Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration. - Related Documents




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343701.0000Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration. Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying ≥ 2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.201829332216
333110.9998Impact of Wastewater Treatment on the Prevalence of Integrons and the Genetic Diversity of Integron Gene Cassettes. The integron platform allows the acquisition, expression, and dissemination of antibiotic resistance genes within gene cassettes. Wastewater treatment plants (WWTPs) contain abundant resistance genes; however, knowledge about the impacts of wastewater treatment on integrons and their gene cassettes is limited. In this study, by using clone library analysis and high-throughput sequencing, we investigated the abundance of class 1, 2, and 3 integrons and their corresponding gene cassettes in three urban WWTPs. Our results showed that class 1 integrons were most abundant in WWTPs and that wastewater treatment significantly reduced the abundance of all integrons. The WWTP influents harbored the highest diversity of class 1 integron gene cassettes, whereas class 3 integron gene cassettes exhibited highest diversity in activated sludge. Most of the gene cassette arrays detected in class 1 integrons were novel. Aminoglycoside, beta-lactam, and trimethoprim resistance genes were highly prevalent in class 1 integron gene cassettes, while class 3 integrons mainly carried beta-lactam resistance gene cassettes. A core class 1 integron resistance gene cassette pool persisted during wastewater treatment, implying that these resistance genes could have high potential to spread into environments through WWTPs. These data provide new insights into the impact of wastewater treatment on integron pools and highlight the need for surveillance of resistance genes within both class 1 and 3 integrons.IMPORTANCE Wastewater treatment plants represent a significant sink and transport medium for antibiotic resistance bacteria and genes spreading into environments. Integrons are important genetic elements involved in the evolution of antibiotic resistance. To better understand the impact of wastewater treatment on integrons and their gene cassette contexts, we conducted clone library construction and high-throughput sequencing to analyze gene cassette contexts for class 1 and class 3 integrons during the wastewater treatment process. This study comprehensively profiled the distribution of integrons and their gene cassettes (especially class 3 integrons) in influents, activated sludge, and effluents of conventional municipal wastewater treatment plants. We further demonstrated that while wastewater treatment significantly reduced the abundance of integrons and the diversity of associated gene cassettes, a large fraction of integrons persisted in wastewater effluents and were consequentially discharged into downstream natural environments.201829475864
343820.9998Dynamics of class 1 integrons in aerobic biofilm reactors spiked with antibiotics. Class 1 integrons are strongly associated with the dissemination of antibiotic resistance in bacteria. However, little is known about whether the presence of antibiotics affects the abundance of integrons and antibiotic resistance genes during biological wastewater treatment. To explore the roles of class 1 integrons in spreading antibiotic resistance genes in environmental compartments, the dynamics of integrons were followed in biofilm reactors treating synthetic wastewater respectively spiked with streptomycin (STM) and oxytetracycline (OTC). The relative abundance of the integron-integrase gene (intI1) increased 12 or 29-fold respectively when treated with STM or OTC, under incrementally increasing dosage regimes from 0 to 50 mg L(-1). Significant increases in intI1 abundance initially occurred at an antibiotic dose of 0.1 mg L(-1). At the beginning of the experiment, 51% to 64% of integrons carried no gene cassettes. In STM and OTC spiked systems, there was a significant increase in the proportion of integrons that contained resistance gene cassettes, particularly at intermediate and higher antibiotic concentrations. Gene cassettes encoding resistance to aminoglycosides, trimethoprim, beta-lactam, erythromycin, and quaternary ammonium compounds were all detected in the treated systems. Three tetracycline resistance genes (tetA, tetC, tetG) were significantly correlated with the abundance of intI1 (p < 0.01), despite no tet resistance being present as a gene cassette. Genome sequencing of isolates showed synteny between the tet resistance genes and intI1, mediated through linkage to transposable elements including Tn3, IS26 and ISCR3. Class 1 integrons appeared to be under positive selection in the presence of antibiotics, and might have actively acquired new gene cassettes during the experiment.202032474215
535430.9998Cultivation-dependent and high-throughput sequencing approaches studying the co-occurrence of antibiotic resistance genes in municipal sewage system. During the past years, antibiotic-resistant bacteria (ARB) leading for the spreading of antibiotic resistance genes (ARGs) became a global problem, especially multidrug-resistant (MDR) bacteria are considered the prime culprit of antibiotic resistance. However, the correlation between the antibiotic-resistant phenotype and the ARG profiles remains poorly understood. In the present study, metagenomic functional screening and metagenomic analysis of coliforms were combined to explore the phenotype and genotype of the ARBs from municipal sewage. Our results showed that the ARG co-occurrence was widespread in the municipal sewage. The present study also highlighted the high abundance of ARGs from antibiotic resistance coliforms especially the MDR coliforms with ARG level of 33.8 ± 4.2 copies per cell. The ARG profiles and the antibiotic resistance phenotypes of the isolated antibiotic resistant coliforms were also correlated and indicated that the resistance to the related antibiotic (ampicillin, kanamycin, erythromycin, chloramphenicol, and tetracycline) was mostly contributed by the ARGs belonging to the subtypes of β-lactamase, aminoglycoside 3-phosphotransferase, phosphotransferase type 2, chloramphenicol acetyltransferase, tetA, etc.201729034431
336340.9998Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content. Bioactive amounts of antibiotics as well as resistant bacteria reach the soil through manure fertilization. We investigated plasmids that may stimulate the environmental spread and interspecies transfer of antibiotic resistance. After treatment of two soils with manure, either with or without the sulfonamide antibiotic sulfadiazine, a significant increase in copies of the sulfonamide resistance gene sul2 was detected by qPCR. All sul2 carrying plasmids, captured in Escherichia coli from soil, belonged to a novel class of self-transferable replicons. Manuring and sulfadiazine significantly increased the abundance of this replicon type in a chemically fertilized but not in an annually manured soil, as determined by qPCR targeting a transfer gene. Restriction patterns and antibiograms showed a considerable diversity within this novel plasmid group. Analysis of three complete plasmid sequences revealed a conserved 30 kbp backbone with only 36% G+C content, comprised of transfer and maintenance genes with moderate homology to plasmid pIPO2 and a replication module (rep and oriV) of other descent. The plasmids differed in composition of the 27.0-28.3 kbp accessory region, each of which carried ISCR2 and several resistance genes. Acinetobacter spp. was identified as a potential host of such LowGC-type plasmids in manure and soil.200919055690
535550.9998Impact of wastewater treatment processes on antimicrobial resistance genes and their co-occurrence with virulence genes in Escherichia coli. An increase in the frequency of antimicrobial resistance genes (ARGs) in bacteria including Escherichia coli could be a threat to public health. This study investigated the impact of activated sludge and physicochemical wastewater treatment processes on the prevalence of ARGs in E. coli isolates. In total, 719 E. coli were isolated from the influent and effluent (prior to disinfection) of two activated sludge and two physicochemical municipal treatment plants, and genotyped using DNA microarrays. Changes in the abundance of ARGs in the E. coli population were different for the two treatment processes. Activated sludge treatment did not change the prevalence of ARG-possessing E. coli but increased the abundance of ARGs in the E. coli genome while physicochemical treatment reduced both the prevalence of ARG-carrying E. coli as well as the frequency of ARGs in the E. coli genome. Most E. coli isolates from the four treatment plants possessed ARGs of multiple antimicrobial classes, mainly aminoglycoside, β-lactams, quinolone and tetracyclines. In addition these isolates harboured DNA insertion sequence elements including integrase and transposase. A significant positive association was found between the occurrence of ARGs and virulence genotypes.201424380739
314060.9997Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables. Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1-2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and bla(TEM-157) and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.202236371907
344470.9997Multidrug resistance in bacteria associated with leafy greens and soil in urban agriculture systems. Urban farms and community gardens support local food production, though these agroecosystems can contain emerging environmental contaminants that may contribute to the dissemination of antimicrobial resistance (AMR). Our previous research enumerated AMR bacteria associated with leafy vegetable production environments in the greater Washington, D.C. area, identifying >100 isolates with multidrug-resistant (MDR) phenotypes. Here, we performed whole genome sequencing analysis of 87 of these strains recovered from leafy greens (n=29), root zone soil (n=42), and bulk soil (n=16) to comprehensively characterize their MDR genotypes, including taxonomy and any encoded ARGs, stress response genes, and mobile genetic elements (MGEs; e.g., plasmids, phages, conjugative elements). The MDR isolates spanned 4 phyla and 14 genera, with the majority identified as Pseudomonas (n = 29), Serratia (n = 22), Providencia (n = 11), and Bacillus (n = 11). Most of the ARGs were linked to multidrug efflux, while other abundant ARG classes reflected resistance to beta-lactams and tetracyclines. While the genotypes were often conserved within respective species and even genera, the observed phenotypes within taxonomic groups slightly varied, suggesting the potential roles of uncharacterized genetic elements in MDR function. Moreover, all of the MDR isolates encoded at least one gene annotated as a MGE, and there were 19 distinct ARGs located within 5,000 bp upstream or downstream of these sequences, suggesting potential implications for mobilization. Overall, our results indicate that the MDR bacteria in urban agriculture systems, including on fresh produce, are dominated by general soil-associated taxa that carry diverse ARGs and MGEs.202541059364
342280.9997Exploring the Role of Coliform Bacteria in Class 1 Integron Carriage and Biofilm Formation During Drinking Water Treatment. This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacEΔ1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.201627079455
313890.9997Insight into the diversity of antibiotic resistance genes in the intestinal bacteria of shrimp Penaeus vannamei by culture-dependent and independent approaches. Antibiotic resistance genes (ARGs) that distributed in antibiotic resistant bacteria (ARBs) are widespread in aquaculture and have great threats to the aquatic organism as well as to human. However, our understanding about the risk of ARGs to the health of aquatic organism is still limited. In the present study, we got a deep insight into the diversity of ARGs in the intestinal bacteria of shrimp by culture-dependent and independent approaches. Results of the PCR-based detection and culture-dependent analysis indicated that the tetracycline, sulfadiazine, quinolone and erythromycin resistance genes were prevalent in the commercial shrimps that bought from aquatic markets or supermarket. The culture-independent plasmid metagenomic analysis identified 62 different ARGs, which were classified into 21 types, with abundances ranging from 13 to 1418 ppm. The analysis suggested that most of the ARGs come from the plasmids originating from Vibrio (accounted for 2.8-51%) and Aeromonas (accounted for 16-55%), and the Vibrio group was concluded to be the main bacterial pathogen that probably resulted in the shrimp disease. Accordingly, the plasmid metagenomic that focuses on the mobile genetic elements has great potential on the identification of ARGs in complex environments.201930735978
3677100.9997Prevalence of sulfonamide-resistant bacteria, resistance genes and integron-associated horizontal gene transfer in natural water bodies and soils adjacent to a swine feedlot in northern Taiwan. Antibiotics are commonly used in swine feed to treat and prevent disease, as well as to promote growth. Antibiotics released into the environment via wastewater could accelerate the emergence of antibiotic-resistant bacteria and resistance genes in the surrounding environment. In this study, we quantified the occurrence of sulfonamides, sulfonamide-resistant microorganisms and resistance genes in the wastewater from a swine farm in northern Taiwan and its surrounding natural water bodies and soils. Sulfonamide levels were similar in the receiving downstream and upstream river water. However, the prevalence of sulfonamide-resistant bacteria and resistance genes, as analyzed by cultivation-dependent and -independent molecular approaches, was significantly greater in the downstream compared to the upstream river water samples. Barcoded-pyrosequencing revealed a highly diverse bacterial community structure in each sample. However, the sequence identity of the sulfonamide resistance gene sul1 in the wastewater and downstream environment samples was nearly identical (99-100%). The sul1 gene, which is genetically linked to class 1 integrons, was dominant in the downstream water bodies and soils. In conclusion, the increased prevalence of sulfonamide resistance genes in the wastewater from a swine farm, independent of the persistent presence of sulfonamides, could be a potential source of resistant gene pools in the surrounding environment.201424637153
3370110.9997Microbiological contamination and resistance genes in biofilms occurring during the drinking water treatment process. Biofilms are the predominant mode of microbial growth in drinking water systems. A dynamic exchange of individuals occurs between the attached and planktonic populations, while lateral gene transfer mediates genetic exchange in these bacterial communities. Integrons are important vectors for the spread of antimicrobial resistance. The presence of class 1 integrons (intI1, qac and sul genes) was assessed in biofilms occurring throughout the drinking water treatment process. Isolates from general and specific culture media, covering a wide range of environmental bacteria, fecal indicators and opportunistic pathogens were tested. From 96 isolates tested, 9.37% were found to possess genetic determinants of putative antimicrobial resistance, and these occurred in both Gram-positive and Gram-negative bacteria. Class 1 integron integrase gene was present in 8.33% of bacteria, all positive for the qacEΔ1 gene. The sul1 gene was present in 3.12% of total isolates, representing 37.5% of the class 1 integron positive cells. The present study shows that biofilm communities in a drinking water treatment plant are a reservoir of class 1 integrons, mainly in bacteria that may be associated with microbiological contamination. Eight out of nine integron bearing strains (88.8%) were identified based on 16S rRNA gene sequencing as either enteric bacteria or species that may be connected to animal and anthropogenic disturbance.201323247295
3424120.9997Contribution of bacteriophage and plasmid DNA to the mobilization of antibiotic resistance genes in a river receiving treated wastewater discharges. In this study, we quantified eleven antibiotic compounds and nine antibiotic resistance genes (ARGs) in water samples collected upstream and downstream of the discharge point from a municipal wastewater treatment plant (WWTP) into the Ter River. Antibiotics were analyzed by liquid chromatography coupled to mass spectrometry, whereas the concentration of ARGs in bacterial, phage and plasmid DNA fractions was determined by real-time PCR to explore their contribution to environmental antibiotic resistance. WWTP discharges resulted in higher concentrations of antibiotic residues as well as ARGs in water samples collected downstream the impact point. Specifically, genes conferring resistance to macrolides (ermB), fluoroquinolones (qnrS) and tetracyclines (tetW) showed significant differences (p<0.05) between upstream and downstream sites in the three DNA fractions (i.e. bacteria, plasmids and phages). Interestingly, genes conferring resistance to β-lactams (bla(TEM), bla(NDM) and bla(KPC)) and glycopeptides (vanA) only showed significant differences (p<0.05) between upstream and downstream sites in phage and plasmid DNA but not in the bacterial DNA fraction. Our results show for the first time the extent to which phages and plasmids contribute to the mobilization of ARGs in an aquatic environment exposed to chronic antibiotic pollution via WWTP discharges. Accordingly, these mobile genetic elements should be included in further studies to get a global view of the spread of antibiotic resistance.201728551539
5373130.9997Impact of soil supplemented with pig manure on the abundance of antibiotic resistant bacteria and their associated genes. This study was conducted to evaluate the abundance of antibiotic resistant bacteria and their resistance genes from agriculture soil supplemented with pig manure. Uncultivable soil sample was supplemented with pig manure samples under microcosm experimental conditions and plated on Luria-Bertani (LB) agar incorporated with commercial antibiotics. The supplementation of soil with 15% pig manure resulted in the highest increase in the population of antibiotic resistant bacteria (ARB)/multiple antibiotic resistant bacteria (MARB). Seven genera that included Pseudomonas, Escherichia, Providencia, Salmonella, Bacillus, Alcaligenes and Paenalcaligenes were the cultivable ARB identified. A total of ten antibiotic resistant bacteria genes (ARGs) frequently used in clinical or veterinary settings and two mobile genetic elements (MGEs) (Class 1 and Class 2 integrons) were detected. Eight heavy metal, copper, cadmium, chromium, manganese, lead, zinc, iron, and cobalt were found in all of the manure samples at different concentrations. Tetracycline resistance genes were widely distributed with prevalence of 50%, while aminoglycoside and quinolone-resistance gene had 16% and 13%, respectively. Eighteen ARB isolates carried more than two ARGs in their genome. Class 1 integron was detected among all the 18 ARB with prevalence of 90-100%, while Class 2 integron was detected among 11 ARB. The two classes of integron were found among 10 ARB. Undoubtedly, pig manure collected from farms in Akure metropolis are rich in ARB and their abundance might play a vital role in the dissemination of resistance genes among clinically-relevant pathogens.202337308603
3470140.9997A treatment plant receiving waste water from multiple bulk drug manufacturers is a reservoir for highly multi-drug resistant integron-bearing bacteria. The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.201324204801
3471150.9997The prevalence of ampicillin-resistant opportunistic pathogenic bacteria undergoing selective stress of heavy metal pollutants in the Xiangjiang River, China. The emergence of clinically relevant β-lactam-resistant bacteria poses a serious threat to human health and presents a major challenge for medical treatment. How opportunistic pathogenic bacteria acquire antibiotic resistance and the prevalence of antibiotic-resistant opportunistic pathogenic bacteria in the environment are still unclear. In this study, we further confirmed that the selective pressure of heavy metals contributes to the increase in ampicillin-resistant opportunistic pathogens in the Xiangjiang River. Four ampicillin-resistant opportunistic pathogenic bacteria (Pseudomonas monteilii, Aeromonas hydrophila, Acinetobacter baumannii, and Staphylococcus epidermidis) were isolated on Luria-Bertani (LB) agar plates and identified by 16S rRNA sequencing. The abundance of these opportunistic pathogenic bacteria significantly increased in the sites downstream of the Xiangjiang River that were heavily influenced by metal mining activities. A microcosm experiment showed that the abundance of β-lactam resistance genes carried by opportunistic pathogenic bacteria in the heavy metal (Cu(2+) and Zn(2+)) treatment group was 2-10 times higher than that in the control. Moreover, heavy metals (Cu(2+) and Zn(2+)) significantly increased the horizontal transfer of plasmids in pathogenic bacteria. Of particular interest is that heavy metals facilitated the horizontal transfer of conjugative plasmids, which may lead to the prevalence of multidrug-resistant pathogenic bacteria in the Xiangjiang River.202133035873
3245160.9997From Metagenomes to Functional Expression of Resistance: floR Gene Diversity in Bacteria from Salmon Farms. Background. The increase in antibiotic resistance in human-impacted environments, such as coastal waters with aquaculture activity, is related to the widespread use of antibiotics, even at sub-lethal concentrations. In Chile, the world's second largest producer of salmon, aquaculture is considered the main source of antibiotics in coastal waters. In this work, we aimed to characterize the genetic and phenotypic profiles of antibiotic resistance in bacterial communities from salmon farms. Methods. Bacterial metagenomes from an intensive aquaculture zone in southern Chile were sequenced, and the composition, abundance and sequence of antibiotic resistance genes (ARGs) were analyzed using assembled and raw read data. Total DNA from bacterial communities was used as a template to recover floR gene variants, which were tested by heterologous expression and functional characterization of phenicol resistance. Results. Prediction of ARGs in salmon farm metagenomes using more permissive parameters yielded significantly more results than the default Resistance Gene Identifier (RGI) software. ARGs grouped into drug classes showed similar abundance profiles to global ocean bacteria. The floR gene was the most abundant phenicol-resistance gene with the lowest gene counts, showing a conserved sequence although with variations from the reference floR. These differences were recovered by RGI prediction and, in greater depth, by mapping reads to the floR sequence using SNP base-calling. These variants were analyzed by heterologous expression, revealing the co-existence of high- and low-resistance sequences in the environmental bacteria. Conclusions. This study highlights the importance of combining metagenomic and phenotypic approaches to study the genetic variability in and evolution of antibiotic-resistant bacteria associated with salmon farms.202540001366
3360170.9997Gentamicin resistance genes in environmental bacteria: prevalence and transfer. A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm(r)) genes in different non-clinical environments has been performed. We were interested to find out whether Gm(r) genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gm(r) genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, alpha- and beta-Proteobacteria. Gm(r) genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to alpha-, beta- and gamma-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPbeta plasmids. Molecular detection of Gm(r) genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm(r) genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gm(r) genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm(r) genes was not obvious.200219709289
3443180.9997A hybrid DNA sequencing approach is needed to properly link genotype to phenotype in multi-drug resistant bacteria. Antibiotic resistance genes (ARGs) are now viewed as emerging contaminants posing a potential worldwide human health risk. The degree to which ARGs are transferred to other bacteria via mobile genetic elements (MGEs), including insertion sequences (ISs), plasmids, and phages, has a strong association with their likelihood to function as resistance transfer determinants. Consequently, understanding the structure and function of MGEs is paramount to assessing future health risks associated with ARGs in an environment subjected to strong antibiotic pressure. In this study we used whole genome sequencing, done using MinION and HiSeq platforms, to examine antibiotic resistance determinants among four multidrug resistant bacteria isolated from fish farm effluent in Jeju, South Korea. The combined data was used to ascertain the association between ARGs and MGEs. Hybrid assembly using HiSeq and MinION reads revealed the presence of IncFIB(K) and pVPH2 plasmids, whose sizes were verified using pulsed field gel electrophoresis. Twenty four ARGs and 95 MGEs were identified among the 955 coding sequences annotated on these plasmids. More importantly, 22 of 24 ARGs conferring resistance to various antibiotics were found to be located near MGEs, whereas about a half of the ARGs (11 out of 21) were so in chromosomes. Our results also suggest that the total phenotypic resistance exhibited by the isolates was mainly contributed by these putatively mobilizable ARGs. The study gives genomic insights into the origins of putatively mobilizable ARGs in bacteria subjected to selection pressure.202134330011
5359190.9997Metagenomic insights into plasmid-mediated antimicrobial resistance in poultry slaughterhouse wastewater: antibiotics occurrence and genetic markers. Slaughterhouse wastewater represents important convergence and concentration points for antimicrobial residues, bacteria, and antibiotic resistance genes (ARG), which can promote antimicrobial resistance propagation in different environmental compartments. This study reports the assessment of the metaplasmidome-associated resistome in poultry slaughterhouse wastewater treated by biological processes, employing metagenomic sequencing. Antimicrobial residues from a wastewater treatment plant (WWTP) that treats poultry slaughterhouse influents and effluents were investigated through high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Residues from the macrolide, sulfonamide, and fluoroquinolone classes were detected, the latter two persisting after the wastewater treatment. The genetic markers 16S rRNA rrs (bacterial community) and uidA (Escherichia coli) were investigated by RT-qPCR and the sul1 and int1 genes by qPCR. After treatment, the 16S rRNA rrs, uidA, sul1, and int1 markers exhibited reductions of 0.67, 1.07, 1.28, and 0.79 genes copies, respectively, with no statistical significance (p > 0.05). The plasmidome-focused metagenomics sequences (MiSeq platform (Illumina®)) revealed more than 100 ARG in the WWTP influent, which can potentially confer resistance to 14 pharmacological classes relevant in the human and veterinary clinical contexts, in which the qnr gene (resistance to fluoroquinolones) was the most prevalent. Only 7.8% of ARG were reduced after wastewater treatment, and the remaining 92.2% were associated with an increase in the prevalence of ARG linked to multidrug efflux pumps, substrate-specific for certain classes of antibiotics, or broad resistance to multiple medications. These data demonstrate that wastewater from poultry slaughterhouses plays a crucial role as an ARG reservoir and in the spread of AMR into the environment.202439395082