Use of the Ligase Detection Reaction-Polymerase Chain Reaction to Identify Point Mutations in Extended-Spectrum Beta-Lactamases

2000 ◽  
Vol 19 (6) ◽  
pp. 477-480 ◽  
Author(s):  
C. Niederhauser ◽  
L. Kaempf ◽  
I. Heinzer
Keyword(s):  
Polymerase Chain Reaction ◽  
Point Mutations ◽  
Chain Reaction ◽  
Ligase Detection Reaction ◽  
Beta Lactamases ◽  
Extended Spectrum ◽  
Extended Spectrum Beta Lactamases ◽  
Polymerase Chain

Phenotypic and Molecular Detection of BlaNDM gene among Drug-Resistant Klebsiella Isolates

2021 ◽  
Vol 16 ◽  
Author(s):  
Taqdees Malik ◽  
Asma Naim
Keyword(s):  
Polymerase Chain Reaction ◽  
Broad Spectrum ◽  
Therapeutic Option ◽  
Dilution Method ◽  
Chain Reaction ◽  
Klebsiella Species ◽  
Gram Negative ◽  
Beta Lactamases ◽  
Extended Spectrum ◽  
Polymerase Chain

Background: In the past few centuries, a widespread increase in antimicrobial resistance has been observed among Klebsiella species. The antibiotic-resistant strains of the genus Klebsiella are becoming a serious threat in clinical settings due to their involvement in severe invasive and non-invasive infections. The emergence of resistance among these strains is associated with their strong enzymatic activity against several broad-spectrum antibiotics. These enzymes include beta-lactamases, extended-spectrum beta-lactamases (ESBL), AmpC beta-lactamases, and carbapenemases. These resistance enzymes are capable of hydrolyzing various broad-spectrum drugs like extended-spectrum cephalosporin and carbapenems. Objective: The present study was conducted to determine the emerging resistance among Klebsiella strains by identifying the production of carbapenemase enzyme phenotypically and the frequency of the NDM resistance gene by a polymerase chain reaction. Methods: In this study, 236 Gram-negative isolates from different clinical laboratories were subjected to identification. Out of which, 125 isolates were found as Klebsiella species by using standard microbiological techniques. Minimum inhibitory concentrations (MIC) were determined using eight representative antibiotics by the Macro broth dilution method. Phenotypic detection of Carbapenemase producing Klebsiella species was performed by Modified Hodge Test. Phenotypic findings were then checked and compared with genotypic results obtained by using the Polymerase chain reaction (PCR) for the detection of resistance genes responsible for the production of Carbapenemase. Results: In this study, carbapenemase production was found only in 6 (5%) Klebsiella isolates by using the phenotypic method; however 3 isolates out of 125 were screened positive for the gene NDM-1. Conclusion: Since we are considering carbapenems as the last therapeutic option for treating infections, mainly caused by Gram-negative isolates, the prevailing resistance against this drug is widely disseminating. It’s better to evaluate the antibiotic susceptibility, phenotypic screening as well genotypic screening (where possible) for implementing strict antibiotic control policies in health care settings, hospitals, laboratories, etc.

scholarly journals DETEKSI GEN SHV PADA ISOLAT KLINIK Escherichia coli PENGHASIL EXTENDED SPECTRUM BETA−LACTAMASES (ESBLs) DENGAN METODE POLYMERASE CHAIN REACTION (PCR) DARI URIN PASIEN DI RSUD Dr. SOETOMO SURABAYA

2018 ◽  
Vol 11 (2) ◽  
pp. 91-98
Author(s):  
Yulianto Ade Prasetya
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Clinical Microbiology Laboratory ◽  
Beta Lactam ◽  
Chain Reaction ◽  
E Coli ◽  
Beta Lactam Antibiotics ◽  
Extended Spectrum ◽  
Extended Spectrum Beta Lactamases ◽  
Polymerase Chain

 AbstrakEscherichia coli penghasil Extended Spectrum Beta Lactmases (ESBLs) bertanggungjawab terhadap terjadinya wabah infeksi nosokomial, peningkatan morbiditas dan mortalitas, serta peningkatan biaya kesehatan. Enzim yang diproduksi oleh gen SHV dari bakteri mampu menghidrolisis antibiotik sefotaksim dan seftazidim. Tujuan penelitian ini adalah untuk mengetahui prevalensi gen SHV pada isolat klinik E. coli penghasil ESBLs dari urin pasien yang merupakan koleksi Laboratorium Mikrobiologi Klinik RSUD Dr. Soetomo Surabaya pada bulan Januari-Februari 2014. Jenis penelitian yang digunakan adalah observasional deskriptif dengan pendekatan molekuler. Deteksi gen SHV menggunakan metode Polymerase Chain Reaction (PCR) yang kemudian dilakukan elektroforesis dan divisualisasikan pada gel agarose 1,5%. Isolat E. coli yang positif membawa gen SHV ditunjukkan dengan adanya amplikon sebesar 867 bp. Hasil penelitian menunjukkan bahwa dari 30 isolat, sebanyak 12 isolat (40%) positif mengandung gen SHV, dengan prevalensi tertinggi berada di Ruang Instalasi Rawat Jalan. Meropenem dan fosfomisin masih dapat digunakan untuk terapi penyakit yang disebabkan oleh E. coli penghasil ESBLs. Deteksi ESBLs secara genotipik penting dilakukan karena beberapa gen ESBLs menunjukkan resistensi yang berbeda terhadap antibiotik golongan beta laktam. Hasil tersebut memberi informasi kepada pihak rumah sakit manapun untuk mewaspadai prevalensi E. coli penghasil ESBLs melalui pengawasan yang ketat pelaksanaan pemberian antibiotika sesuai tata laksananya.Abstract Extended Spectrum Beta Lactmases (ESBLs) producing−Escherichia coli strains are responsible for the occurrence of nosocomial infection outbreaks, increase of morbidity and mortality, as well as increased healthcare costs. The enzyme produced by the SHV gene from bacteria are able to hydrolyze antibiotics of cefotaxime and cefazazim. The purpose of this study was to detect the presence of SHV gene in clinical ESBLs−producing E. coli isolates from patients’ urines which are collections of Clinical Microbiology Laboratory in Dr. Soetomo Hospital of Surabaya within period of January−February 2014. The research used descriptive observasional design with molecular approach. The SHV gene was detected by using Polymerase Chain Reaction (PCR) method, then the products was performed by electrophoresis and visualized on 1.5% agarose gel. E. coli isolates that positively carrying the SHV gene were demonstrated in the presence of amplicons of 867 bp. The results showed that of 30 isolates, 12 isolates (40%) positively contained the SHV gene, with the highest prevalence being in the Outpatient Installation Room. Meropenem and fosfomycin can still be used for disease therapy caused by ESBLs−producing E. coli. Genotypic detection of ESBLs is important because some ESBLs genes exhibit different resistance to beta−lactam antibiotics. The result provides information to any hospital to be aware of the prevalence of ESBLs−producing E.coli through strict supervision of the implementation of antibiotics according to their administration.

Norwalk-like Virus Sequences Detected by Reverse Transcription-Polymerase Chain Reaction in Mineral Waters Imported into or Bottled in Switzerland

2000 ◽  
Vol 63 (11) ◽  
pp. 1576-1582 ◽  
Author(s):  
CHRISTIAN BEURET ◽  
DOROTHE KOHLER ◽  
THOMAS LÜTHI
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Carbonic Acid ◽  
Point Mutations ◽  
Chemical Properties ◽  
The United States ◽  
Mineral Waters ◽  
Nucleotide Sequence Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

Norwalk-like viruses (NLVs) is a genus belonging to the Caliciviridae. NLVs are transmitted by the fecal-oral and the aerosol route and are the most common cause of outbreaks of nonbacterial gastroenteritis. NLVs are responsible for an estimated 67% of all illnesses caused by known foodborne pathogens and for 96% of nonbacterial gastroenteritis in the United States. Many outbreaks could be associated with the consumption of primarily or secondarily contaminated foods. To our knowledge, no epidemic arising from contaminated mineral water has been reported. We investigated the presence of NLV sequences in 63 mineral waters of 29 different brands that were imported into or bottled in Switzerland. NLV sequences were detected in 21 mineral waters by reverse transcription-seminested polymerase chain reaction. Specimens of two NLV genogroups (gg), gg I and gg II, were randomly present in the contaminated samples. The presence of NLV sequences could not be correlated either with bottle characteristics or with chemical properties like mineralization, pH, or the presence of carbonic acid. Nucleotide sequence analysis of 12 NLV-positive samples revealed several point mutations. All isolated NLV gg I strains have a similarity of 70 to 87% with the common Desert Shield virus (UO4469), and all isolated NLV gg II strains have a similarity of 89 to 93% with the Camberwell virus (U46500). Possible reasons for the presence of NLV sequences in mineral waters are discussed.

scholarly journals Precision of genotyping of Epstein-Barr virus by polymerase chain reaction using three gene loci (EBNA-2, EBNA-3C, and EBER): predominance of type A virus associated with Hodgkin's disease [published erratum appears in Blood 1993 Oct 1;82(7):2268]

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3372-3381 ◽  
Author(s):  
JC Lin ◽  
SC Lin ◽  
BK De ◽  
WC Chan ◽  
BL Evatt ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Point Mutations ◽  
Type A ◽  
Type B ◽  
Chain Reaction ◽  
Barr Virus ◽  
Polymerase Chain ◽  
Epstein Barr

Abstract To precisely determine the genotype of Epstein-Barr virus (EBV) in Hodgkin's disease (HD), we simultaneously analyzed three divergent gene loci (EBNA-2, EBNA-3C, and EBER) that distinguish type A and B viruses. The primers designed to amplify these three gene loci encompass either type-specific deletion sequences (EBNA-2 and EBNA-3C) or type-specific point mutations (EBER) that identify the virus strain based on the sizes of the polymerase chain reaction (PCR)-amplified products or the mobility shifts in single-strand conformation polymorphism analysis. The locations of point mutations were identified by direct sequencing of the PCR-amplified DNA. We analyzed 15 EBV-infected cell lines and found a good correlation between EBNA-2 and EBNA-3C typing results. In contrast, approximately 33% of the cell lines analyzed maintained type A sequences in EBNA-2 and EBNA-3C genes while carrying type B sequences in the EBER region. Data obtained from analysis of cell lines served as a reference for studying HD samples. EBV DNA was detected in about 70% of HD. Among the EBV-positive samples, 56% were associated with type A virus, 13% with type B, and 31% with dual viral sequences. Thus, type A virus is predominant in HD. Based on the histology, the frequencies of EBV positivity were 83%, 71%, and 33% for mixed cellularity, nodular sclerosis, and lymphocyte predominance, respectively. The detection of high frequency of both type A and B sequences in HD may provide a lead in investigating the role of dual viral infection in EBV pathogenesis.

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