Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets

2007 ◽  
Vol 21 (2) ◽  
pp. 125-133 ◽  
Author(s):  
Holger Volkmann ◽  
Thomas Schwartz ◽  
Silke Kirchen ◽  
Carmen Stofer ◽  
Ursula Obst
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Antibiotic Resistance Genes ◽  
Cross Reaction ◽  
Bacterial Antibiotic Resistance ◽  
Total Dna ◽  
Wastewater Samples

scholarly journals The Transferable Resistome of Produce

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Khald Blau ◽  
Antje Bettermann ◽  
Sven Jechalke ◽  
Eva Fornefeld ◽  
Yann Vanrobaeys ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Blot Hybridization ◽  
Antibiotic Resistance Genes ◽  
Human Pathogens ◽  
Content Type ◽  
E Coli

ABSTRACTProduce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets in Germany were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (TET)-resistantEscherichia coliwere isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistantE. coliisolates, transconjugants, and total community DNA (TC-DNA) from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons, and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for IncI and IncF plasmids. TET-resistantE. coliisolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids andblaCTX-M-1. From mixed salad and cilantro, IncF, IncI1, and IncP-1β plasmids were captured exogenously. Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing intoE. colisuggests that they could transfer to gut bacteria as well.IMPORTANCEProduce is one of the most popular food commodities. Unfortunately, leafy greens can be a reservoir of transferable antibiotic resistance genes. We found that IncF and IncI plasmids were the most prevalent plasmid types inE. coliisolates from produce. This study highlights the importance of the rare microbiome associated with produce as a source of antibiotic resistance genes that might escape cultivation-independent detection, yet may be transferred to human pathogens or commensals.

scholarly journals Using Real-Time PCR to Investigate Some of Antibiotic Resistance Genes from Streptococcus agalactiae Isolates from ewe Mastitis cases in Nineveh province

2020 ◽  
Vol 17 (3(Suppl.)) ◽  
pp. 0931
Author(s):  
Ayman Mohamed Jaber Albanna ◽  
Aseel A. H. Al-Layla
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Resistance Genes ◽  
Streptococcus Agalactiae ◽  
Real Time Pcr ◽  
Bacterial Resistance ◽  
Single Gene ◽  
Antibiotic Resistance Genes ◽  
Antibiotics Resistance ◽  
Dilution Method

In this study, from a total of 856 mastitis cases in lactating ewes, only 34 Streptococcus agalactiae isolates showed various types of resistance to three types of antibiotics (Penicillin, Erythromycin and Tetracycline). St. agalactiae isolates were identified according to the standard methods, including a new suggested technique called specific Chromogenic agar. It was found that antibiotic bacterial resistance was clearly identified by using MIC-microplate assay (dilution method). Also, by real-time PCR technique, it was determined that there were three antibiotics genes resistance ( pbp2b, tetO and mefA ). The high percentage of isolate carried of a single gene which was the Tetracycline (20.59%) followed by percentage Penicillin was (17.65%) and the lowest was in Erythromycin (11.77%). However, there were many isolates that carried two genes of antibiotics resistance represented by Penicillin and Erythromycin with collective present of 38.22%, and for the Penicillin and Tetracycline, the percentage was found to be 11.77%. In contrast, no common gene with two antibiotics (Erythromycin and  Tetracycline) was detected. On the other hand, it was found that no bacterial sharing with three kinds of antibiotic resistance genes ( pbp2b, tetO and mefA ). This study has revealed that the St. agalactiae isolates did induce recurrent mastitis in lactating Iraqi's ewes. 

Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan)

2004 ◽  
Vol 56 (2) ◽  
pp. 277-286 ◽  
Author(s):  
Holger Volkmann ◽  
Thomas Schwartz ◽  
Petra Bischoff ◽  
Silke Kirchen ◽  
Ursula Obst
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Municipal Wastewater ◽  
Antibiotic Resistance Genes

scholarly journals Metagenomic Sequencing and Quantitative Real-Time PCR for Fecal Pollution Assessment in an Urban Watershed

2021 ◽  
Vol 3 ◽  
Author(s):  
Kyle D. Brumfield ◽  
Joseph A. Cotruvo ◽  
Orin C. Shanks ◽  
Mano Sivaganesan ◽  
Jessica Hey ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Fecal Contamination ◽  
Antibiotic Resistance Genes ◽  
Fecal Pollution ◽  
Storm Event ◽  
Urban Watershed ◽  
Quantitative Real Time Pcr

Microbial contamination of recreation waters is a major concern globally, with pollutants originating from many sources, including human and other animal wastes often introduced during storm events. Fecal contamination is traditionally monitored by employing culture methods targeting fecal indicator bacteria (FIB), namely E. coli and enterococci, which provides only limited information of a few microbial taxa and no information on their sources. Host-associated qPCR and metagenomic DNA sequencing are complementary methods for FIB monitoring that can provide enhanced understanding of microbial communities and sources of fecal pollution. Whole metagenome sequencing (WMS), quantitative real-time PCR (qPCR), and culture-based FIB tests were performed in an urban watershed before and after a rainfall event to determine the feasibility and application of employing a multi-assay approach for examining microbial content of ambient source waters. Cultivated E. coli and enterococci enumeration confirmed presence of fecal contamination in all samples exceeding local single sample recreational water quality thresholds (E. coli, 410 MPN/100 mL; enterococci, 107 MPN/100 mL) following a rainfall. Test results obtained with qPCR showed concentrations of E. coli, enterococci, and human-associated genetic markers increased after rainfall by 1.52-, 1.26-, and 1.11-fold log10 copies per 100 mL, respectively. Taxonomic analysis of the surface water microbiome and detection of antibiotic resistance genes, general FIB, and human-associated microorganisms were also employed. Results showed that fecal contamination from multiple sources (human, avian, dog, and ruminant), as well as FIB, enteric microorganisms, and antibiotic resistance genes increased demonstrably after a storm event. In summary, the addition of qPCR and WMS to traditional surrogate techniques may provide enhanced characterization and improved understanding of microbial pollution sources in ambient waters.

scholarly journals Environmental Spread of New Delhi Metallo-β-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh

2017 ◽  
Vol 83 (15) ◽  
Author(s):  
Mohammad Aminul Islam ◽  
Moydul Islam ◽  
Rashedul Hasan ◽  
M. Iqbal Hossain ◽  
Ashikun Nabi ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Tap Water ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
New Delhi ◽  
Multidrug Resistant Bacteria ◽  
Content Type ◽  
Wastewater Samples

ABSTRACT Resistance to carbapenem antibiotics through the production of New Delhi metallo-β-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the bla NDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among bla NDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including bla CTX-M-1 (80%), bla CTX-M-15 (63%), bla TEM (76%), bla SHV (33%), bla CMY-2 (16%), bla OXA-48-like (2%), bla OXA-1 (53%), and bla OXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying bla NDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community. IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the bla NDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

scholarly journals Evaluation of residual antibacterial potency in antibiotic production wastewater using a real-time quantitative method

2015 ◽  
Vol 17 (11) ◽  
pp. 1923-1929 ◽  
Author(s):  
Hong Zhang ◽  
Yu Zhang ◽  
Min Yang ◽  
Miaomiao Liu
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Environmental Impacts ◽  
Resistance Genes ◽  
Quantitative Method ◽  
Antibiotic Production ◽  
Antibiotic Resistance Genes ◽  
Antibiotic Activity ◽  
Wastewater Discharge ◽  
Related Substances

While antibiotic pollution has attracted considerable attention due to its potential in promoting the dissemination of antibiotic resistance genes in the environment, the antibiotic activity of their related substances has been neglected, which may underestimate the environmental impacts of antibiotic wastewater discharge.

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