scholarly journals Surveillance of potential pathogens and antibiotic resistance in wastewater and surface water from Boston, USA and Vellore, India using long-read metagenomic sequencing

2021 ◽  
Author(s):  
Erica R Fuhrmeister ◽  
Lee E Voth-Gaeddert ◽  
Angeline Metilda ◽  
Albert Tai ◽  
Rebecca E Batorsky ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Surface Water ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Metagenomic Sequencing ◽  
Antibiotic Resistant ◽  
Short Read ◽  
Long Reads ◽  
Long Read

Environmental sampling (wastewater) could be an efficient surveillance strategy to capture global emerging trends in the spread of antibiotic resistance. Long-read DNA sequencing can resolve the genetic context of antibiotic resistance genes (ARGs) and is a promising tool for non- culture-based monitoring of antibiotic-resistant pathogens and ARGs in environmental samples, but has not been rigorously validated against conventional methods. We tested long-read sequencing using the portable Nanopore MinION for surveying pathogens, ARGs, and antibiotic- resistant pathogens in municipal wastewater, hospital wastewater, and surface water collected from Boston, USA and Vellore, India. We compared detection of enteric pathogens by assembly of long reads, with and without short-read polishing, and unassembled raw long reads for ARGs to multiplex real-time PCR. Using real-time PCR as a benchmark, long-read metagenomics was 49% sensitive and 75% specific at pathogen detection in assembled contigs, and 16% sensitive and 100% specific at detecting 28 clinically relevant resistance genes in raw long reads. Short- read polishing did not substantially improve pathogen identification or impact ARG identification in the assembled contigs, demonstrating that short-read polishing is not required, which greatly reduces costs. The high specificity of ARG detection supports portable long-read sequencing as a valuable tool to profile ARGs and antibiotic-resistant pathogens for environmental surveillance programs.

Characterization of Antibiotic Resistance E. Coli and Antibiotic Resistance Genes in Aquatic Environment of Taihu Lake, China

2013 ◽  
Vol 295-298 ◽  
pp. 630-634 ◽  
Author(s):  
Ni Ni Han ◽  
Song He Zhang ◽  
Pei Fang Wang ◽  
Chao Wang
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Surface Water ◽  
Resistance Genes ◽  
Taihu Lake ◽  
Antibiotic Resistance Genes ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Realtime Pcr

The aims of this study are to evaluate multiple antibiotic resistant Escherichia coli isolated from surface water and to investigate the presence and distribution antibiotic resistance genes (ARGs) in sediments of Taihu Lake. The results show that the presentence of four ARGs concentrations in the sediments of the lake was in sequence: strB>qnrB>strA>qnrS, as determined by realtime-PCR technique. The southwest and east areas of Taihu Lake were polluted seriously than other areas from all kinds of antibiotics. The screening Escherichia coli had a higher resistance to streptomycin, tetracycline and ampicillin than other four antibiotics, and had a lowest resistance to levofloxacin.

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.

Prevalence of antibiotic resistance genes in antibiotic-resistant Escherichia coli isolates in surface water of Taihu Lake Basin, China

2015 ◽  
Vol 22 (15) ◽  
pp. 11412-11421 ◽  
Author(s):  
Song He Zhang ◽  
Xiaoyang Lv ◽  
Bing Han ◽  
Xiucong Gu ◽  
Pei Fang Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Surface Water ◽  
Resistance Genes ◽  
Taihu Lake ◽  
Antibiotic Resistance Genes ◽  
Lake Basin ◽  
Antibiotic Resistant ◽  
Taihu Lake Basin

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. 

Evaluation of five antibiotic resistance genes in wastewater treatment systems of swine farms by real-time PCR

2014 ◽  
Vol 496 ◽  
pp. 116-121 ◽  
Author(s):  
Chi-Wei Tao ◽  
Bing-Mu Hsu ◽  
Wen-Tsai Ji ◽  
Tsui-Kang Hsu ◽  
Po-Min Kao ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Wastewater Treatment ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Antibiotic Resistance Genes ◽  
Swine Farms ◽  
Wastewater Treatment Systems

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 From the Farms to the Dining Table: The Distribution and Molecular Characteristics of Antibiotic-Resistant Enterococcus spp. in Intensive Pig Farming in South Africa

2021 ◽  
Vol 9 (5) ◽  
pp. 882
Author(s):  
Sasha Badul ◽  
Akebe L. K. Abia ◽  
Daniel G. Amoako ◽  
Keith Perrett ◽  
Linda A. Bester ◽  
...  
Keyword(s):  
South Africa ◽  
Antibiotic Resistance ◽  
Real Time ◽  
Real Time Pcr ◽  
Diffusion Method ◽  
Antibiotic Resistant ◽  
Pig Farming ◽  
Enterococcus Spp ◽  
Food Animals ◽  
The Continuum

Foodborne pathogens, including antibiotic-resistant species, constitute a severe menace to food safety globally, especially food animals. Identifying points of concern that need immediate mitigation measures to prevent these bacteria from reaching households requires a broad understanding of these pathogens’ spread along the food production chain. We investigated the distribution, antibiotic susceptibility, molecular characterization and clonality of Enterococcus spp. in an intensive pig production continuum in South Africa, using the farm-to-fork approach. Enterococcus spp. were isolated from 452 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) using the IDEXX Enterolert®/Quanti-Tray® 2000 system. Pure colonies were obtained on selective media and confirmed by real-time PCR, targeting genus- and species-specific genes. The susceptibility to antibiotics was determined by the Kirby–Bauer disk diffusion method against 16 antibiotics recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. A total of 284 isolates, consisting of 79.2% E. faecalis, 6.7% E. faecium, 2.5% E. casseliflavus, 0.4% E. gallinarum, and 11.2% other Enterococcus spp., were collected along the farm-to-fork continuum. The isolates were most resistant to sulfamethoxazole-trimethoprim (78.8%) and least resistant to levofloxacin (5.6%). No resistance was observed to vancomycin, teicoplanin, tigecycline and linezolid. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. Also, 78% of the isolates were multidrug-resistant. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3′)-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most detected virulence gene was gelE. Clonality revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, mainly isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. The results suggest that intensive pig farming could serve as a reservoir of antibiotic-resistant bacteria that could be transmitted to occupationally exposed workers via direct contact with animals or consumers through animal products/food. This highlights the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.

scholarly journals Finding the right fit: A comprehensive evaluation of short-read and long-read sequencing approaches to maximize the utility of clinical microbiome data

2021 ◽  
Author(s):  
Jeanette L. Gehrig ◽  
Daniel M. Portik ◽  
Mark D. Driscoll ◽  
Eric Jackson ◽  
Shreyasee Chakraborty ◽  
...  
Keyword(s):  
Active Ingredient ◽  
Taxonomic Resolution ◽  
23S Rrna ◽  
Metagenomic Sequencing ◽  
Short Read ◽  
Microbiome Analysis ◽  
Long Reads ◽  
Microbiome Research ◽  
Long Read ◽  
Microbiome Data

A longstanding challenge in human microbiome research is achieving the taxonomic and functional resolution needed to generate testable hypotheses about the gut microbiome's impact on health and disease. More recently, this challenge has extended to a need for in-depth understanding of the pharmacokinetics and pharmacodynamics of clinical microbiome-based interventions. Whole genome metagenomic sequencing provides high taxonomic resolution and information on metagenome functional capacity, but the required deep sequencing is costly. For this reason, short-read sequencing of the bacterial 16S ribosomal RNA (rRNA) gene is the standard for microbiota profiling, despite its poor taxonomic resolution. The recent falling costs and improved fidelity of long-read sequencing warrant an evaluation of this approach for clinical microbiome analysis. We used samples from participants enrolled in a Phase 1b clinical trial of a novel live biotherapeutic product to perform a comparative analysis of short-read and long-read amplicon and metagenomic sequencing approaches to assess their value for generating informative and actionable clinical microbiome data. Comparison of ubiquitous short-read 16S rRNA amplicon profiling to long-read profiling of the 16S-ITS-23S rRNA amplicon showed that only the latter provided strain-level community resolution and insight into novel taxa. Across all methods, overall community taxonomic profiles were comparable and relationships between samples were conserved, highlighting the accuracy of modern microbiome analysis pipelines. All methods identified an active ingredient strain in treated study participants, though detection confidence was higher for long-read methods. Read coverage from both metagenomic methods provided evidence of active ingredient strain replication in some treated participants. Compared to short-read metagenomics, approximately twice the proportion of long reads were assigned functional annotations (63% vs. 34%). Finally, similar bacterial metagenome-assembled genomes (MAGs) were recovered across short-read and long-read metagenomic methods, although MAGs recovered from long reads were more complete. Overall, despite higher costs, long-read microbiome characterization provides added scientific value for clinical microbiome research in the form of higher taxonomic and functional resolution and improved recovery of microbial genomes compared to traditional short-read methodologies.

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