Tao Yu

Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river.

ABSTRACT We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as Gammaproteobacteria. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (tet) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with tet(A) being the most common (67.0%), followed by tet(W), tet(C), tet(J), tet(L), tet(D), tet(Y), and tet(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the tet genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.

Bacterial community characteristics under long-term antibiotic selection pressures

To investigate bacterial community characteristics under long-term antibiotic selection pressures, water samples from the upstream and the downstream sections of two rivers individually receiving the treated penicillin G and oxytetracycline production wastewater, as well as the anaerobic and the aerobic effluent of the penicillin G production wastewater treatment plant, were taken and analyzed. Antibiotic resistance ratios of bacterial communities in water samples were estimated by culture-based analysis. The majority of bacterial colonies (approximately 55%-70%) in both downstream rivers and the aerobic effluent showed resistance to 80 μg/ml of antibiotics tested, while the resistance ratios were less than 10% and 5% respectively for both upstream rivers. Six 16S rRNA gene clone libraries were constructed with 355 sequences and 215 OTUs totally obtained representing 465 clones. The antibiotic stresses seemed not reduce the diversities of bacterial communities in antibiotic containing water samples compared to those in the two reference upstream rivers. Bacterial groups present in the two reference upstream rivers were common residents in freshwater ecosystems, with the dominant groups as the phyla Proteobacteria including Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, as well as Actinobacteria and Bacteroidetes. The phyla Proteobacteria and Firmicutes were dominant in all antibiotic containing water samples, with the clones belonged to Deltaproteobacteria and Epsilonproteobacteria significantly abundant, as well as Gram-positive low GC bacteria in the classes Clostridia and Bacilli. It thus seemed that Deltaproteobacteria, Epsilonproteobacteria, Clostridia and Bacilli might be specifically associated with antibiotic containing environments.

Microbial community compositional analysis for series reactors treating high level antibiotic wastewater.

A full-scale biosystem consisting of two anaerobic reactors (HA and BF1) and four aerobic ones (BF2-BF4 and OD) in succession and receiving antibiotic-bearing (mainly streptomycin) wastewater was used for studying the impacts of antibiotics on microbial community structures. Significant decreases of streptomycin (from 3955 ± 1910 to 23.1 ± 4.7 μg L(-1)) and COD(Cr) were observed along the treatment process. Cloning results show that the anaerobic reactors (HA and BF1) were dominated with Deltaproteobacteria (51%) mainly affiliated with sulfate-reducing bacteria (SRB), while the aerobic BF2 receiving streptomycin of 408.6 ± 59.7 μg L(-1) was dominated with Betaproteobacteria (34%), Deltaproteobacteria (31%) and Bacteroidetes (14%). Gammaproteobacteria (15.9-22.4%), Betaproteobacteria (10.0-20.3%), and Bacteroidetes (4.5-29.7%) became the major bacterial groups in aerobic BF3-OD receiving streptomycin of ≤83 ± 13 μg L(-1). Archaea affiliated with Methanomethylovorans hollandica-like methylotroph was abundant in HA and BF1 (archaea/bacteria, 0.54-0.40; based on specific gene copy number), suggesting the coexistence of SRB and methanogens in degrading pollutants. Fungi were abundant (fungi/bacteria, 0.15; based on specific gene copy number) with the dominance of Ascomycota (clone ratio of Ascomycota/eukarya, 25.5%) in BF2, suggesting that fungi could be an important player in pollutant removal under high levels of antibiotics. This study demonstrates that under high antibiotic levels, wastewater treatment communities may maintain system stability through adjusting bacterial, archaeal, and eukaryal compositions.

Nitrifier characteristics in submerged membrane bioreactors under different sludge retention times

Three submerged membrane bioreactors (MBRs) were operated continuously for 230 days by feeding with synthetic inorganic wastewater (NH(4)(+)-N, 100 mg L(-1)) under different solids retention times (SRTs. M(30d), 30 days; M(90d), 90 days; M(infinite), no sludge purge) to examine the influence of SRT on nitrification performance and microbial characteristics. All the reactors could oxidize NH(4)(+)-N to NO(3)(-)-N effectively without accumulation of NO(2)(-)-N. M(30d) with the shortest SRT showed significantly higher specific ammonium oxidizing rate (SAOR, 0.22 kg NH(4)(+)-N kg(-1) MLSS day(-1)) and specific nitrate forming rate (SNFR, 0.13 kg NO(3)(-)-N kg(-1) MLSS day(-1)) than the other two MBRs (0.12-0.14 kg NO(3)(-)-N kg(-1) MLSS day(-1) and 0.042-0.068kg NO(3)(-)-N kg(-1) MLSS day(-1), respectively). Short SRT led to low extracellular polymeric substances (EPS) concentration and long operating cycle. The nitrite oxidizing bacteria (NOB) ratios by both the fluorescence in situ hybridization (FISH) (3.6% for M(30d) and 2.1-2.2% for M(90d) and M(infinite)) and MPN (1.4 x 10(7)cells g(-1) MLSS for M(30d) and 6.2 x 10(5) and 2.7 x 10(4)cells g(-1) MLSS for M(90d) and M(infinite)) analyses showed that M(30d) favored the accumulation of NOB, which was in accordance with the SNFR result. However, the ammonia oxidizing bacteria (AOB) ratios (3.5%, 3.2% and 4.9% for M(30d), M(90d) and M(infinite)) were not in accordance with the SAOR result. PCR-DGGE, clone library and FISH results showed that the fast-growing Nitrosomonas and Nitrobacter sp. were the dominant AOB and NOB, respectively for M(30d), while considerable slow-growing Nitrosospira and Nitrospira sp. existed in M(infinite), which might be an important reason why M(infinite) had a low SAOR and SNFR.

The management of undesirable cyanobacteria blooms in channel catfish ponds using a constructed wetland: Contribution to the control of off-flavor occurrences

An exploratory study on the management of undesirable cyanobacteria blooms with respect to off-flavor problems using an integrated vertical-flow constructed wetland (CW) was performed at a small commercial-scale channel catfish farm from 2004 to 2007. The results of the three-year experiment indicated that water treatment by the CW could reduce the possibility of dominance by undesirable cyanobacteria species that often cause off-flavor problems. A detailed investigation in 2007, showed that the concentrations of geosmin, MIB (2-methylisoborneol), and β-cyclocitral in the water of the recirculating pond (4.3ngL(-1), U.D. (undetected) and 0.2ngL(-1), respectively) treated by the CW were significantly lower than those in the control pond (152.6ngL(-1), 63.3ngL(-1) and 254.8ngL(-1), respectively). In addition, the relationships among the cyanobacteria species, the off-flavor compounds and ten environmental variables were explored by canonical correspondence analysis (CCA). The results showed that Oscillatoria sp., Oscillatoria kawamurae and Microcystis aeruginosa were the main sources of off-flavor compounds in the catfish ponds. The successful manipulation of undesirable cyanobacteria species potentially resulted in lower concentrations of odorous compounds in the water of the recirculating pond. An investigation of the concentrations of geosmin and MIB in catfish fillets showed that the levels of odorous compounds were below the OTC (odor threshold concentration) values in the recirculating pond but were above the OTC values from July to October in the control pond. Water recycling by the CW could potentially be one of the best management practices to control off-flavor occurrences in aquaculture.

Emergence of plasmid-mediated quinolone resistance genes in clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa in Henan, China

A total of 216 clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa were collected from a general hospital in Henan, China, and screened for the plasmid-mediated quinolone resistance (PMQR) determinants. The presence of β-lactamase genes and mutations in quinolone resistance-determining regions were investigated among the PMQR-positive isolates.

Efflux pump-mediated benzalkonium chloride resistance in Listeria monocytogenes isolated from retail food

In this study, efflux pump-mediated benzalkonium chloride (BC) resistance, including plasmid-encoded (Qac protein family and BcrABC) and chromosome-borne efflux pumps, was investigated in Listeria monocytogenes from retail food in China. Among the 59 L. monocytogenes strains, 13 (22.0%) strains were resistant to BC. The PCR results showed that bcrABC was harbored by 2 of 13 BC resistant strains. However, none of the qac genes were detected among the 59 strains. The bcrABC was absent in both of the plasmid cured strains, indicating that this BC resistance determinant was plasmid-encoded in the two bcrABC-positive strains. In the presence of reserpine, most of the bcrABC-negative strains had decreases in the MICs of BC, suggesting the existence of other efflux pumps and their role in BC resistance. After exposed to reserpine, the reduction in BC MICs was observed in the two cured strains, indicating that efflux pumps located on chromosome was also involved in BC resistance. Our findings suggest that food products may act as reservoirs for BC resistant isolates of L. monocytogenes and plasmid- and chromosome-encoded efflux pumps could mediate the BC resistance of L. monocytogenes, which is especially relevant to the adaption of this organism in food-related environments with frequent BC use.

Removal efficiency and balance of nitrogen in a recirculating aquaculture system integrated with constructed wetlands

The nitrogen (N) balance for aquaculture is an important aspect, especially in China, and it is attributed to the eutrophication in many freshwater bodies. In recent years, constructed wetlands (CWs) have been widely used in wastewater treatment and ecosystem restoration. A recirculating aquaculture system (RAS) consisting of CWs and 4 fish ponds was set up in Wuhan, China. Channel catfish (Ictalurus punctatus) fingerlings were fed for satiation daily for 168 days with 2 diets containing 5.49 % and 6.53 % nitrogen, respectively. The objectives of this study were to investigate the N budget in the RAS, and try to find out the feasibility of controlling N accumulation in the fish pond. It is expected that the study can provide a mass balance for the fate of N in the eco-friendly treatment system to avoid eutrophication. The results showed that the removal rates of ammonia (NH+ 4-N), sum of nitrate & nitrite (NO− X -N), and total nitrogen (TN) by the CWs were 20–55%, 38–84 % and 39–57 %, respectively. Denitrification in the CWs was the main pathway of nitrogen loss (41.67 %). Nitrogen accumulation in pond water and sediment accounted for 3.39 % and 12.65 % of total nitrogen loss, respectively. The nitrogen removal efficiency and budget showed that the CW could be used to control excessive nitrogen accumulation in fish ponds. From the viewpoint of the nitrogen pollution control, the RAS combined with the constructed wetland can be applied to ensure the sustainable development for aquaculture.

Characterization and horizontal transfer of class 1 integrons in Escherichia coli isolates from cooked meat products

INTRODUCTION Escherichia coli is a commensal bacterium in humans, animals, and the environment that is one of the microorganisms commonly resistant to antimicrobials. Cooked meat products, which are popular in China, are easily contaminated by E. coli during processing and storage. METHODOLOGY In this study, a total of 75 E. coli isolates from cooked meat products in Henan province, China, were assayed for the presence of and horizontal transfer of class 1 integrons. RESULTS Class 1 integrons were detected in 11 (14.7%) of these isolates, and contained four groups of resistance gene cassettes, including dfrA17-aadA5, dfrA1-aadA1, dfrA12-orfF-aadA2, and an uncommon array of aacA4-catB8-aadA1. The transfer frequency of selected integron-positve donors ranged from 10(-6) to 10(-4) transconjugants per recipient cell, and the integron-containing DNA from the donors could be transferred to E. coli J53Azr with the transformation frequency of 10(-7) to 10(-5). CONCLUSIONS Class 1 integrons could be transferred to recipient E. coli J53 by conjugation and natural transformation. These findings suggest the role of commensal E. coli isolates from cooked meats as an important reservoir for integrons and the possible transfer of antimicrobial resistance genes to humans via the food chain.

Antimicrobial resistance, class 1 integrons, and horizontal transfer in Salmonella isolated from retail food in Henan, China.

INTRODUCTION Salmonellosis remains one of the most frequently occurring foodborne diseases worldwide, especially in developing countries. The increasing prevalence of multidrug resistance among Salmonella isolates from food has been an emerging problem in China. METHODOLOGY In this study, a total of 638 food samples including raw meat, seafood, vegetables, and cooked meat were collected in Henan province of China between July 2007 and August 2008 to determine the prevalence of Salmonella. These isolates were subjected to serotyping, antimicrobial susceptibility, presence of class 1 integrons, and horizontal transfer of integrons. RESULTS The overall percentage of Salmonella prevalence was 9.7% (n = 62). Among these isolates, S. Anatum and S. Senftenberg were most common, and high rates of antimicrobial resistance were observed to sulfamethoxazole (90.3%), trimethoprim/sulfamethoxazole (87.1%), streptomycin (29.0%), and ciprofloxacin (25.8%). Class 1 integrons were detected in 16.1% of these isolates, and contained gene cassettes dfrA12-aadA2, dfrA1-aadA1, and dfrA1. Three Salmonella isolates could transfer their integrons and resistance genes to Escherichia coli by conjugation. CONCLUSIONS Our findings indicate that the mobile DNA elements could play an important role in the dissemination of resistance determinants among those Salmonella isolates.