Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. - Related Documents




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355201.0000Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids. In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes.200818557938
285610.9998Multiresistant Enterobacteriaceae with class 1 and class 2 integrons in a municipal wastewater treatment plant. In this study, 1832 strains of the family Enterobacteriaceae were isolated from different stages of a municipal wastewater treatment plant, of which 221 (12.1%) were intI-positive. Among them 61.5% originated from raw sewage, 12.7% from aeration tank and 25.8% from the final effluent. All of the intI-positive strains were multiresistant, i.e. resistant to at least three unrelated antimicrobials. Although there were no significant differences in resistance range, defined as the number of antimicrobial classes to which an isolate was resistant, between strains isolated from different stages of wastewater treatment, for five β-lactams the percentage of resistant isolates was the highest in final effluent, which may reflect a selective pressure the bacteria are exposed to, and the possible route of dissemination of β-lactam resistant strains to the corresponding river. The sizes of the variable part of integrons ranged from 0.18 to 3.0 kbp and contained up to four incorporated gene cassettes. Sequence analysis identified over 30 different gene cassettes, including 24 conferring resistance to antibiotics. The highest number of different gene cassettes was found in bacteria isolated from the final effluent. The gene cassettes were arranged in 26 different resistance cassette arrays; the most often were dfrA1-aadA1, aadA1, dfrA17-aadA5 and dfrA12-orfF-aadA2. Regarding the diversity of resistance genes and the number of multiresistant bacteria in the final effluent, we concluded that municipal sewage may serve as a reservoir of integron-embedded antibiotic resistance genes.201222507248
289220.9998Characterization and transferability of class 1 integrons in commensal bacteria isolated from farm and nonfarm environments. This study assessed the distribution of class 1 integrons in commensal bacteria isolated from agricultural and nonfarm environments, and the transferability of class 1 integrons to pathogenic bacteria. A total of 26 class 1 integron-positive isolates were detected in fecal samples from cattle operations and a city park, water samples from a beef ranch and city lakes, and soil, feed (unused), manure, and compost samples from a dairy farm. Antimicrobial susceptibility testing of class 1 integron-positive Enterobacteriaceae isolates from city locations displayed multi-resistance to 12-13 out of the 22 antibiotics tested, whereas class 1 integron-positive Enterobacteriaceae isolates from cattle operations only displayed tetracycline resistance. Most class 1 integrons had one gene cassette belonging to the aadA family that confers resistance to streptomycin and spectinomycin. One isolate from a dog fecal sample collected from a city dog park transferred its class 1 integron to a strain of Escherichia coli O157:H7 at a frequency of 10(-7) transconjugants/donor by in vitro filter mating experiments under the stated laboratory conditions. Due to the numerous factors that may affect the transferability testing, further investigation using different methodologies may be helpful to reveal the transferability of the integrons from other isolates. The presence of class 1 integrons among diverse commensal bacteria from agricultural and nonfarm environments strengthens the possible role of environmental commensals in serving as reservoirs of antibiotic resistance genes.201020704511
295330.9998Diverse Mobile Genetic Elements and Conjugal Transferability of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Escherichia coli Isolates From Penaeus vannamei and Pork From Large Markets in Zhejiang, China. High prevalence rates of sulfonamide resistance genes sul1, sul2, and sul3 have been observed in Gram-negative bacteria isolated from humans, domestic animals, and aquaculture species worldwide. We investigated the distribution characteristics, location, conjugative transferability, and genetic environments of sul genes from Escherichia coli isolates collected from Penaeus vannamei and pork samples from three large markets in Zhejiang, China. The prevalence rates of sul genes in sulfonamide-resistant E. coli isolates from P. vannamei and pork samples were 90.0 and 88.6%, respectively, and the prevalence of sul1 and sul2 was significantly higher than that of sul3 (p < 0.05). Twenty-four representative sul-positive E. coli isolates were analyzed in detail. Southern blot hybridization confirmed that sul genes of E. coli isolates were located on plasmids and/or chromosomes. Transfer of resistance through conjugation was observed in all 18 E. coli isolates harboring sul genes on plasmids. Replicon typing identified seven different incompatibility groups and IncF was the dominant replicon type among sul gene-containing plasmids from both sources. PCR walking analysis indicated that 87.5% (35/40) of sul gene-related fragments carried insertion sequences (ISs) belonging to a variety of families in diverse sites, with IS26 occurring most frequently. In addition, the sul1 gene was detected mainly in fragments carrying class 1 integrons. Co-location on the same fragment with resistance genes that may contribute to the persistence and dissemination of sul1 and/or sul2 genes. The diversity of mobile genetic elements and resistance genes adjacent to sul3 was much lower than those adjacent to sul1 and sul2, especially those located in chromosomes, which reduced the transmission potential of the sul3 gene. In conclusion, combined with the results of clonal relatedness analysis by PFGE and MLST of 24 representative E. coli isolates from P. vannamei and pork samples, it showed that a small number of sul genes were vertically transmitted among E. coli from P. vannamei and that horizontal gene transfer was likely the main transmission mechanism of sul genes from both sources. Our results provide important information to better understand the risk of transmission of sul genes from seafood and meat to humans.201931428076
355440.9998Transmissible Plasmids and Integrons Shift Escherichia coli Population Toward Larger Multiple Drug Resistance Numbers. Transmissible plasmids and integrons may play important roles in the persistence and spread of antibiotic-resistant bacteria throughout aquatic environment by accumulating antibiotic resistance genes (ARG). Class 1 and class 2 integron (intI), mobilization (mob), sulfamethoxazole resistance (sul), and trimethoprim resistance (dfr) genes were PCR-amplified and confirmed through DNA sequencing following plasmid extraction from 139 antibiotic-resistant Escherichia coli. E. coli had previously been recovered from wastewater treatment plant effluent and receiving stream water in Northwest Arkansas and isolates had expressed resistance to one to six antibiotics. Almost half of the total isolates (47%) carried putatively transmissible plasmids with mob(F12) gene as the most frequently detected mobilization gene. When two or three mob genes were detected per isolate, there was a significant shift in the population toward larger multiple drug resistance (MDR) number. Class 1 and/or 2 integrons were prevalent (46%), and the presence of integron significantly shifted the isolate population toward larger MDR number. More isolates carried single or coexistence of two or three sul genes (99.3%), and single or a combination up to five dfr genes (89.3%) than had exhibited in vitro resistance to the respective antibiotics. These findings indicate not only the role of the wastewater treatment effluent and the stream environment in coaccumulation of ARG with transmissible plasmids and integrons in multiple antibiotic-resistant E. coli populations but also suggest that density of sul and dfr resistance genes within an isolate may serve as a biomarker for mobile MDR in general.201829058514
355550.9998Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non-aquaculture sites. Antimicrobial resistance (AR) detected by disc diffusion and antimicrobial resistance genes detected by DNA hybridization and polymerase chain reaction with amplicon sequencing were studied in 124 marine bacterial isolates from a Chilean salmon aquaculture site and 76 from a site without aquaculture 8 km distant. Resistance to one or more antimicrobials was present in 81% of the isolates regardless of site. Resistance to tetracycline was most commonly encoded by tetA and tetG; to trimethoprim, by dfrA1, dfrA5 and dfrA12; to sulfamethizole, by sul1 and sul2; to amoxicillin, by blaTEM ; and to streptomycin, by strA-strB. Integron integrase intl1 was detected in 14 sul1-positive isolates, associated with aad9 gene cassettes in two from the aquaculture site. intl2 Integrase was only detected in three dfrA1-positive isolates from the aquaculture site and was not associated with gene cassettes in any. Of nine isolates tested for conjugation, two from the aquaculture site transferred AR determinants to Escherichia coli. High levels of AR in marine sediments from aquaculture and non-aquaculture sites suggest that dispersion of the large amounts of antimicrobials used in Chilean salmon aquaculture has created selective pressure in areas of the marine environment far removed from the initial site of use of these agents.201424612265
283860.9998On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer. The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets, and the horizontal transfer potential of extended-spectrum β-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying β-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a β-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified: bla(CTX-M-1), bla(CTX-M-14), bla(CTX-M-15), bla(CTX-M-27), bla(CTX-M-55), and bla(PER-1). bla(CTX-M-15) was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including sul1, str(A), str(B), erm(B), erm(F), intI1, aadA, incW, bla(PSE), and bla(OXA-20), were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however, erm(B) and erm(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for in vitro conjugation of plasmids conferring resistance to extended-spectrum β-lactams and other classes of antibiotics into E. coli CV601. IMPORTANCE Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.202133931422
592770.9998The prevalence of, associations between and conjugal transfer of antibiotic resistance genes in Escherichia coli isolated from Norwegian meat and meat products. OBJECTIVES: To investigate the distribution of, associations between and the transferability of antimicrobial resistance genes in resistant Escherichia coli strains isolated from Norwegian meat and meat products. METHODS: The 241 strains investigated were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET) during the years 2000-2003. PCR was carried out for detection of resistance genes. Conjugation experiments were carried out with the resistant isolates from meat as donor strains and E. coli DH5alpha as the recipient strain. Statistical analyses were performed with the SAS-PC-System version 9.1 for Windows. RESULTS: Resistance genes common in pathogenic E. coli were frequently found among the isolates investigated. Strains harbouring several genes encoding resistance to the same antimicrobial agent were significantly (P < 0.0001) more frequently multiresistant than others. Strong positive associations were found between the tet(A) determinant and the genetic elements sul1, dfrA1 and aadA1. Negative associations were found between resistance genes encoding resistance to the same antimicrobial agent: tet(A)/tet(B), sul1/sul2 and strA-strB/aadA1. The resistance genes were successfully transferred from 38% of the isolates. The transfer was more frequent from resistant isolates harbouring class 1 integrons (P < 0.001). CONCLUSIONS: Acquired resistance played a major role in conferring resistance among the isolates investigated. The possibility of transferring resistance increases both by increased multiresistance and by the presence of class 1 integrons. The conjugation experiments suggest that tet(A) and class 1 integrons are often located on the same conjugative plasmid.200616931539
289680.9997Resistance gene patterns of tetracycline resistant Escherichia coli of human and porcine origin. Resistance transfer from animals to humans (and vice versa) is a frequently discussed topic in human and veterinary medicine, albeit relevant studies focus mainly on phenotypic antibiotic resistance. In order to get a comparative insight regarding the distribution of selected resistance genes [tet(A/B/C/D/M/K/L/O/S/W/Z), sulI, II, III, str(A/B), aad(A)] in Escherichia coli of different origins, phenotypically tetracycline resistant isolates of porcine and human origin (n=137 and 152) were investigated using PCR. The most common gene was tet(A) in porcine, but tet(B) in human isolates (>55%). Tet(C/M/D) were rare (1-7%); tet(K/L/O/S/W/Z) were not detected. Co-occurrence of tet(A) and tet(B) was more frequent in human strains (11% vs. 2%). 88% of the porcine isolates had one, and 9% had two tet-genes. By contrast, only 69% of the human strains had one tet-gene, whereas 17% were carriers of two tet-determinants. The most common sulfonamide resistance gene was represented by sulII (40% in porcine, 62% in human isolates), followed by sulI. SulIII was present in eight isolates. Streptomycin resistance was mostly mediated by str(A)/str(B) in porcine, and by str(A)/str(B)/aad(A) in human strains (35% each). In one E. coli of human origin, 7 resistance genes were simultaneously detected. Co-occurrence of 5 or 6 resistance genes was more present in human strains, whereas porcine isolates carried more often only 1-4 genes. The huge diversities between gene patterns of bacteria of human and porcine origin indicate that genetic transfers between microorganisms from different sources are less frequent than transfers within populations of the same source.201019939589
289390.9997Antibiotic-resistant bacteria associated with retail aquaculture products from Guangzhou, China. This study examined the prevalence of antibiotic-resistant (ART) bacteria and representative antibiotic resistance (AR)-encoding genes associated with several aquaculture products from retail markets in Guangzhou, China. ART commensal bacteria were found in 100% of the products examined. Among 505 multidrug-resistant isolates examined, close to one-fourth contained intI and sul1 genes: 15% contained sul2 and 5% contained tet (E). Incidences of β-lactamase-encoding genes bla(TEM), bla(CMY) and erythromycin resistance determinants ermB and ermC were 4.5, 1.7, 1.3, and 0.3%, respectively. Most of the ART isolates identified from the rinse water were Aeromonas spp.; those from intestines belonged to the Enterobacteriaceae. Plasmid-associated intI and AR-encoding genes were identified in several ART isolates by Southern hybridization. Three multidrug resistance-encoding plasmids were transferred into Escherichia coli DH5 a by chemical transformation and led to acquired AR in the transformants. In addition, the AR traits in many isolates were quite stable, even in the absence of selective pressure. Further studies are needed to reveal risk factors associated with the aquaculture production chain for targeted AR mitigation.201323433377
5924100.9997In vivo transfer of an incFIB plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1. Transfer of resistance genes from bacteria from food producing animals to human pathogens is a potential risk to human health. The aim of this study was to determine in vivo transfer of a plasmid harbouring a class 1 integron containing gene cassettes dfrA1-aadA1 from Salmonella to Escherichia coli and the influence of the use of antimicrobials on this transfer. Thirty four-day-old SPF chickens colonized with E. coli K12 were divided into 3 groups of 10 animals each, and placed in separate isolators. Eleven days after inoculation with E. coli K12 the chickens were inoculated orally with 10(4)CFU of S. enterica spp. enterica serovar Typhimurium containing a plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1. Two days after the administration of S. Typhimurium 1 group was treated orally with doxycycline, 1 group orally with trimethoprim/sulphamethoxazole and 1 group remained untreated (control group). E. coli K12, S. Typhimurium and the transconjugants were isolated from cloacal samples on selective MacConkey agar plates. Transfer of the plasmid was confirmed by plasmid characterization, PCR, PFGE and hybridization. Plasmid mediated transfer of a class 1 integron was observed almost immediately after inoculation with S. Typhimurium. Treatment of the chickens with antibiotics had neither a positive nor a negative effect on the transfer rates. In addition to the resistance genes located on the integron, resistance genes encoding for tetracycline and amoxicillin resistance transferred from the donor strain as well. The resistance genes and the integron were located on a 130 kb sized IncFIB plasmid. Our data demonstrate in vivo transfer of an IncFIB plasmid harbouring a class 1 integron containing gene cassettes dfrA1-aadA1 from Salmonella to E. coli, with or without selective pressure of antibiotics in chickens.200919264430
2017110.9997Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant. OBJECTIVES: To investigate the presence and distribution of integron-carrying bacteria from a slaughterhouse wastewater treatment plant (WWTP). METHODS: Enterobacteriaceae and aeromonads were isolated at different stages of the wastewater treatment process and screened for the presence of integrase genes by dot-blot hybridization. Integrase-positive strains were characterized in terms of phylogenetic affiliation, genetic content of integrons and antimicrobial resistance profiles. Plasmid location of some integrons was established by Southern-blot hybridization. Strains containing integron-carrying plasmids were selected for mating experiments. RESULTS: Integrase genes were present in all samples, including the final effluent. The global prevalence was determined to be 35%, higher than in other aquatic environments. Forty-two integrase-positive isolates were further characterized. Nine distinct cassette arrays were found, containing genes encoding resistance to beta-lactams (bla(OXA-30)), aminoglycosides (aadA1, aadA2, aadA13, aadB), streptothricin (sat1, sat2), trimethoprim (dfrA1, dfrA12), a putative esterase (estX) and a protein with unknown function (orfF). Gene cassette arrays aadA1, dfrAI-aadA1 and estX-sat2-aadA1 were common to aeromonads and Enterobacteriaceae. The class 2 integron containing an estX-sat2-aadA1 cassette array was detected for the first time in Aeromonas sp. Nearly 12% (5 out of 43) of intI genes were located in plasmids. intI genes from isolates MM.1.3 and MM.1.5 were successfully conjugated into Escherichia coli at frequencies of 3.79 x 10(-5) and 5.46 x 10(-5) per recipient cell, respectively. CONCLUSIONS: Our data support the hypothesis that WWTPs constitute a potential hot spot for horizontal gene transfer and for selection of antimicrobial resistance genes among aquatic bacteria. Moreover, water discharges represent a possible risk for dissemination of undesirable genetic traits.200717913715
1940120.9997Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture. A total of 273 Escherichia coli isolates from raw and treated municipal wastewaters were investigated to evaluate the frequency and persistence of antibiotic resistance and to detect the occurrence of conjugative R plasmids and integrons. The highest resistance rates were against ampicillin (22.71%), tetracycline (19.41%), sulfamethoxazole (16.84%) and streptomycin (14.28%). Multiple antibiotic resistance was present in 24.17% of the isolates. Several multiple antibiotic-resistant isolates proved to be able to transfer en bloc their resistance patterns by conjugative R plasmids with different molecular sizes and restriction profiles. Class 1 integrons of 1 or 1.5 kbp were found in 5 out of 24 representative multiresistant E. coli isolates. Although wastewater treatments proved to be effective in eliminating Salmonella spp. and in reaching WHO microbiological standards for safe use of wastewater in agriculture, they were ineffective in reducing significantly the frequency of plasmid-mediated multiple antibiotic resistance in surviving E. coli. Since multiple antibiotic-resistant bacteria carrying integrons and conjugative R plasmids can constitute a reservoir of antibiotic-resistance genes in wastewater reclaimed for irrigation, risks for public health should be considered. Bacterial strains carrying R plasmids and integrons could contaminate crops irrigated with reclaimed wastewater and transfer their resistances to the consumers' intestinal bacteria.200919240351
2929130.9997Occurrence of antibiotic resistance and characterization of resistance genes and integrons in Enterobacteriaceae isolated from integrated fish farms in South China. Antibiotics are still widely applied in animal husbandry to prevent diseases and used as feed additives to promote animal growth. This could result in antibiotic resistance to bacteria and antibiotic residues in animals. In this paper, Enterobacteriaceae isolated from four integrated fish farms in Zhongshan, South China were tested for antibiotic resistance, tetracycline resistance genes, sulfonamide resistance genes, and class 1 integrons. The Kirby-Bauer disk diffusion method and polymerase chain reaction (PCR) assays were carried out to test antibiotic susceptibility and resistance genes, respectively. Relatively high antibiotic resistance frequencies were found, especially for ampicillin (80%), tetracycline (52%), and trimethoprim (50%). Out of 203 Enterobacteriaceae isolates, 98.5% were resistant to one or more antibiotics tested. Multiple antibiotic resistance (MAR) was found highest in animal manures with a MAR index of 0.56. Tetracycline resistance genes (tet(A), tet(C)) and sulfonamide resistance genes (sul2) were detected in more than 50% of the isolates. The intI1 gene was found in 170 isolates (83.7%). Both classic and non-classic class 1 integrons were found. Four genes, aadA5, aadA22, dfr2, and dfrA17, were detected. To our knowledge, this is the first report for molecular characterization of antibiotic resistance genes in Enterobacteriaceae isolated from integrated fish farms in China and the first time that gene cassette array dfrA17-aadA5 has been detected in such fish farms. Results of this study indicated that fish farms may be a reservoir of highly diverse and abundant antibiotic resistant genes and gene cassettes. Integrons may play a key role in multiple antibiotic resistances posing potential health risks to the general public and aquaculture.201121975604
2897140.9997The Role of Flies in Disseminating Plasmids with Antimicrobial-Resistance Genes Between Farms. Dissemination of antimicrobial resistance is a major global public health concern. To clarify the role of flies in disseminating antimicrobial resistance between farms, we isolated and characterized tetracycline-resistant Escherichia coli strains isolated from flies and feces of livestock from four locations housing swine (abattoir, three farms) and three cattle farms. The percentages of isolates from flies resistant to tetracycline, dihydrostreptomycin, ampicillin, and chloramphenicol (80.8%, 61.5%, 53.8%, and 50.0%, respectively) and those from animal feces (80.5%, 78.0%, 41.5%, and 46.3%, respectively) in locations housing swine were significantly higher than those from cattle farms (p<0.05). The rates of resistance in E. coli derived from flies reflected those derived from livestock feces at the same locations, suggesting that antimicrobial resistance spreads between livestock and flies on the farms. The results of pulsed-field gel electrophoresis (PFGE) analysis showed that, with a few exceptions, all E. coli isolates differed. Two pairs of tetracycline-resistant strains harbored similar plasmids with the same tetracycline-resistance genes, although the origin (fly or feces), site of isolation, and PFGE patterns of these strains differed. Therefore, flies may disseminate the plasmids between farms. Our results suggest that flies may be involved not only in spreading clones of antimicrobial-resistant bacteria within a farm but also in the widespread dissemination of plasmids with antimicrobial resistance genes between farms.201526061440
2065150.9997Exogenous plasmid capture to characterize tetracycline-resistance plasmids in sprouts obtained from retail in Germany. This study aimed to characterize antibiotic-resistance plasmids present in microorganisms from sprout samples using exogenous plasmid capture. Fresh mung bean sprouts were predominantly colonized by bacteria from the phyla Proteobacteria and Bacteroidetes. To capture plasmids, a plasmid-free Escherichia (E.) coli CV601 strain, containing a green fluorescent protein gene for selection, was used as the recipient strain in exogenous plasmid capture experiments. Transconjugants were selected on media containing cefotaxime or tetracycline antibiotics. While no cefotaxime-resistant transconjugants were obtained, 40 tetracycline-resistant isolates were obtained and sequenced by Illumina NextSeq short read and Nanopore MinION long read sequencing. Sequences were assembled using Unicycler hybrid assembly. Most of the captured long plasmids carried either the tet(A) or tet(D) resistance gene, belonged to the IncFI or IncFII replicon types, and were predicted as conjugative. While the smaller plasmids contained the tet(A) tetracycline resistance gene as well as additional quinolone (qnrS1), sulfonamide (sul1) and trimethoprim (dfrA1) resistance genes, the larger plasmids only contained the tet(D) resistance gene. An exception was the largest 192 kbp plasmid isolated, which contained the tet(D), as well as sulfonamide (sul1) and streptomycin (aadA1) resistance genes. The smaller plasmid was isolated from different sprout samples more often and showed a 100% identity in size (71,155 bp), while the 180 kbp plasmids showed some smaller or larger differences (in size between 157,683 to 192,360 bp). This suggested that the plasmids obtained from the similar sprout production batches could be clonally related. Nanopore MinION based 16S metagenomics showed the presence of Enterobacter (En.) cloacae, En. ludwigii, En. kobei, Citrobacter (C.) werkmanii, C. freundii, Klebsiella (K.) oxytoca and K. pneumonia, which have previously been isolated from fresh produce in Germany. These bacteria may harbor antibiotic resistance genes on plasmids that could potentially be transferred to similar genera. This study demonstrated that bacteria present in sprouts may act as the donors of antibiotic resistance plasmids which can transfer resistance to other bacteria on this product via conjugation.202540012786
2894160.9997Related antimicrobial resistance genes detected in different bacterial species co-isolated from swine fecal samples. A potential factor leading to the spread of antimicrobial resistance (AR) in bacteria is the horizontal transfer of resistance genes between bacteria in animals or their environment. To investigate this, swine fecal samples were collected on-farm and cultured for Escherichia coli, Salmonella enterica, Campylobacter spp., and Enterococcus spp. which are all commonly found in swine. Forty-nine of the samples from which all four bacteria were recovered were selected yielding a total of 196 isolates for analysis. Isolates were tested for antimicrobial susceptibility followed by hybridization to a DNA microarray designed to detect 775 AR-related genes. E. coli and Salmonella isolated from the same fecal sample had the most AR genes in common among the four bacteria. Genes detected encoded resistance to aminoglycosides (aac(3), aadA1, aadB, and strAB), β-lactams (ampC, ampR, and bla(TEM)), chloramphenicols (cat and floR), sulfanillic acid (sul1/sulI), tetracyclines (tet(A), tet(D), tet(C), tet(G), and tet(R)), and trimethoprim (dfrA1 and dfh). Campylobacter coli and Enterococcus isolated from the same sample frequently had tet(O) and aphA-3 genes detected in common. Almost half (47%) of E. coli and Salmonella isolated from the same fecal sample shared resistance genes at a significant level (χ², p < 0.0000001). These data suggest that there may have been horizontal exchange of AR genes between these bacteria or there may be a common source of AR genes in the swine environment for E. coli and Salmonella.201121385089
3553170.9997Genetic redundancy and persistence of plasmid-mediated trimethoprim/sulfamethoxazole resistant effluent and stream water Escherichia coli. Antibiotic resistant bacteria may persist in effluent receiving surface water in the presence of low (sub-inhibitory) antibiotic concentrations if the bacteria possess multiple genes encoding resistance to the same antibiotic. This redundancy of antibiotic resistance genes may occur in plasmids harboring conjugation and mobilization (mob) and integrase (intI) genes. Plasmids extracted from 76 sulfamethoxazole-trimethoprim resistant Escherichia coli originally isolated from effluent and an effluent-receiving stream were used as DNA template to identify sulfamethoxazole (sul) and trimethoprim (dfr) resistances genes plus detect the presence of intI and mob genes using PCR. Sulfamethoxazole and trimethoprim resistance was plasmid-mediated with three sul (sul1, sul2 and sul3 genes) and four dfr genes (dfrA12, dfrA8, dfrA17, and dfrA1 gene) the most prevalently detected. Approximately half of the plasmids carried class 1 and/or 2 integron and, although unrelated, half were also transmissible. Sampling site in relationship to effluent input significantly affected the number of intI and mob but not the number of sul and dfr genes. In the presence of low (sub-inhibitory) sulfamethoxazole concentration, isolates persisted regardless of integron and mobilization gene designation, whereas in the presence of trimethoprim, the presence of both integron and mobilization genes made isolates less persistent than in the absence of both or the presence of a gene from either group individually. Regardless, isolates persisted in large concentrations throughout the experiment. Treated effluent containing antibiotic resistant bacteria may be an important source of integrase and mobilization genes into the stream environment. Sulfamethoxazole-trimethoprim resistant bacteria may have a high degree of genetic redundancy and diversity carrying resistance to each antibiotic, although the role of integrase and mobilization genes towards persistence is unclear.201627455416
2829180.9997Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. The prevalence of selected streptomycin (Sm)-resistance genes, i.e. aph (3''), aph (6)-1d, aph (6)-1c, ant (3'') and ant (6), was assessed in a range of pristine as well as polluted European habitats. These habitats included bulk and rhizosphere soils, manure from farm animals, activated sludge from wastewater treatment plants and seawater. The methods employed included assessments of the prevalence of the genes in habitat-extracted DNA by PCR, followed by hybridisation with specific probes, Sm-resistant culturable bacteria and exogenous isolation of plasmids carrying Sm-resistance determinants. The direct DNA-based analysis showed that aph (6)-1d genes were most prevalent in the habitats examined. The presence of the other four Sm-modifying genes was demonstrated in 58% of the tested habitats. A small fraction of the bacterial isolates (8%) did not possess any of the selected Sm-modifying genes. These isolates were primarily obtained from activated sludge and manure. The presence of Sm-modifying genes in the isolates often coincided with the presence of IncP plasmids. Exogenous isolation demonstrated the presence of plasmids of 40-200 kb in size harbouring Sm-resistance genes from all the environments tested. Most plasmids were shown to carry the ant (3'') gene, often in combination with other Sm-resistance genes, such as aph (3'') and aph (6)-1d. The most commonly found Sm-modifying gene on mobile genetic elements was ant (3''). Multiple Sm-resistance genes on the same genetic elements appeared to be the rule rather than the exception. It is concluded that Sm-resistance genes are widespread in the environmental habitats studied and often occur on mobile genetic elements and ant (3'') was most often encountered.200219709288
3557190.9997Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water. Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacEΔ1 gene at the 3' conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans.201626991286