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A total of 150 cloacal swabs were collected from desi chickens, 217 Enterobacteriaceae isolates were identified. The phenotypic antimicrobial resistance among Enterobacteriaceae was studied for 14 selected antibiotics by disc diffusion method. The selection of antibiotics was based on usage of antibiotics in commercial poultry farms and also based on priority of critically important antibiotics in humans. All Enterobacteriaceae isolates were subjected to multiplex PCR - I and II for detection of blaTEM, blaSHV and blaOXA genes and blaCTX-M, group 1 and 2 genes. Predominant β- Lactamase genes in gut microbiota of desi chicken include blaTEM (90.55%) followed by blaCTX-M group I (25.86%) and blaSHV (9.44%) genes. All the samples were found to be negative for blaOXA and blaCTX-M group 2 genes
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Background: Extended Spectrum Beta Lactamases(ESBLs) are rapidly evolving group of β-lactamaseenzymes that are of particular concern to clinicians andepidemiologists. Most ESBLs have been evolved bygenetic mutation from blaTEM and blaSHV genes, andare well described in Klebsiella pneumoniae. Aim andObjective: To investigate the ESBL genotypes in K.pneumoniae isolates from Respiratory Tract Infections(RTIs). Material and Methods: Clinical isolates of K.pneumoniae were obtained from RTI -sputum samples.Antimicrobial susceptibility was determined by KirbyBauer disc diffusion method. ESBL was detectedphenotypically and multiplex Polymerase ChainReaction (PCR) specific for blaTEM, blaSHV andblaCTX-M genes was performed to identify genotypes.Results: During the 19 months period, a total of 212 ofK. pneumoniae were found from RTIs. Of these 212isolates, 60 isolates (28.3%) were ESBL producers byphenotypic method. Of these 212 isolates, 96 wererandomly selected for multiplex PCR for blaTEM,blaSHV and blaCTX-M genes. The findings ofmultiplex PCR showed that 24 isolates (25%)possessed blaTEM gene and only 4 isolates (4.1%)possessed each blaSHV and blaCTX-M gene alone.Isolates having both blaTEM+blaSHV genes were 20(20.8%), and both blaTEM+blaCTX-M genes were 12(12.5%); and isolate possessing all threeblaTEM+blaSHV+blaCTX-M genes were 20 (20.8%).The overall prevalence of blaTEM, blaSHV andblaCTX-M genes in this study was 79.1%, 45.8% and37.5% respectively. Imipenem was most effectiveantibiotic. Conclusion: Spread of ESBL producing K.pneumoniae is a major concern, as it causes limitationsto optimal treatment. Multiplex PCR can be used as arapid method to identify ESBL genotypes in K.pneumoniae. It will prove valuable for surveillance andestablishing the treatment line against drug resistantorganisms, thus saving precious time and resources. Inour study blaTEM genotype was most prevalent.
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Background: Urinary tract infections (UTIs) represent one of the most common diseases that are encountered in clinical practice and are caused mainly by Escherichia coli (E. coli). Aims: The objectives of this study were to identify and compare the blaTEM, blaSHV and blaCTX-M as marker of beta-lactamase genes in E. coli strains isolated from patients with UTIs collecting from King Abdul-Aziz hospital in Taif region, Saudi Arabia. Study Design: In vitro experimental and molecular study. Place and Duration of Study: Genetic engineering and biotechnology unit, Taif University, from September, 2016 to November, 2017. Methodology: Beta-lactame antibiotics are prescribed in most infectious disease including UTIs. Twenty one isolates identified as E. coli using microbial identification and confirmed by 16S rDNA. Results: These isolates were susceptible to Imipenem (100%), Ampicillin (90%) and Cefoxitin, but resistant to Cefepime (38%). Existance of selected bla-genes (blaTEM, blaSHV and blaCTX-M) were detected in the 21 isolates by PCR. Moreover, phylogeny tree was drawn based on 16S rDNA sequence. The results of this study show significant differences in susceptibility to different beta-lactam antibiotics among the bla-genes in E. coli isolates. Conclusion: Therefore, our findings instead of our data provide some new epidemiological information about the clonal nature of E. coli isolated from patients with UTIs in Taif region, KSA.
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Objective To investigate the carbapenem-resistant mechanism of one strain of Klebsiella pneumoniae( KPN) and one strain of Pseudomonas aeruginosa( PAE) .Methods The identity of the isolates was confirmed by using MALDI-TOF mass spectrometry, 16S rDNA and special gene amplification .The minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by VITEK 2 Compact System .CarbaNP Antimicrobial susceptibility testing , plasmid extraction, electroporation experiment , PCR amplification, and cloning and sequencing were carried out to analyze the en-coding gene of β-lactamases.Results The types of β-lactamases of the KPN were blaKPC-2 and blaSHV, confirmed by se-quencing of the PCR products , and that of the PAE were blaKPC-2 .Only blaKPC-2 was displayed in both transformants .All of the results of CarbaNP were type A .Conclusion Both strains of KPN and PAE resisting to carbapenem produce a plasmid-mediated carbapenemase blaKPC-2 , which belongs to Bush group 2f, class A β-lactamase.The extended-spectrum β-lacta-mases gene encoding blaSHV of the KPN might be located in the chromosome or not in the plasmid carrying with blaKPC-2 .
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Aim: This study was carried out to determine the presence of blaTEM, blaSHV and blaCTX-M genes in extended-spectrum β-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 E. coli and 219 K. pneumoniae isolates were recovered during the period between August 2009 and July 2010. Kirby-Bauer disk diffusion method was performed to determine the antibiotic resistance pattern. Screening and phenotypic confi rmatory test for ESBL production were performed using standard disc diffusion methods. Each of the initial ESBL screening test isolate was investigated for the presence of blaTEM, blaSHV and blaCTX-M genes via polymerase chain reaction (PCR) using gene-specifi c primers. Results: P henotypic confi rmatory test able to detect ESBL production in 73.58% of E. coli and 67.24% of K. pneumoniae. However, PCR amplifi cation showed the presence of one or more ESBL genes in each of the initial ESBL screening positive isolate. Among three ESBL genotypes, the most prevalent genotype was found to be blaCTX-M in E. coli (88.67%) and blaTEM in K. pneumoniae (77.58%) ESBL producing isolates. Majority of ESBL producing isolates possess more than one ESBL genes. Conclusion: T his study constituted a primer report on high prevalence of blaTEM and blaCTX-M genes in ESBL producing isolates of E. coli and K. pneumoniae and denotes the need of more extensive studies on these antibiotic genes to determine the magnitude of the problem of antibiotic resistance exiting in this locality.
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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 profi le in bacteria isolated from urinary tract infection samples. Materials and Methods: In this study, 298 Gram negative bacteria isolated from 6739 urine specimens were identifi ed by biochemical standard tests. Antimicrobial susceptibility testing was performed by the disk diffusion method. Extendedspectrum 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.
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El objetivo de este estudio fue identificar los genes blaTEM, blaSHV y blaCTX-M en aislados clínicos de enterobacterias productoras de b-lactamasas de espectro extendido (BLEE), recolectadas entre septiembre y noviembre de 2005. Además de la resistencia a las cefalosporinas de tercera generación, los aislados también mostraron resistencia a cloranfenicol (59,2%) amikacina (37,0%) y gentamicina (40,7%) y se mostraron sensibles a imipenem y meropenem. Nueve cepas lograron transferir la resistencia a las cefalosporinas de tercera generación, así como la producción de BLEE. En los aislados clínicos se detectaron los genes blaSHV, blaTEM y blaCTX-M, donde los tipos blaTEM-1, blaSHV-1, blaSHV-5 blaSHV-5-2a y blaCTX-M-1 fueron los prevalentes; mientras que en las transconjugantes sólo se detectaron blaTEM-1, blaSHV-5 y blaSHV-5-2a. Se identificaron en total siete tipos de genes, de los cuales cinco eran codificantes de enzimas tipo BLEE, lo que demuestra que en el centro hospitalario la resistencia a las cefalosporinas de tercera generación es debida a diversas enzimas.
The objective of the present investigation was to identify the blaTEM, blaSHV and blaCTX-M genes on extended-spectrum b-lactamases (ESBL) producing Enterobacteriaceae from clinical isolates, collected between September and November 2005. In addition to third-generation cephalosporin resistance, the isolates also showed resistance to chloramphenicol (59.2%), amikacin (37.0%) and gentamicin (40.7%), and demonstrated sensitivity to imipenem and meropenem. Nine strains were capable of transferring third-generation cephalosporin resistance, as well as the production of ESBL. In the clinical isolates, the genes blaSHV, blaTEM and blaCTX-M were detected, being more prevalent the types blaTEM-1, blaSHV-1, blaSHV-5 blaSHV-5-2a and blaCTX-M-1; while in the trans-conjugated only blaTEM-1, blaSHV-5 y blaSHV-5-2a were found. In total, seven types of genes were identified, five of which were codifying genes for ESBL-type enzymes. This demonstrates that in the hospital center, resistance to third-generation cephalosporin is mediated by several enzymes.
Subject(s)
Humans , Bacterial Proteins/genetics , Cross Infection/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/genetics , Genes, Bacterial , beta-Lactamases/genetics , Bacterial Proteins/physiology , Cross Infection/genetics , DNA, Bacterial/genetics , Enterobacter/drug effects , Enterobacter/enzymology , Enterobacter/genetics , Enterobacteriaceae Infections/genetics , Enterobacteriaceae/drug effects , Enterobacteriaceae/enzymology , Enterobacteriaceae/isolation & purification , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , Substrate Specificity , beta-Lactamases/physiologyABSTRACT
BACKGROUND: Among Gram-negative pathogens in Korea, the incidence of resistance to thirdgeneration cephalosporins is becoming an ever-increasing problem. This study was designed to determine the prevalence of third-generation cephalosporins-resistant Escherichia coli and Klebsiella pneumoniae isolates from patients in a tertiary care hospital in Busan, Korea, and to characterize the mechanism of resistance. METHODS: A total of 710 E. coli and 237 K. pneumoniae non-duplicate isolates were collected from patients in Kosin Medical Center in 1999. Antimicrobial susceptibilities were tested by the disk diffusion method. Extended-spectrum beta-lactamase (ESBL) production was determined by the double disk synergy test. MICs were determined by the agar dilution method. Searches for blaTEM, blaSHV, and blaCMY genes in cefotaxime-resistant or intermediate isolates were performed by PCR amplification. PCR products were used to determine the sequence of resistance genes by the dideoxy-chain termination method. RESULTS: Seven percent of E. coli and 25% of K. pneumoniae isolates were resistant to cefotaxime. Among the isolates with decreased susceptibility to cefotaxime, 69% (18/26) of E. coli and 80% (20/25) of K. pneumoniae isolates showed positive results in double disk synergy test. Banding patterns of PCR amplification showed that the blaTEM, blaSHV, and blaCMY genes were harboured by 71% (20/28), 86% (24/28) and 14% (4/28) of isolates with decreased susceptibility to cefotaxime,respectively. Seventy-one percent (20/28) of the isolates contained more than two types of beta- lactamase genes. Nucleotide sequence analysis of PCR products revealed that blaSHV-12 and blaTEM-1b were the dominant types of beta-lactamase gene. In addition, we also identified blaTEM-52, blaSHV-5, and a new ESBL gene named blaTEM-17b. CONCLUSIONS: Third-generation cephalosporins-resistant E. coli and K. pneumoniae are wide spread in Kosin Medical Center, Busan, Korea. Most of the isolates with decreased susceptibility to cefotaxime had blaTEM and/or blaSHV, and some isolates harboured blaCMY genes that may confer resistance against cephamycins. The spread of these beta-lactamase genes could compromise the future usefulness of third-generation cephalosporins for the treatment of infections caused by E. coli and K. pneumoniae.