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Clinical the rapid rise of antibiotic resistance is one of the major public health problem facing humanity. In recent years, the widespread contamination of the environment (especially water environment) in antibiotic-resistant bacteria has aroused widespread concern. Environment resistant bacteria increase was mainly due to human activities, such as hospitals, municipal, and aquaculture effluent discharge. Environment resistant bacteria can infect humans through drinking water, aquatic products, as well as direct contact means; and these resistant through horizontal gene transfer will carry resistance genes spread to the sensitivity of the pathogen, resulting in environmental health risks. Environmental resistant object of study includes two elements, a target location, target species. At present, people in the water environment of the target location is usually water samples were collected along the basin, the target species is often a small number of specific species (such as E. coli), research water samples the resistant species abundance and their resistance gene. However, less sediment samples of the highest species richness in the water environment, the diversity of different species of the same place is a drug-resistant and drug resistance gene is also inadequate. Extended-spectrum beta-lactamases (ESBLs) is a hydrolyzable three generations of cephalosporin streptozotocin monocyclic beta-lactam antibiotics, a large class of beta-lactamase, mainly by Klebsiella pneumoniae and Escherichia coli generated. ESBLs-producing E. coli is the most common clinical multi-drug-resistant one in the hospital environment and community environment has a higher incidence. People found ESBLs-producing strains from aquatic environments, but has not been reported so far on their diversity in aquatic sediments. Therefore, in order to further grasp the mechanism of resistance of ESBLs-producing bacteria in the aquatic environment, and population characteristics as the target object to the urban river sediments to study the bacteria producing ESBLs and ESBLs genetic diversity. Meanwhile, this paper also application 16SrDNA-based T-RFLP technology, microbial community structure analysis of urban river sediments in the cefotaxime role of biodiversity change, information antibiotics Environmental Microbiology. To elucidate in Guangzhou, a diversity of of hospital wastewater polluted urban river sediments ESBLs bacteria and resistance genes; bacterial community structure in the sediments under the action of different concentrations of cefotaxime diversity changes. Method 1, the sediment samples collected from the river section of the Guangzhou, Beijing, about 1 km away from a large downstream of the top three hospitals. Culture and non-culture methods to study the mechanism of resistance of ESBLs diversity. In order to be separated as much as possible out of a variety of types of bacteria, we selected a variety of medium, including NA NA 10-fold dilution of the culture medium (NA10-1), EMB, SS, TCBS, PIA, AMB, EA and R2a; screening stage of the study in a medium were added to the p-lactam antibiotic ampicillin (30 mg L-1) or cefotaxime (60 mg L-1), in order to reduce bias errors generated due to the type of antibiotic. Tablet coating method bacterial isolates, plus antibiotic medium plates, according to the growth of bacteria form randomly picked 200-300 drug-resistant strains were isolated and purified, and further ESBLs phenotype detection and identification of bacteria. Resistant bacteria ESBLs phenotypic assays by three steps: (1) using the CLSI recommended ESBLs-producing bacteria phenotype screening test; (2) CLSI phenotypic confirmatory test; (3) PHOENIX-100 automatic identification / susceptibility Determination of MIC values, confirm once again screened strains producing extended-spectrum beta-lactamase enzyme. ESBLs-producing strains identified by PHOENIX-100 automatic identification / susceptibility system biochemical identification and 16SrDNA-sequencing strain type of bacteria. In addition, according to the MIC values ??corresponding to common antibiotics PHOENIX-100 automatic identification / susceptibility measured observation the These ESBLs-producing strains of multi-drug resistant case. At the same time, by polymerase chain reaction (PCR) technique ESBLs-producing strains ESBLs gene types, including blaTEM, blaSHV, blaIMp, blaOXA and blaCTX-M. This article also used the Ultraclean Soil DNA kit to extract sediment total DNA, and combined with the TA clone library sequencing, analysis of the sediment microbial diversity of the CTX-M type ESBLs gene from the perspective of non-culture. 2, sediment samples used in this study bacterial community structure diversity in ibid., respectively, said 30g of the sediment samples in mg L-1, 6.4 mg L-1, 64 mg L-1 and 320 mg L-1 of cephalosporin cefotaxime the role 0D, 3D, 7d, 14d and 21d, and then take 2g each treatment group in different sampling times, respectively, said all samples; based SDS directly extracted soil DNA extraction of total DNA of each sample; Finally, based 16SrDNA T-RFLP techniques, collection of T-RFLP patterns obtained on the fragment size, peak area and peak height, combined with the sampling time or the concentration of antibiotics, bacterial community structure diversity in the statistical analysis; and diversity coefficient statistical formulas to calculate OUT, species richness, diversity index (Shannon-Weiner index) and species, and combines ANOVA statistical analysis SPSS statistical software for different time points of bacterial community structure diversity; same time, by nested PCR analysis diversity in the different time points resistance gene blaCTX-M. 1, resistant isolates of ESBLs Phenotype: beta lactam antibiotics on a variety of medium plate colony growth morphology, the study of randomly picked 245 anti-p-lactam antibiotic resistant. After phenotypic screening and confirmation experiments, the 76 resistant preliminary confirmed as ESBLs-producing strains. 76 strains producing ESBLs results determined by PHOENIX-100 MIC values, 56 last confirmed as ESBLs-producing strains. ESBLs-producing strains of bacteria were identified: 56 ESBLs-producing strains of bacteria of the intestinal rod Branch 39, in which the case (31) for Escherichia coli, Klebsiella (2), zig-zag genus (1) gas Aeromonas spp (4). In addition, we also found that pale spp (7) Pseudochrobactrum the genus saccharolyticum (3) and Acinetobacter ESBLs-producing strains of the genus (9). 3, multi-drug resistant strains producing ESBLs: These strains producing ESBLs in addition to beta-lactam antibiotics ampicillin, piperacillin and aztreonam, and the first, second, third and fourth generation cephalosporins cefazolin the morpholine, ceftazidime be, cefotaxime and cefepime high resistance (resistance rate of more than 90%), other types of antibiotic resistance is not high (resistance rates are lower than 50%), but is still subject to the carbapenem antibiotic imipenem and meropenem inhibited (resistance rate of less than 3%). The 4, ESBLs genotype of detection: 56 strains producing ESBLs the PCR electrophoresis and sequencing results can be seen, of blaTEM and blaCTX-M-positive strains were 22 and 45, and blaOXA and blaIMP-positive strains of the only one, blaSHV can not be detected. Further, according to the results of the BLASTN sequencing comparison, we blaCTX-M gene isoforms, respectively, with A, B, C, D, E said, A subtype 22, the sequence of blaCTX-M27 (AB545827) 96% similarity; and eight subtypes B, and blaCTX-M27 (AB545827) 100% similar; C subtype 5 blaCTx-M64 (AB284167) 100% similarity; and 6 subtype D, and blaCTX-M3 (GU125712) 100% similar; subtype E strains blaKluY-3 (AY623934), 95% similarity. 22 blaTEM strain genetic subtypes are with blaTEM 1 39 (GQ149344) 100% consistent. BlaCTX-M type ESBLs gene library: randomly picked 60 positive clones were sequenced, 47 positive clones obtained the right the blaCTX of-M type ESBLs gene sequences, these gene sequences using BLASTN sequencing comparison, for 13 species blaCTX-M subtypes, and the formation of two major blaCTX-M subtypes clustering branching. 6, the the cefotaxime city river sediment microbial community structure and diversity of blaCTX-M: It can be seen from the clustering results, all samples were divided into two main bacterial community structure clustering branches, the first sub- branches for all of the samples before and after the third day, and second branches of the 7th day, 14 day and 21-day samples. Further observation PCA analysis results, you can see the laboratory processing time can cause bacterial community structure have a significant change, and the presence and concentration of the bacterial community structure changes with antibiotics, there was no significant correlation. In addition, the different time points of bacterial community diversity index by ANOVA statistical analysis, each sample with the the laboratory treatment time, were no significant differences in bacterial species and a variety of the relative number of bacterial community diversity , but the relative degrees of bacterial species away significantly. Results, further species of different time points coefficient OneWay ANOVA analysis results visible, 7d and 14d, 3d and 21d and 14d and 21d bacteria relative degree difference significant. Seen by nested PCR electrophoresis results, all sampling time are detected blaCTX-1, blaCTX-2 and blaCTX-8, were detected only in the role of DNA (0d) blaCTX-25 in the active and inactive 3d detected. Conclusion This article reveals a the hospital wastewater contaminated urban river sediments highly diverse ESBLs resistance mechanisms. On the one hand, the sediments contain many different species of ESBL bacteria, Enterobacteriaceae both known, there has not been reported Brucella Moraxellacea Division ESBL-producing bacteria; On the other hand, these ESBL bacteria carries the type of drug resistance genes, in particular for the main resistance gene blaCTX-M type. bldCTX-M both identical with the reported sequence for known genes, the type having a different degree of variation in the potential of the new genes. Sediments highly abundant resistant case of resistance genes together to form highly rich diversity of ESBL-producing repository. This study shows that urban rivers may provide an important potential source of infection in the community of ESBL-producing bacteria. Urban river sediment microbial community structure, this paper analyzed through the establishment of cefotaxime role model diversity in flora, failed to find a significant change in the role of antibiotics, also found no significant differences in microbial community structure. PCR results showed that the resistance gene blaCTX-M with laboratory processing time, the decline of diversity. Therefore, this paper reveals, river sediments have been exposed to the discharge of waste water pollution, which is rich in a large number of drug-resistant group, the overall microbial community structure on the role of antibiotics is not sensitive. And microbial community structure of non-situ carried out research, laboratory processing time is the most significant factor.
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