polymerase chain reaction (PCR). Conventional PCR was performed to detect seven aminoglycoside modifying enzyme genes namely aac(6')-Ib, aac(6')-Ic, aac(3)-Ia, aac(3)-Ib, aac(3)-IIa, ant(2'')-Ia, and ant(4'')-IIa. RESULTS: Most common multidrug-resistant isolate was Klebsiella pneumoniae (35%) followed by Escherichia coli (30%). Among the 102 selected isolates all harboured blaTEM gene, 71 (69.6%) harboured blaCTX-M gene and 48 (47%) blaSHV gene. Among the selected isolates 60% showed the presence of AmpC genes. Most common aminoglycosie modifying enzyme gene was AAC 6' Ib (51%) followed by ANT 2" Ia (36%). CONCLUSION: This study suggests a wider use of molecular methods using specific PCR amplification of resistance genes. It would be beneficial to perform the molecular identification of antimicrobial resistance genes to effectively monitor and manage antibiotic resistance, administer appropriate antimicrobial medication, practice antimicrobial stewardship and improve hospital infection control procedures.202439734850
2204180.9997Multidrug resistance pattern of bacterial agents isolated from patient with chronic sinusitis. BACKGROUND: Treatment of chronic sinusitis is complicated due to increase of antibiotic-resistant bacteria. The aim of this study was to determine the multidrug resistance (MDR) pattern of the bacteria causing chronic sinusitis in north of Iran. METHODS: This cross-sectional study was carried out on patients with chronic sinusitis. Bacterial susceptibility to antimicrobial agents was determined according to the CLSI 2013 standards. Double-disk synergy (DDS) test was performed for the detection of extended-spectrum beta-lactamase (ESBL) producing bacteria; also methicillin-resistant Staphylococcus (MRSA) strains were identified by MRSA screen agar. The MDR isolates were defined as resistant to 3 or more antibiotics. Data were analyzed using SPSS 17 software. Descriptive statistics was used to describe the features of the data in this study. RESULTS: The rate of ESBL-producing bacteria was 28.75-37.03% among enterobacteriaceae and the rate of MRSA was 42.75%-60% among Staphylococcus strains. The most detectable rate of the MDR bacterial isolates was Gram-negative bacteria 39 (76.47%) and Enterobacter spp. 19(70.37%) was the most multidrug resistant isolate among Gram negative bacteria. Also 36 (73.46%) of the gram positive bacterial isolated were multidrug resistance and Staphylococcus aureus 9(90%) was the most MDR among Gram positive bacteria. CONCLUSION: Antimicrobial resistance is increasing in chronic bacterial sinusitis. The emergence of MRSA and ESBL bacteria causing chronic sinusitis is increasing.201627386063
1469190.9997Investigation of Bacterial Infections and Antibiotic Resistance Patterns Among Clinical Isolates in the Center of Iran. Introduction: Bacterial infection is a considerable problem in hospitals. Thus, this study was executed to appraise the rampancy of bacterial infections, antimicrobial susceptibility patterns, and molecular characterization of isolates among patients in Bafgh Hospital in Yazd, Iran, in 2020. Methods: In the current study, we surveyed 103 isolates of 400 clinical specimens from early March 2020 to September 2020 in Bafgh Hospital. We assessed phenotypic traits and antibiotic resistance with standard microbiological methods. Phenotypic methods were also performed to identify extended-spectrum beta-lactamases (ESBLs) in Gram-negative bacilli, inducible clindamycin resistance, and methicillin resistance in Staphylococcus according to CLSI guidelines. Molecular identification of isolates was done by conventional PCR 16S rRNA gene sequencing. Furthermore, we investigated the prevalence of resistant genes including bla (TEM), bla (PER-2), bla (CTX-M), bla (SHV), and bla (VEB-1) in Gram-negative bacteria and the mecA gene in staphylococcal species. Results: From 400 different clinical specimens, 103 isolates of Gram-positive and Gram-negative bacteria were isolated. Based on phenotypic and molecular methods, most common isolates were Escherichia coli (53 isolates), followed by Klebsiella spp. (18 isolates), and Staphylococcus aureus (16 isolates). The highest resistance was found in Gram-positive bacteria to erythromycin (66.67%) and penicillin (55.56%), while considering Gram-negative bacteria, the most resistant was cefixime (49.41%) and trimethoprim-sulfamethoxazole (47.05%). In addition, out of 16 S. aureus isolates, 62.5% and 17.65% were resistant to methicillin and clindamycin, respectively. Among 83 Gram-negative isolates, 22.89% were ESBL-positive. The prevalence of bla (SHV), bla (PER2), bla (TEM), bla (CTX-M), and bla (VEB-1) genes was 78.31%, 59.03%, 40.96%, 30.12%, and 0%, respectively. Conclusions: The outbreak of bacterial infections is relatively high in hospitals. Recognizing risk agents for bacterial infections and restricting the administration of multidrug-resistant antibiotics is a substantial measure that must be taken to prevent patient mortality.202540822981