scholarly journals Multiplex Asymmetric PCR-Based Oligonucleotide Microarray for Detection of Drug Resistance Genes Containing Single Mutations in Enterobacteriaceae

2007 ◽  
Vol 51 (10) ◽  
pp. 3707-3713 ◽  
Author(s):  
Ling-Xiang Zhu ◽  
Zhi-Wei Zhang ◽  
Dong Liang ◽  
Di Jiang ◽  
Can Wang ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Annealing Temperature ◽  
Point Mutations ◽  
Epidemiological Studies ◽  
Clinical Diagnostics ◽  
Gram Negative Bacteria ◽  
Asymmetric Pcr ◽  
Unrelated Sequence ◽  
Allele Specific ◽  
Reference Strains

ABSTRACT A multiplex asymmetric PCR (MAPCR)-based microarray method was developed for the detection of 10 known extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamase genes in gram-negative bacteria and for the typing of six important point mutations (amino acid positions 35, 43, 130, 179, 238, and 240) in the bla SHV gene. The MAPCR is based on a two-round reaction to promote the accumulation of the single-stranded amplicons amenable for microarray hybridization by employing multiple universal unrelated sequence-tagged primers and elevating the annealing temperature at the second round of amplification. A strategy to improve the discrimination efficiency of the microarray was constituted by introducing an artificial mismatch into some of the allele-specific oligonucleotide probes. The microarray assay correctly identified the resistance genes in both the reference strains and some 111 clinical isolates, and these results were also confirmed for some isolates by direct DNA sequence analysis. The resistance genotypes determined by the microarray correlated closely with phenotypic MIC susceptibility testing. This fast MAPCR-based microarray method should prove useful for undertaking important epidemiological studies concerning ESBLs and plasmid-mediated AmpC enzymes and could also prove invaluable as a preliminary screen to supplement phenotypic testing for clinical diagnostics.

scholarly journals DNA Microarray for Detection of Macrolide Resistance Genes

2006 ◽  
Vol 50 (6) ◽  
pp. 2038-2041 ◽  
Author(s):  
Marco Cassone ◽  
Marco M. D'Andrea ◽  
Francesco Iannelli ◽  
Marco R. Oggioni ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Dna Microarray ◽  
Resistance Genes ◽  
Dna Sequences ◽  
Bacterial Species ◽  
Glass Slide ◽  
Macrolide Resistance ◽  
Gram Negative Bacteria ◽  
Dna Templates ◽  
Bacterial Genes ◽  
Reference Strains

ABSTRACT A DNA microarray was developed to detect bacterial genes conferring resistance to macrolides and related antibiotics. A database containing 65 nonredundant genes selected from publicly available DNA sequences was constructed and used to design 100 oligonucleotide probes that could specifically detect and discriminate all 65 genes. Probes were spotted on a glass slide, and the array was reacted with DNA templates extracted from 20 reference strains of eight different bacterial species (Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus haemolyticus, Escherichia coli, and Bacteroides fragilis) known to harbor 29 different macrolide resistance genes. Hybridization results showed that probes reacted with, and only with, the expected DNA templates and allowed discovery of three unexpected genes, including msr(SA) in B. fragilis, an efflux gene that has not yet been described for gram-negative bacteria.

scholarly journals Evaluation of an Expanded Microarray for Detecting Antibiotic Resistance Genes in a Broad Range of Gram-Negative Bacterial Pathogens

2012 ◽  
Vol 57 (1) ◽  
pp. 458-465 ◽  
Author(s):  
Roderick Card ◽  
Jiancheng Zhang ◽  
Priya Das ◽  
Charlotte Cook ◽  
Neil Woodford ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Antimicrobial Agents ◽  
Antibiotic Resistance Genes ◽  
Clinical Diagnostics ◽  
British Society ◽  
Gram Negative Bacteria ◽  
Predictive Values ◽  
Gram Negative ◽  
Positive Results

ABSTRACTA microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.

Extended Spectrum Beta Lactamase (ESBL), bla TEM,bla SHVand bla CTX-M,resistance genes in Community and Healthcare Associated Gram Negative Bacteria from Osun State, Nigeria

2020 ◽  
Vol 20 ◽  
Author(s):  
Ganiyat Shitta ◽  
Olufunmilola Makanjuola ◽  
Olusolabomi Adefioye ◽  
Olugbenga Adekunle Olowe
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Multiple Antibiotic Resistance ◽  
Esbl Production ◽  
Gram Negative ◽  
Extended Spectrum Beta Lactamase ◽  
Healthcare Associated ◽  
Esbl Producers

Background: Extended Spectrum Beta Lactamase (ESBL) production in gram negative bacteria confers multiple antibiotic resistance, adversely affecting antimicrobial therapy in infected individuals. ESBLs result from mutations in β-lactamases encoded mainly by the bla TEM,bla SHVand bla CTX-Mgenes. The prevalence of ESBL producing bacteria has been on the increase globally especially its upsurge among isolates from community-acquired infections. Aim: To determine ESBL prevalence and identify ESBL genes among clinical isolates in Osun State, Nigeria. Material and Methods: A cross-sectional study was carried out from August 2016 –July 2017 in Osun State, Nigeria. Three hundred and sixty Gram negative bacteria recovered from clinical samples obtained from both community and healthcare associated infections were tested. They included147 Escherichia coli(40.8%), 116 Klebsiella spp(32.2%), 44 Pseudomo-nas aeruginosa(12.2%) and23 Proteus vulgaris (6.4%) isolates. Others were Acinetobacter baumannii, Serratia rubidae, Citrobacter spp, Enterobacter spp and Salmonella typhi. Disk diffusion antibiotic susceptibility testing was carried out, isolates were screened for ESBL production and confirmed using standard laboratory procedures. ESBLs resistance genes were identified by Polymerase Chain Reaction (PCR). Results: All isolates demonstrated multiple antibiotic resistance. Resistance to ampicillin, amoxicillin with clavulanate and erythromycin was 100%, whereas resistance to Imipenem was very low (5.0%). : Overall prevalence of ESBL producers was 41.4% with Klebsiellaspp as the highest ESBL producing Enterobacteriacaea. ESBL producers were more prevalent among the hospital pathogens than community pathogens, 58% vs 29.5% (p=0.003). ESBL genes were detected in all ESBL producers with the blaCTX-Mgene predominating (47.0%) followed by blaTEM(30.9%) and blaSHVgene was the least, 22.1%. The blaCTX-Mgene was also the most prevalent in the healthcare pathogens (62%) but it accounted for only 25% in those of community origin. Conclusion: A high prevalence of ESBL producing gram negative organisms occurs both in healthcare and in the community in our environment with the CTX-M variant predominating. Efforts to control spread of these pathogens should be addressed.

scholarly journals Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuan Wu ◽  
Lin Yang ◽  
Wen-Ge Li ◽  
Wen Zhu Zhang ◽  
Zheng Jie Liu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Genes ◽  
Point Mutations ◽  
Evolutionary Process ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Clostridioides Difficile ◽  
Hospitalized Elderly ◽  
Great Heterogeneity ◽  
High Level

Abstract Background Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates. Results Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes. Conclusions We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.

Novel point mutations in sulfadoxine resistance genes of Plasmodium falciparum from India

Acta Tropica ◽  
2009 ◽  
Vol 110 (1) ◽  
pp. 75-79 ◽  
Author(s):  
Shilpi Garg ◽  
Vishal Saxena ◽  
Swarna Kanchan ◽  
Pooja Sharma ◽  
Siddharth Mahajan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Resistance Genes ◽  
Point Mutations

scholarly journals Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

2003 ◽  
Vol 69 (12) ◽  
pp. 7145-7152 ◽  
Author(s):  
Zhonghua Ma ◽  
Michael A. Yoshimura ◽  
Themis J. Michailides
Keyword(s):  
Dna Sequences ◽  
Point Mutations ◽  
Brown Rot ◽  
Stone Fruit ◽  
Monilinia Fructicola ◽  
Tubulin Gene ◽  
Benzimidazole Fungicides ◽  
Specific Pcr ◽  
Allele Specific ◽  
Fruit Orchards

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

Determination of carbapenemase genes in clinical isolates using Cepheid Xpert Carba-R

2021 ◽  
pp. 16-19
Author(s):  
N. I. Gabrielyan ◽  
V. G. Kormilitsyna ◽  
V. K. Zaletaeva ◽  
A. V. Krotevich ◽  
I. A. Miloserdov ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Infection Control ◽  
Resistance Genes ◽  
Carbapenem Resistance ◽  
Critical Issue ◽  
Sensitivity Study ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Carbapenem Resistant

Detection of carbapenem resistance genes is a critical issue for hospitals due to possible recommendations for infection control and targeted therapy. The Cepheid Xpert instrument, a Carba-R test for the detection and differentiation of five common carbapenemase genes, was tested from September 2020 to February 2021. As part of the approbation, 20 tests were provided. This review presents the results of the approbation of a relatively regular sensitivity study on Siemens WalkAway‑96 plus. Cepheid Xpert Carba-R analysis has been shown to be an accurate and fast tool for detecting colonization by carbapenem-resistant gram-negative bacteria, which can help limit the spread of these organisms in hospitals.

scholarly journals Multiple Sclerosis in French Canadians: Evidence for HLA Class II Susceptibility and Resistance Genes

1990 ◽  
Vol 17 (4) ◽  
pp. 382-386 ◽  
Author(s):  
D.G. Haegert ◽  
M. Michaud ◽  
G.S. Francis
Keyword(s):  
Multiple Sclerosis ◽  
Resistance Genes ◽  
Rflp Analysis ◽  
Class Ii ◽  
Hla Class Ii ◽  
Extended Haplotype ◽  
Allele Specific ◽  
Relationship Of ◽  
The Relationship ◽  
Susceptibility And Resistance

ABSTRACT:HLA class II DRBI, DQB1 and DQA1 gene probes were used to study DNA from unrelated French Canadian multiple sclerosis (MS) patients and controls by restriction fragment length polymorphism (RFLP) analysis. An MS-associated and linked series of allele-specific RFLPs or allogenotypes was identified among this relatively homogeneous ethnic group; the allogenotypes include DRwl5, DQw6 and a DQA1 allogenotype termed DQαlb. An additional allogenotype which cross-hybridizes with DQA1 and is termed DQA2 upper (DQA2U), was shown not only to be part of the MS-associated extended haplotype, but also to be independently associated with MS in DRwl5-negative patients. Conversely a second DQA2 allogenotype, termed DQA2 lower (DQA2L) and a DQB1 allogenotype (DQw7) linked to DQA2L showed negative correlations with MS. It seems likely that the relationship of the HLA class II gene region to MS is complex and that MS susceptibility may reflect interaction between disease susceptibility and resistance genes.

scholarly journals Comparison of antimicrobial resistance in Gram negative bacteria isolated from effluents in coastal districts of Karnataka, India

2017 ◽  
Vol 07 (04) ◽  
pp. 016-020
Author(s):  
Juliet Roshini Mohan Raj ◽  
Rajeshwari Vittal ◽  
Santosh Kogaluru Shivakumaraswamy ◽  
Vijaya Kumar Deekshit ◽  
Indrani Karunasagar
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Industrial Effluents ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cleaning Agents ◽  
Hospital Effluents ◽  
Antimicrobial Resistance Genes ◽  
Domestic Effluents ◽  
Different Sources

AbstractDownstream water systems provide for a conducive environment for horizontal gene transfer. The objective of this study was to determine the burden of antimicrobial resistance in waste water effluents from different sources and their impact on human health. Gram negative bacteria were isolated from 30 samples each of industrial, hospital and domestic effluents. The antimicrobial susceptibility of the 367 isolates from 90 effluent samples was determined by disc diffusion test and presence of antimicrobial resistance genes by polymerase chain reaction. Resistance to ampicillin was 62% in hospital effluents and was higher than that recorded for industrial and domestic effluents. While the highest percentage of resistance to tetracycline was observed in isolates from industrial effluents (42%) a low of 9.5% was observed in hospital effluents. Antimicrobial resistance determinants present on mobile genetic elements were observed in a small fraction (~10%) of the resistant isolates. The resistance profile of isolates in effluents reflect the practices of different industries. Resistant isolates in domestic effluents could be a reflection of the indiscriminate use of antibiotics andthat many of the contents of disinfectants and cleaning agents routinely used may contain structural analogs of antimicrobials used in therapy. Though by phenotypic test a higher prevalence of antimicrobial resistance was recorded the genotypic study revealed the prevalence to be low. This could be due to the limited number of antimicrobial resistance genes included in this study.

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