intI1 Type Mobile Genetic Elements Co-selected Antibiotic-Resistant Genes in Untreated Hospital Wastewaters

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26–ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1–IS26–IS903–ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and β-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.

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Hospital wastewater effluent: hot spot for antibiotic resistant bacteria

Journal of Water Sanitation and Hygiene for Development ◽

10.2166/washdev.2020.086 ◽

2020 ◽

Vol 10 (2) ◽

pp. 171-178 ◽

Cited By ~ 1

Author(s):

Urška Rozman ◽

Darja Duh ◽

Mojca Cimerman ◽

Sonja Šostar Turk

Keyword(s):

Hot Spot ◽

Gene Families ◽

Resistant Bacteria ◽

Hospital Wastewater ◽

Antibiotic Resistant Bacteria ◽

Antibiotic Resistant ◽

Pcr Screening ◽

Beta Lactamases ◽

Resistant Genes ◽

Hospital Wastewaters

Abstract Hospital wastewaters are highly complex effluents acting as a hotspot for antibiotic resistant bacteria. Especially, Gram-negative bacteria bearing multiple antibiotic resistant genes are increasingly found in hospital wastewaters. The aim of this study was to evaluate the presence of extended spectrum beta-lactamases (ESBL) and carbapenemase producing Enterobacteriaceae in hospital wastewaters from one Slovenian and two Austrian hospitals, as well as the occurrence of antibiotic resistant genes encoding for VIM, KPC, NDM, CTXM and OXA beta-lactamases in isolates from hospital wastewater. The results indicated high levels of ESBL producing Enterobacteriaceae in ranges up to 107 cfu/mL. Carbapenemase producing Enterobacteriaceae and OXA 48-type CPE were present in ranges up to 105 cfu/mL. Out of 89 multiplied polymerase chain reaction (PCR) amplicons, only 36 were positive for different β-lactamase gene families, among those only three isolates were identified as multiresistant. The dominant ESBL family was CTXM in 19 different isolates. This was followed by 10 OXA-48 positive isolates and 10 VIM positive isolates. KPC or NDM carbapenemases were not identified with PCR screening of the isolates. Hospital wastewaters serve as a reservoir for nearly all clinically important antibiotic resistances. The importance of evaluating such potential environmental reservoirs is especially evident when outbreak cases could not be linked to an epidemiological source.

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Molecular tools to detect the IncJ elements: A family of integrating, antibiotic resistant mobile genetic elements

Journal of Microbiological Methods ◽

10.1016/j.mimet.2005.10.004 ◽

2006 ◽

Vol 66 (1) ◽

pp. 32-42 ◽

Cited By ~ 19

Author(s):

Barry M. McGrath ◽

John A. O'Halloran ◽

Anna V. Piterina ◽

J. Tony Pembroke

Keyword(s):

Mobile Genetic Elements ◽

Molecular Tools ◽

Antibiotic Resistant ◽

Genetic Elements

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A novel method to isolate free-floating extracellular DNA from wastewater for quantitation and metagenomic profiling of mobile genetic elements and antibiotic resistance genes

10.1101/2020.05.01.072397 ◽

2020 ◽

Cited By ~ 1

Author(s):

David Calderón-Franco ◽

Mark C. M. van Loosdrecht ◽

Thomas Abeel ◽

David G. Weissbrodt

Keyword(s):

Activated Sludge ◽

Mobile Genetic Elements ◽

Extracellular Dna ◽

Metagenomic Sequencing ◽

Effluent Water ◽

Isolation Method ◽

Genetic Elements ◽

Resistant Genes ◽

Treated Effluent ◽

Diethylaminoethyl Cellulose

AbstractAntibiotic resistant genes (ARGs) and mobile genetic elements (MGEs) can be found in the free-floating extracellular DNA (eDNA) fraction of microbial systems. These xenogenic components can generate bacterial cells resistant to one or more antibiotics by natural transformation. Because of low concentration in wastewater, the obtaining of a high quality and a high yield of eDNA extract is challenging. We developed a method using chromatography to isolate eDNA without causing cell lysis (often unchecked) from complex wastewater matrices. The chromatographic step involved a diethylaminoethyl-cellulose-monolithic column to capture the eDNA found in cell-free filtered wastewater samples (e.g. influent wastewater, activated sludge and treated effluent wastewaster). Free-floating eDNA yields from 1 L of influent, activated sludge and treated effluent water reached 12.5 ± 1.9 μg, 12.3 ± 1 μg and 5.6 ± 2.9 μg of raw eDNA and 9.0 ± 0.7 μg, 5.6 ± 0.46 μg and 2.6 ± 1.3 μg of purified eDNA, respectively. In order to check the suitability of free-floating eDNA extracts for molecular analysis, qPCR and metagenomics were performed. eDNA extracts from treated effluent water were analyzed by qPCR to quantify a selected panel of ARGs and MGEs. Microbiome, resistome, and mobilome profiles from activated sludge free-floating eDNA were measured by metagenomic sequencing. Between iDNA and eDNA fractions, qPCR showed differences of 0.94, 1.11, 1.92 and 1.32 log10 gene copies mL−1 for sulfonamides resistant genes (sul1 and sul2), β-lactamase resistance gene blaCTXM, and the class 1 integron-integrase (intI1) MGE, respectively. These differences highlighted the crucial need for an isolation method to discern both iDNA and eDNA to understand ARGs persistence and quantity in complex cultures. The eDNA yields obtained from 1 L of activated sludge (3.6 g of total suspended solids L−1) samples were substantially higher than the amount of DNA template needed for high-throughput sequencing (>1 μg) in service facilities. Subsystems classification showed that the eDNA metagenome was mainly composed by MGEs (65.1%). The 35.9% rest related to traditional functional genetic signatures. It was the first time the resistome from the eDNA fraction was analyzed showing lower number of primary aligned reads when compared to the iDNA and a predominance of aminoglycosides and β-lactamams. Metagenome results showed that eDNA can not be discarded as a pool of ARGs and MGEs for horizontal gene transfer. This novel isolation method was powerful to elucidate the molecular compositions of free-floating eDNA fractions in complex environmental samples such as wastewater environments at different microbial densities. Data obtained using this extraction method will foster xenogenic and microbial risk assessments across urban and natural water systems. This will support water authorities in the delineation of measures to adopt at wastewater treatment plants to remove them and safeguard environmental and public health.Graphical abstractPicture created with BioRender

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