Sulfonamide Resistance Genes Sul1 Research Articles

Distribution and relationship between antimicrobial resistance genes and heavy metals in surface sediments of Taihu Lake, China.

Heavy metals, pharmaceuticals, and other wastes released into the environment can significantly influence environmental antibiotic resistance. We investigated the occurrence of 22 antimicrobial resistance genes (ARGs) and 10 heavy metal concentrations, and the relationship between ARGs and heavy metals in surface sediment from seven sites of Lake Taihu. The results showed significant correlations (p < 0.05) between sediment ARG levels, especially for tetracycline and sulfonamides (e.g., tet(A), tet(D), tet(E), tet(O), sul1, sul2 and int-1) and specific heavy metals (Fe, Mn, Cr, Cu, Zn, among others) in the Lake. In the surface sediments, heavy metals showed an interaction with resistance genes, but the strength of interaction was diminished with increasing depth. For most of the heavy metals, the concentration of elements in the top sediments was higher than that in other depths. Tetracycline resistance genes (tet(A), tet(B), tet(D), tet(E) and tet(O), β-lactam resistance genes (SHV, TEM, CTX, OXA and OXY) and sulfonamide resistance genes (sulA, sul1, sul2, sul3 and int-1) were detected. They showed a trend which inferred a statistically significant increase followed by decreases in the relative abundance of these ARGs (normalized to 16S rRNA genes) with increasing depth. This study revealed that tet(A), tet(O), TEM, OXY, int-1, sul1 and sul3 were widespread in surface sediments with high abundance, indicating that these genes deserve more attention in future work.

Antimicrobial Resistance in Bordetella bronchiseptica.

Bordetella bronchiseptica is involved in respiratory tract infections mainly in dogs and pigs but may also cause infections in humans. Valid and representative data on antimicrobial susceptibility of B. bronchiseptica is rare. Approved antimicrobial susceptibility testing methods have been published, but very few clinical breakpoints are available. The MIC values are low for most agents but high for β-lactam antibiotics and macrolides. Information on the genetic basis of resistance is scarce. For a small number of isolates that are resistant or show elevated MICs, the molecular basis of resistance was identified. Three tetracycline resistance genes, tet(A), tet(C), and tet(31), coding for major facilitator superfamily efflux pumps, were identified. Two other major facilitator superfamily exporter genes confer resistance to chloramphenicol (cmlB1) or to chloramphenicol and florfenicol (floR). Two class B chloramphenicol acetyltransferase genes (catB1 and catB3), which confer resistance to nonfluorinated phenicols by enzymatic inactivation, have been identified in B. bronchiseptica. Like the trimethoprim resistance genes dfrA1 and dfrB1, which code for trimethoprim-insensitive dihydrofolate reductases, the genes catB1 and catB3 were located on gene cassettes and found in class 1 integrons also harboring the sulfonamide resistance gene sul1. In addition, the gene sul2 has also been detected. Both sul1 and sul2 code for sulfonamide-insensitive dihydropteroate synthases. A gene cassette harboring the β-lactamase gene blaOXA-2 was also identified, whereas β-lactam resistance in B. bronchiseptica seems to be more likely due to reduced influx in combination with the species-specific β-lactamase encoded by blaBOR-1. The resistance genes were mostly located on conjugative plasmids.

The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1

Aquaculture, one of the most important food production practices worldwide, faces serious challenges of mitigating the detrimental impacts of intensive farming on the environment and increased prevalence of antibiotic resistance. To develop an environment-friendly aquaculture system, a land-based and farm-scale sequentially integrated multi-trophic aquaculture (IMTA) system was established for farming Chanos chanos in southwestern Taiwan. In this system, fishes are cultured in combination with organic extractive shellfish and inorganic extractive seaweed. This study aimed to evaluate the prevalence of sulfonamide-resistant bacteria, microbial community structure, and occurrence of sulfonamide resistance genes in the IMTA and traditional aquaculture systems. Water and sediment samples were collected before raising and after harvesting C. chanos. Our results showed that the occurrence of sulfonamide-resistant phenotypes in the IMTA system was comparable with that in influent seawater, while the traditional system exhibited a high sulfonamide resistance rate. Additionally, the traditional system resulted in a deviation of the bacterial community structure from that of seawater. In the water samples from the IMTA system and influent seawater, Proteobacteria and Bacteroidetes were the two dominant phyla, representing approximately 75% and 15% of the community, respectively. In the traditional system, Actinobacteria, constituting 39% of the community, was the dominant bacterial phylum. Thirty-one sulfonamide-resistant bacterial species were isolated. In conclusion, a sequentially IMTA system showed superior ability to maintain the prevalence of antibiotic resistance and the integrity of the bacterial community structure compared to the traditional farming system, representing a potentially valuable aquaculture system for preserving the sustainability of the marine environment.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 β-lactamases. In addition to the β-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, β-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Antibiotics and microbial resistance in Brazilian soils under manure application

AbstractAnimal manure is commonly used in agriculture as fertiliser. This practice could represent, however, a risk of soil degradation through the accumulation of pharmaceutical residues or resistant microorganisms. Thus, 12 pharmaceuticals (11 antibiotics and 1 nonsteroidal anti‐inflammatory drug) were quantified in soil samples from 15 sites regularly fertilised with different animal manures (dairy cow, pig or poultry litter manure) and from forest sites in southern Brazil. This study (a) investigated the impacts of Brazilian animal waste application on antibiotic levels and induced bacterial resistance of soils and (b) determined whether one type of waste is more polluting than others. Three bacterial resistance genes, namely, sul1, qnr A, and erm, were also quantified to determine the impacts of manure application on the resistance of soil microbial communities. The effect of manure application was confirmed by the presence of antibiotic residues (up to 38.3 μg kg−1) and bacterial resistance modifications of the bacterial community (up to 8.97 × 10−3 copies of genes per bacteria) in the studied soils. Soils amended with pig manure presented the highest antibiotic concentrations. However, soils subjected to dairy cow grazing over long periods of time presented the highest concentrations of sulfonamide resistance gene (sul1). Our results lead us to infer that the type of manure can induce a “specific pollution fingerprint.”

Establishment of a Multiplex Loop-Mediated Isothermal Amplification Method for Rapid Detection of Sulfonamide Resistance Genes (sul1, sul2, sul3) in Clinical Enterobacteriaceae Isolates from Poultry.

Antimicrobial resistance genes play an important role in mediating resistance to sulfonamide in Gram-negative bacteria. While PCR is the current method to detect sulfonamide resistance genes (sul1, sul2, sul3), it is time-consuming and costly and there is an urgent need to develop a more convenient, simpler and rapid test for the sul. In this study, we describe a multiplex loop-mediated isothermal amplification (m-LAMP) assay we developed for the rapid and simultaneous detection of three sul. This m-LAMP assay successfully detected seven reference strains with different sul genotypes, but was negative for nine sul-negative reference strains. The m-LAMP products were verified by HinfI restriction enzyme digestion and the detection limit of the test was 0.5 pg genomic DNA per reaction. Testing 307 sulfonamide-resistant Enterobacteriaceae clinical isolates with the m-LAMP revealed all were positive for the sul with sul2 (79.5%) and sul1 (64.5%) being most prevalent, and sul3 the least (12.1%). Of the Enterobacteriaceae isolates tested, the Salmonella Indiana, a newly emerging serovar resistant to numerous antimicrobials, were most commonly positive with 33% having sul3.

Photosynthetic purple sulfur bacterium Marichromatium purpuratum RuA2 induces changes in water quality parameters, the occurrence of sulfonamide resistance gene and microbial community structure of marine aquaculture

Traditional methods to combat disease with antimicrobial agents in aquaculture have caused serious concern and alternative approaches using probiotics have been sought. A photosynthetic purple sulfur bacteria Marichromatium purpuratum RuA2 that thrives at salt concentrations of 0.5% to 5.0% and has a more temperate tolerance for oxygen was isolated from a marine fish pond located on the southwestern shore of Qigu, Taiwan. The effects of M. purpuratum RuA2 on the water quality parameters, the occurrence of the sulfonamide resistance gene sul1 and the microbial community were investigated in situ. Supplementation with strain RuA2 resulted in an increased removal rate of total organic carbon and nitrate ions, as well as a lower occurrence of sul1, in the bacterial community. The abundance and diversity of the bacterial community in the control and M. purpuratum RuA2-supplemented water samples were investigated. Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Planctomycetes were identified to be the top five predominant phyla in the control and M. purpuratum RuA2-supplemented water samples. However, supplementation with M. purpuratum RuA2 increased the diversity of the bacterial community composition. Fourteen unique genera were detected solely in strain RuA2-supplemented water samples and the relative abundance of seven shared genera was associated with supplementation with strain RuA2. In conclusion, M. purpuratum RuA2 showed a desirable capacity for improving water quality, reducing the occurrence of the resistance gene sul1 and advantageous effects on the microbial community structure. Tolerant to salinity and oxygen, M. purpuratum RuA2 exhibits an attractive potential for resolving the challenges facing aquaculture today.

Tetracyclines, sulfonamides and quinolones and their corresponding resistance genes in the Three Gorges Reservoir, China

The Three Gorges Project significantly impacted water quality and ecological balance in this area. The special engineered aquatic environment could be an important reservoir for antibiotic resistance genes (ARGs). Fifteen ARGs corresponding to three groups of antibiotics (tetracyclines, sulfonamides and quinolones) were determined in surface water, soil and sediment in this study. Total concentrations of antibiotics ranged from 21.55 to 536.86ng/L, 3.69 to 438.76ng/g, 15.78 to 213.84ng/g in water, soil and sediment, respectively. Polymerase chain reaction (PCR) of ARGs revealed the presence of two sulfonamide resistance genes (sul1, sul2), five tetracycline resistance genes (tetA, tetB, tetM, tetQ, tetG) and class 1 integron gene (intI1) in all samples. And the relative abundance of sulfonamide resistance genes was generally higher than tetracycline resistance genes in three matrices. Significant correlations (p<0.05) were found between the concentrations of intI1 and ARGs (tetA, tetB, tetM, tetQ, tetG, sul1, sul2), indicating intI1 may facilitate the proliferation and propagation of these genes. Redundancy analysis (RDA) showed distribution of ARGs was related to the certain antibiotics residues, which may exert selective pressure on bacteria and thus enrich the abundance of ARGs. The results of this study could provide useful information for both better understanding and management of the contamination caused by ARGs and related antibiotics in engineered aquatic environments.

Detection of virulence genes and plasmid replicons in Salmonella enterica subsp. enterica, which were allocated during 2014–2017 on the territory of Ukraine

The article presents the results of the identification of virulence and antibiotic resistance genes, as well as plasmid replicons in isolates of Salmonella enterica subsp. enterica, which were isolated on the territory of Ukraine during 2014–2017. Also, plasmid replicons were identified for further determination of the type of plasmids contained in the genome of the isolates. The ability of Salmonella to obtain new properties is well known. The most common way is gaining of genetic information as a result of conjugation. In this case, genes encoding factors of pathogenicity, adhesion, virulence, or antibiotic resistance are localized on mobile genetic elements: replicons, transposons, or plasmids. The purpose of the work was to investigate isolates for the presence of genes of virulence, antibiotic resistance and replicons of plasmids in 50 isolates of Salmonella enterica subsp. enterica, isolated during 2014–2017 in Ukraine. The research was carried out by polymerase chain reaction with primers for 10 target genes/loci (invA, agfB, sefA, prt, sul1, 5'-3'CS, tetG, pN, pFIA, pFIIA) followed by visualization in agarose gel electrophoresis. It was found that 100% of all strains (50/50) had invA and agfB genes. SefA and prt genes were identified in 44% (22/50) and 58% (29/50) isolates, respectively. The sulfonamide resistance gene sul1 was detected in only four isolates, including 2 S. enteritidis isolates from Odesa and Kyiv oblasts, one isolate S. Virchow, and one unidentified isolate. The resistance gene for tetG tetracyclines was found only in 34% of isolates (17/50). The conservative sequence of integron In104 was detected in 52% of isolates (26/50). Replicon plasmid pN was detected in 68% of isolates (34/50), pFIIA – in 44% (22/50). Replicon pFIA is found in 8% (4/50) isolates. Solving the problem of non-typhoid salmonellosis is possible by controlling the epidemiological and epizootiological situation. According to WHO, more than 90 million cases of non-typhoid infection are reported annually. The cause of disease were mostly Enteritidis and Typhimurium. The variety and widespread distribution of salmonella is due primarily to their ability to adapt to the organism that they infect. The results obtained are important for determining the pathogenicity of isolates circulating in a particular area, as well as expanding the possibilities for tracking the source of infection.

Prevalence and Molecular Characterization of Antimicrobial Resistance in Escherichia coli Isolated from Raw Milk and Raw Milk Cheese in Egypt

The goal of this study was to examine antimicrobial resistance and characterize the implicated genes in 222 isolates of Escherichia coli from 187 samples of raw milk and the two most popular cheeses in Egypt. E. coli isolates were tested for susceptibility to 12 antimicrobials by a disk diffusion method. Among the 222 E. coli isolates, 66 (29.7%) were resistant to one or more antimicrobials, and half of these resistant isolates showed a multidrug resistance phenotype (resistance to at least three different drug classes). The resistance traits were observed to tetracycline (27.5%), ampicillin (18.9%), streptomycin (18.5%), sulfamethoxazole-trimethoprim (11.3%), cefotaxime (4.5%), kanamycin (4.1%), ceftazidime (3.6%), chloramphenicol (2.3%), nalidixic acid (1.8%), and ciprofloxacin (1.4%). No resistance to fosfomycin and imipenem was observed. Tetracycline resistance genes tetA, tetB, and tetD were detected in 53 isolates, 9 isolates, and 1 isolate, respectively, but tetC was not detected. Aminoglycoside resistance genes strA, strB, aadA, and aphA1 were detected in 41, 41, 11, and 9 isolates, respectively. Sulfonamide resistance genes sul1, sul2, and sul3 were detected in 7, 25, and 3 isolates, respectively. Of 42 ampicillin-resistant isolates, blaTEM, blaCTX-M, and blaSHV were detected in 40, 9, and 3 isolates, respectively, and 10 (23.8%) ampicillin-resistant isolates were found to produce extended-spectrum β-lactamase. Each bla gene of extended-spectrum β-lactamase-producing E. coli was further subtyped to be blaCTX-M-15, blaCTX-M-104, blaTEM-1, and blaSHV-12. The class 1 integron was also detected in 28 resistant isolates, and three different patterns were obtained by PCR-restriction fragment length polymorphism. Sequencing analysis of the variable region revealed that four isolates had dfrA12/orfF/aadA2, two had aadA22, and one had dfrA1/aadA1. These data suggest that antimicrobial-resistant E. coli are widely distributed in the milk production and processing environment in Egypt and may play a role in dissemination of antimicrobial resistance to other pathogenic and commensal bacteria.

Discovery of the fourth mobile sulfonamide resistance gene

BackgroundOver the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context.ResultsUp to 89% of identified open reading frames encode known resistance genes, or variations thereof (> 1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1–3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe.ConclusionsOur findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.

Metagenomic profiling of ARGs in airborne particulate matters during a severe smog event

Information is currently limited regarding the distribution of antibiotic resistance genes (ARGs) in smog and their correlations with airborne bacteria. This study characterized the diversity and abundance of ARGs in the particulate matters (PMs) of severe smog based on publicly available metagenomic data, and revealed the occurrence of 205 airborne ARG subtypes, including 31 dominant ones encoding resistance to 11 antibiotic types. Among the detectable ARGs, tetracycline, β-lactam and aminoglycoside resistance genes had the highest abundance, and smog and soil had similar composition characteristics of ARGs. During the smog event, the total abundance of airborne ARGs ranged from 4.90 to 38.07ppm in PM2.5 samples, and from 7.61 to 38.49ppm in PM10 samples, which were 1.6–7.7 times and 2.1–5.1 times of those in the non-smog day, respectively. The airborne ARGs showed complicated co-occurrence patterns, which were heavily influenced by the interaction of bacterial community, and physicochemical and meteorological factors. Lactobacillus and sulfonamide resistance gene sul1 were determined as keystones in the co-occurrence network of microbial taxa and airborne ARGs. The results may help to understand the distribution patterns of ARGs in smog for the potential health risk evaluation.

Antimicrobial resistance and resistance genes in Salmonella strains isolated from broiler chickens along the slaughtering process in China

A total of 189 Salmonella isolates were recovered from 627 samples which were collected from cecal contents of broilers, chicken carcasses, chicken meat after cutting step and frozen broiler chicken products along the slaughtering process at a slaughterhouse in Sichuan province of China. The Salmonella isolates were subjected to antimicrobial susceptibility testing to 10 categories of antimicrobial agents using the Kirby–Bauer disk diffusion method. Those antibiotics-resistant isolates were further investigated for the occurrence of resistance genes, the presence of class 1 integron as well as the associated gene cassettes, and the mutations within the gyrA and parC genes. Consequently, the prevalence of Salmonella was 30.14% (47.96% for cecal content, 18.78% for chicken carcasses, 31.33% for cutting meat and 14.00% for frozen meat, respectively). The predominant serotypes were S. Typhimurium (15.34%) and S. Enteritidis (69.84%). High resistance rates to the following drugs were observed: nalidixic acid (99.5%), ampicillin (87.8%), tetracycline (51.9%), ciprofloxacin (48.7%), trimethoprim/sulfamethoxazole (48.1%), and spectinomycin (34.4%). Antimicrobial resistance profiling showed that 60.8% of isolates were multidrug resistant (MDR), and MDR strains increased from 44.7% to 78.6% along the slaughtering line. 94.6% (n=157) of beta-lactam-resistant isolates harbored at least one resistance gene of blaTEM or blaCTX-M. The relatively low prevalence of aminoglycoside resistance genes (aac(3)-II, aac(3)-IV, and ant(2″)-I) was found in 49 (66.2%) of antibiotic-resistant isolates. The tetracycline resistance genes (tet(A), tet(B), tet(C), and tet(G) and sulfonamide resistance genes (sul1, sul2, and sul3) were identified in 84 (85.7%) and 89 (97.8%) antibiotic-resistant isolates respectively. floR was identified in 44 (97.8%) florfenicol-resistant isolates. Class 1 integron was detected in 37.4% (n=43) of the MDR isolates. Two different gene cassettes, blaOXA-30-aadA1 (19 isolates) and blaOXA-30-aadA1/drfA1-orfC (2 isolates), were identified in class 1 integron-positive isolates. 97.9% (184/188) of quinolone-resistant isolates had at least one mutation within gyrA or parC. Overall, antimicrobial resistance showed an increasing trend along the slaughtering process. The results showed that broiler chicken products in the slaughterhouse were contaminated with MDR Salmonella, which might originate from food producing animals to some extent, and cross-contamination during slaughter, and facilitate the dissemination of the resistance genes to consumers along the production chain, which suggests importance of controlling Salmonella during slaughter for public health, underlying strict hygiene method and HACCP management to reduce cross-contamination.

The treatment of wastewater containing pharmaceuticals in microcosm constructed wetlands: the occurrence of integrons (int1\u20132) and associated resistance genes (sul1\u20133, qacE\u03941)

The aim of this study was to analyze the occurrence of sulfonamide resistance genes (sul1–3) and other genetic elements as antiseptic resistance gene (qacEΔ1) and class 1 and class 2 integrons (int1–2) in the upper layer of substrate and in the effluent of microcosm constructed wetlands (CWs) treating artificial wastewater containing diclofenac and sulfamethoxazole (SMX), which is a sulfonamide antibiotic. The bacteria in the substrate and in the effluents were equipped with the sul1–2, int1, and qacEΔ1 resistance determinants, which were introduced into the CW system during inoculation with activated sludge and with the soil attached to the rhizosphere of potted seedlings of Phalaris arundinacea ‘Picta’ roots (int1). By comparing the occurrence of the resistance determinants in the upper substrate layer and the effluent, it can be stated that they neither were lost nor emerged along the flow path. The implications of the presence of antibiotic resistance genes in the effluent may entail a risk of antibiotic resistance being spread in the receiving environment. Additionally, transformation products of SMX were determined. According to the obtained results, four (potential) SMX transformation products were identified. Two major metabolites of SMX, 2,3,5-trihydroxy-SMX and 3,5-dihydroxy-SMX, indicated that SMX may be partly oxidized during the treatment. The remaining two SMX transformation products (hydroxy-glutathionyl-SMX and glutathionyl-SMX) are conjugates with glutathione, which suggests the ability of CW bacterial community to degrade SMX and resist antimicrobial stress.

Environmental analysis of typical antibiotic-resistant bacteria and ARGs in farmland soil chronically fertilized with chicken manure

Antibiotics and the corresponding resistant bacteria and resistance genes (ARGs) are generally considered emerging pollutants. To assess the impacts of tetracycline (TC) and sulfonamide (SA) antibiotics that are eliminated with fecaluria as drug prototypes, farmland soil used to research long-term fertilization with chicken manure was collected at four sites in Shandong Province. In this study, the rates of bacterial drug resistance to the same antibiotic decreased with an increase in the concentration of that antibiotic, and the resistance rates to TCs were lower than those to SAs. PCR of ARGs revealed that the ARGs detected at the highest frequency were the TC resistance genes tetW and tetO and the SA resistance genes sul1 and sul2. Real-time qPCR showed that the quantities of ARGs in farmland soil fertilized with chicken manure were significantly greater compared with the control soil. Moreover, significant correlations (R2=0.9525, p<0.05) between the number of sul ARGs and the total SA concentration were observed in all of the soil samples. In summary, this study showed that SAs can induce the appearance of ARGs and pollute the soil environment.

Characterization of a blaNDM‑1‑harboring plasmid from a Salmonellaenterica clinical isolate in China.

The plasmid-mediated transmission of antibiotic resistance genes has been reported to be involved in the development of antibiotic resistance in bacteria, and poses a serious threat for the success of bacterial infection treatment and human health worldwide. The present study used a 454 GS-FLX pyrosequencing system to determine the ~140 kb nucleotide sequence of plasmid pHS36-NDM, which was identified in a Salmonella Stanley isolate from the stool sample of an 11-month-old girl at Lishui Central Hospital, China, and which contains a New Delhi metallo-β-lactamase-1 (NDM-1) carbapenem resistance gene (blaNDM-1). The 181 open reading frames encode proteins with functions including replication, stable inheritance, antibiotic resistance and mobile genetic elements. Both horizontal transfer and passage stability-related genes were identified in pHS36-NDM, including a conserved type 4 secretion system and stbA (stable plasmid inheritance protein A). Two multidrug resistance gene islands were identified: The ISEcp1-blaCMY transposition unit which contains a CMY-6 β-lactamase gene (blaCMY-6) and a quaternary ammonium compound resistance gene (sugE); and the intI1-ISCR27 accessory region, which contained a trimethoprim resistance gene (dfrA12), two aminoglycoside resistance genes (aadA2 and rmtC), a truncated quaternary ammonium compound resistance gene (qacE∆1), a sulfonamide resistance gene (sul1), the blaNDM-1 carbapenemase and a bleomycin resistance gene (bleMBL). pHS36-NDM shared high homology with other blaNDM-1-containing plasmids reported in Sweden, Italy and Japan. However, no previous international travel history was documented for the patient and her family, even to neighboring cities. Furthermore, pHS36-NDM is of a different incompatibility group to other published blaNDM-1-carrying plasmids reported in China, with low homology in the surrounding structure of blaNDM-1. The present study will facilitate the understanding of the underlying resistance and dispersal mechanism of pHS36-NDM, and will deepen our recognition of the ongoing spread of the blaNDM-1-containing plasmids.

Inactivation of Antibiotic Resistant Bacteria and Resistance Genes by Ozone: From Laboratory Experiments to Full-Scale Wastewater Treatment.

Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

Characterization of biocide-tolerant bacteria isolated from cheese and dairy small-medium enterprises

A collection of 120 bacterial isolates from small medium enterprises involved in the production of cow milk and the manufacture of goat cheese were screened for sensitivity to biocides benzalkonium chloride (BC), cetrimide (CT), hexadecylpyridinium chloride (HDP), triclosan (TC), hexachlorophene (CF) and poly-(hexamethylen guanidinium) hydrochloride (PHMG). Nineteen isolates were selected according to biocide tolerance and identified by 16S rDNA sequencing as Lactococcus sp. (6) Enterococcus sp. (1), Lactobacillus sp. (4), Bacillus sp. (1) Escherichia sp. (5), Enterobacter sp. (1) and Helicobacter sp. (1). These were further characterised regarding antimicrobial resistance phenotype and genotype. Several isolates were multiply (3 or more) tolerant to biocides or resistant to antibiotics, but only two Escherichia sp. isolates and Enterobacter sp. were multiply resistant to biocides and antibiotics. Statistical analysis of biocide tolerance and antibiotic resistance revealed significant positive correlations between different biocides and between biocides and antibiotics. The biocide tolerance genes most frequently found were qacEΔ1 and qacA/B. The sulfonamide resistance gene sul1 was found in two Escherichia sp. isolates and in Enterobacter sp., all of which also carried qacEΔ1. Beta-lactam (blaCTX-M, blaPSE) and tetracycline resistance genes [tet(A), tet(C) and tet(D)] were detected. Efflux pump genes acrB and mdfA were found in most Gram-negative isolates. Results from the study suggest that exposure to biocides can indirectly select for antibiotic resistance.

Abundance of Class 1 Integron-Integrase and Sulfonamide Resistance Genes in River Water and Sediment Is Affected by Anthropogenic Pressure and Environmental Factors.

In this study, we determined the presence of class 1 integron-integrase gene in culturable heterotrophic bacteria isolated from river water and sediment sampled upstream and downstream of a wastewater treatment plant effluent discharge. Moreover, we quantified intI1 and sulfonamide resistance genes (sul1 and sul2) in the water and sediment using qPCR. There was no correlation between the results from water and sediment samples, which suggests integron-containing bacteria are differentially retained in these two environmental compartments. The discharge of treated wastewater significantly increased the frequency of intI1 among culturable bacteria and the gene copy number in river water, and increased the number of sul1 genes in the sediment. We also observed seasonal differences in the frequency of the class 1 integron-integrase gene among culturable heterotrophs as well as intI1 copy number in water, but not in sediment. The results suggest that the abundance of class 1 integrons in aquatic habitat depends on anthropogenic pressure and environmental factors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00248-016-0843-4) contains supplementary material, which is available to authorized users.

Removal of antibiotics and antibiotic resistance genes from domestic sewage by constructed wetlands: Effect of flow configuration and plant species

This study aims to investigate the removal of antibiotics and antibiotic resistance genes (ARGs) in raw domestic wastewater by various mesocosm-scale constructed wetlands (CWs) with different flow configurations or plant species including the constructed wetland with or without plant. Six mesocosm-scale CWs with three flow types (surface flow, horizontal subsurface flow and vertical subsurface flow) and two plant species (Thaliadealbata Fraser and Iris tectorum Maxim) were set up in the outdoor. 8 antibiotics including erythromycin-H2O (ETM-H2O), monensin (MON), clarithromycin (CTM), leucomycin (LCM), sulfamethoxazole (SMX), trimethoprim (TMP), sulfamethazine (SMZ) and sulfapyridine (SPD) and 12 genes including three sulfonamide resistance genes (sul1, sul2 and sul3), four tetracycline resistance genes (tetG, tetM, tetO and tetX), two macrolide resistance genes (ermB and ermC), two chloramphenicol resistance genes (cmlA and floR) and 16S rRNA (bacteria) were determined in different matrices (water, particle, substrate and plant phases) from the mesocosm-scale systems. The aqueous removal efficiencies of total antibiotics ranged from 75.8 to 98.6%, while those of total ARGs varied between 63.9 and 84.0% by the mesocosm-scale CWs. The presence of plants was beneficial to the removal of pollutants, and the subsurface flow CWs had higher pollutant removal than the surface flow CWs, especially for antibiotics. According to the mass balance analysis, the masses of all detected antibiotics during the operation period were 247,000, 4920–10,600, 0.05–0.41 and 3500–60,000μg in influent, substrate, plant and effluent of the mesocosm-scale CWs. In the CWs, biodegradation, substrate adsorption and plant uptake all played certain roles in reducing the loadings of nutrients, antibiotics and ARGs, but biodegradation was the most important process in the removal of these pollutants.