scholarly journals The transferable resistome of produce

2018 ◽  
Author(s):  
Khald Blau ◽  
Antje Bettermann ◽  
Sven Jechalke ◽  
Eva Fornefeld ◽  
Yann Vanrobaeys ◽  
...  
Keyword(s):  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Blot Hybridization ◽  
Antibiotic Resistance Genes ◽  
Southern Blot Hybridization ◽  
Pcr Primers ◽  
Human Pathogens ◽  
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 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 (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, IncH11, IncU and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids andblaCTX-M-1. From mixed salad and cilantro, IncF, Inc11, 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.

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.

Dissemination of the blaCTX-M-15 gene among Enterobacteriaceae via outer membrane vesicles

2020 ◽  
Vol 75 (9) ◽  
pp. 2442-2451
Author(s):  
Martina Bielaszewska ◽  
Ondřej Daniel ◽  
Helge Karch ◽  
Alexander Mellmann
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Resistance Genes ◽  
Blot Hybridization ◽  
Acquired Resistance ◽  
Antibiotic Resistance Genes ◽  
Membrane Vesicles ◽  
Southern Blot Hybridization ◽  
Outer Membrane Vesicles ◽  
Laboratory Conditions

Abstract Background Bacterial outer membrane vesicles (OMVs) are an emerging source of antibiotic resistance transfer but their role in the spread of the blaCTX-M-15 gene encoding the most frequent CTX-M ESBL in Enterobacteriaceae is unknown. Objectives To determine the presence of blaCTX-M-15 and other antibiotic resistance genes in OMVs of the CTX-M-15-producing MDR Escherichia coli O104:H4 outbreak strain and the ability of these OMVs to spread these genes among Enterobacteriaceae under different conditions. Methods OMV-borne antibiotic resistance genes were detected by PCR; OMV-mediated transfer of blaCTX-M-15 and the associated blaTEM-1 was quantified under laboratory conditions, simulated intraintestinal conditions and under ciprofloxacin stress; resistance to antibiotics and the ESBL phenotype were determined by the CLSI disc diffusion methods and the presence of pESBL by plasmid profiling and Southern blot hybridization. Results E. coli O104:H4 OMVs carried blaCTX-M-15 and blaTEM-1 located on the pESBL plasmid, but not chromosomal antibiotic resistance genes. The OMVs transferred blaCTX-M-15, blaTEM-1 and the associated pESBL into Enterobacteriaceae of different species. The frequencies of the OMV-mediated transfer were significantly increased under simulated intraintestinal conditions and under ciprofloxacin stress when compared with laboratory conditions. The ‘vesiculants’ (i.e. recipients that received the blaCTX-M-15- and blaTEM-1-harbouring pESBL via OMVs) acquired resistance to cefotaxime, ceftazidime and cefpodoxime and expressed the ESBL phenotype. They were able to further spread pESBL and the blaCTX-M-15 and blaTEM-1 genes via OMVs. Conclusions OMVs are efficient vehicles for dissemination of the blaCTX-M-15 gene among Enterobacteriaceae and may contribute to blaCTX-M-15 transfer in the human intestine.

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 Whole Genome Sequencing of Extended-Spectrum- and AmpC- β-Lactamase-Positive Enterobacterales Isolated From Spinach Production in Gauteng Province, South Africa

2021 ◽  
Vol 12 ◽  
Author(s):  
Loandi Richter ◽  
Erika M. du Plessis ◽  
Stacey Duvenage ◽  
Mushal Allam ◽  
Arshad Ismail ◽  
...  
Keyword(s):  
South Africa ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Irrigation Water ◽  
Production Systems ◽  
Antibiotic Resistance Genes ◽  
Whole Genome ◽  
Human Pathogens ◽  
E Coli ◽  
Encoding Gene

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum β-lactamase- (ESBL) and/or AmpC β-lactamase- (AmpC) producing Enterobacterales in irrigation water and associated irrigated fresh produce represents risks related to the environment, food safety, and public health. In South Africa, information about the presence of ESBL/AmpC-producing Enterobacterales from non-clinical sources is limited, particularly in the water–plant-food interface. This study aimed to characterize 19 selected MDR ESBL/AmpC-producing Escherichia coli (n=3), Klebsiella pneumoniae (n=5), Serratia fonticola (n=10), and Salmonella enterica (n=1) isolates from spinach and associated irrigation water samples from two commercial spinach production systems within South Africa, using whole genome sequencing (WGS). Antibiotic resistance genes potentially encoding resistance to eight different classes were present, with blaCTX-M-15 being the dominant ESBL encoding gene and blaACT-types being the dominant AmpC encoding gene detected. A greater number of resistance genes across more antibiotic classes were seen in all the K. pneumoniae strains, compared to the other genera tested. From one farm, blaCTX-M-15-positive K. pneumoniae strains of the same sequence type 985 (ST 985) were present in spinach at harvest and retail samples after processing, suggesting successful persistence of these MDR strains. In addition, ESBL-producing K. pneumoniae ST15, an emerging high-risk clone causing nosocomical outbreaks worldwide, was isolated from irrigation water. Known resistance plasmid replicon types of Enterobacterales including IncFIB, IncFIA, IncFII, IncB/O, and IncHI1B were observed in all strains following analysis with PlasmidFinder. However, blaCTX-M-15 was the only β-lactamase resistance gene associated with plasmids (IncFII and IncFIB) in K. pneumoniae (n=4) strains. In one E. coli and five K. pneumoniae strains, integron In191 was observed. Relevant similarities to human pathogens were predicted with PathogenFinder for all 19 strains, with a confidence of 0.635–0.721 in S. fonticola, 0.852–0.931 in E. coli, 0.796–0.899 in K. pneumoniae, and 0.939 in the S. enterica strain. The presence of MDR ESBL/AmpC-producing E. coli, K. pneumoniae, S. fonticola, and S. enterica with similarities to human pathogens in the agricultural production systems reflects environmental and food contamination mediated by anthropogenic activities, contributing to the spread of antibiotic resistance genes.

scholarly journals Unusual Events Involved in Banana bunchy top virus Strain Evolution

2009 ◽  
Vol 99 (7) ◽  
pp. 812-822 ◽  
Author(s):  
Hui-Chuan Fu ◽  
Jer-Ming Hu ◽  
Ting-Hsuan Hung ◽  
Hong-Ji Su ◽  
Hsin-Hung Yeh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Replication Initiation ◽  
Common Region ◽  
High Sequence Identity ◽  
Banana Bunchy Top Virus ◽  
Stem Loop ◽  
Integral Component

Banana bunchy top virus (BBTV) can be transmitted by aphids and consists of at least six integral components (DNA-R, -U3, -S, -M, -C, and -N). Several additional replication-competent components (additional Reps) are associated with some BBTV isolates. A collected BBTV strain (TW3) that causes mild symptoms was selected to study the processes in BBTV evolution. Southern blot hybridization, polymerase chain reaction (PCR), and real-time PCR did not detect DNA-N in TW3. Real-time PCR quantification of BBTV components revealed that, except for the copy number of TW3 DNA-U3, each detected integral component of BBTV TW3 was at least two orders lower than that of the severe strains. No infection was observed in plants inoculated with aphids, which were first given acquisition access to the TW3-infected banana leaves. Recombination analysis revealed recombination between the integral component TW3 DNA-U3 and the additional Rep DNA-Y. All BBTV integral components contain a replication initiation region (stem-loop common region) that share high sequence identity. Sequence alignment revealed that TW3 DNA-R, -S, -M, and -C all have a stem-loop common region containing a characteristic 9-nucleotide deletion found only in all reported DNA-N. Our data suggest that the additional Rep DNAs can serve as sources of additional genetic diversity for integral BBTV components.

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.

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