Sub-lethal concentration of sulfamethoxazole affects the growth performance of milkfish (Chanos chanos), the microbial composition of antibiotic-resistant bacteria and the prevalence of sulfonamide-resistance genes in mariculture.

To investigate the impacts of sub-lethal concentrations of antibiotic agents in mariculture, culturable approach and DNA based detection were employed to isolate and analyse resistant bacteria and resistant genes in this study. Milkfish (Chanos chanos), the target rearing animal was exposed to sulfamethoxazole (SMX; 2 mg/L) for 8 weeks and resulted in reduced survival rate and weight gain to 61.9 % and 28.4 %, respectively compared to control milkfish (p < 0.001). The composition of SMX-resistant bacteria isolated from the culture water and the gastrointestinal tracts of milkfish underwent changes in response to SMX treatment with a reduced diversity. The prevalence of SMX resistant genes sul in bacterial isolates was elevated from 2.8 % of control to 100 % of SMX-administrated water. Exposure to SMX at a sub-lethal dosage enhanced the prevalence of resistance genes sul1 and sul2 in resistant bacteria, thus implying high frequency of resistance dissemination in the marine environment and surrounding ecosystems.

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene.

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.

Degradation of di-n-butyl phthalate by newly isolated Ochrobactrum sp.

A gram negative isolate designated JDC-41 was obtained from river sludge using mixtures of phthalate esters as the sole source and energy. The isolate was identified as Ochrobactrum sp. based on its 16S rRNA gene sequence. Over 87% of supplied di-n-butyl phthalate (DBP) was degraded by JDC-41 in a pH neutral mineral salts medium at 30 degrees C within 48 h. Increased DBP (50-500 mg/L) in the culture correspondingly increased degradation half-life from 3.83 to 18.12 h. DBP induced cells more rapidly degraded DBP.

Characterization of a Sequential UV Photolysis-Biodegradation Process for Treatment of Decabrominated Diphenyl Ethers in Sorbent/Water Systems.

Decabrominated diphenyl ether (BDE-209) is a primary component of the brominated flame retardants used in a variety of industrial and domestic applications. BDE-209 bioaccumulates in aquatic organisms and has been identified as an emerging contaminant that threatens human and ecosystem health. Sequential photolysis-microbial biodegradation processes were utilized here to treat BDE-209 in clay- or soil-water slurries. The removal efficiency of BDE-209 in the clay-water slurries was high; i.e., 96.5%, while that in the soil-water slurries was minimal. In the clay-water slurries the first order rate constants for the UV photolysis and biodegradation of BDE-209 were 0.017 1/day and 0.026 1/day, respectively. UV wavelength and intensity strongly influenced the BDE-209 photolysis and the subsequent biodegradation of photolytic products. Facultative chemotrophic bacteria, including Acidovorax spp., Pseudomonas spp., Novosphingobium spp. and Sphingomonas spp., were the dominant members of the bacterial community (about 71%) at the beginning of the biodegradation; many of these organisms have previously been shown to biodegrade BDE-209 and other polybrominated diphenyl ether (PBDE) congeners. The Achromobacter sp. that were isolated (NH-2; NH-4; NH-6) were especially effective during the BDE-209 degradation. These results indicated the effectiveness of the sequential UV photolysis and biodegradation for treating certain BDE-209-contaminated solids; e.g., clays; in bioreactors containing such solids as aqueous slurries. Achieving a similar treatment effectiveness for more heterogeneous solids containing natural organic matter, e.g., surface solids, appears to be significantly more difficult. Further investigations are needed in order to understand the great difference between the clay-water or soil-water slurries.

Effects of sulfamethoxazole and sulfamethoxazole-degrading bacteria on water quality and microbial communities in milkfish ponds.

Intensive farming practices are typically used for aquaculture. To prevent disease outbreaks, antibiotics are often used to reduce pathogenic bacteria in aquaculture animals. However, the effects of antibiotics on water quality and microbial communities in euryhaline fish culture ponds are largely unknown. The aim of this study was to investigate the interactions between sulfamethoxazole (SMX), water quality and microbial communities in milkfish (Chanos chanos) culture ponds. The results of small-scale milkfish pond experiments indicated that the addition of SMX decreased the abundance of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and photosynthetic bacteria. Consequently, the levels of ammonia and total phosphorus in the fish pond water increased, causing algal and cyanobacterial blooms to occur. In contrast, the addition of the SMX-degrading bacterial strains A12 and L effectively degraded SMX and reduced the levels of ammonia and total phosphorus in fish pond water. Furthermore, the abundances of AOB, NOB and photosynthetic bacteria were restored, and algal and cyanobacterial blooms were inhibited. This study demonstrate the influences of SMX on water quality and microbial community composition in milkfish culture ponds. Moreover, the use of the bacterial strains A12 and L as dual function (bioaugmentation and water quality maintenance) beneficial bacteria was shown to provide an effective approach for the bioremediation of SMX-contaminated euryhaline milkfish culture ponds.

Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells.

Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca(2+)-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca(2+)](c)]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca(2+) signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca(2+) signaling coupled with nAChRs and voltage-operated Ca(2+) channels in a dose-dependent manner, with IC(50)s of 1 and 5.9 microM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca(2+)](c) in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, partially suppressed the [Ca(2+)](c) rise induced by NP, but NP totally blocked the [Ca(2+)](c) rise induced by thapsigargin. This illustrates that NP can cause Ca(2+) release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca(2+) signaling coupled with nAChRs but increased that coupled with voltage-operated Ca(2+) channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca(2+) channels, and Ca(2+)-induced Ca(2+) release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca(2+)-ATPase inhibitor.

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.

Bacterial communities associated with aerobic degradation of polybrominated diphenyl ethers from river sediments.

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants and have therefore drawn much environmental concern. We aimed to compare aerobic degradation of different PBDE congeners under various treatments and reveal the bacterial community associated with PBDE degradation in sediment. Results of this study indicate that degradation rates of BDE-15 were enhanced 45.1 and 81.3 % with the addition of suspended and microencapsulated Pseudomonas sp., respectively. However, the degradation rates of BDE-28, BDE-47, BDE-99, and BDE-100 did not differ among experimental treatments. Degradation rates of PBDE congeners were in the order of BDE-15 > BDE-28 > BDE-47 > BDE-99 > BDE-100. Using a pyrosequencing-based metagenomic approach, we found that addition of various treatments altered the microbial community composition in the sediment. Twenty-four bacterial genera associated with degradation of PBDEs; six are the core bacterial genera common among PBDE degraders. The diverse bacterial composition among different PBDE congener degradation indicates different combinations of bacteria involved in degradation of different PBDE congeners.

Suitability of the traditional microbial indicators and their enumerating methods in the assessment of fecal pollution of subtropical freshwater environments.

Twenty-four freshwater sampling sites (11 river water, 6 spring water, and 7 groundwater) were selected from 4 sampling areas located in the northern and central parts of Taiwan. A total of 125 water samples were collected during a 5-month sampling period, and the numbers of total heterotrophic bacteria, total coliforms, fecal coliforms, enterococci, Aeromonas hydrophila, and Salmonella spp. were enumerated. Besides the traditional membrane filtration method, total coliforms and Escherichia coli were also simultaneously enumerated using the Colilert (Quanti-Tray/2000) method. On average, 94% and 80% of the water samples assessed with the Colilert method had equal or higher total coliform and E. coli counts, respectively, as compared with the membrane filtration method. Furthermore, when m-FC agar was used to enumerate fecal coliforms, 18% of the samples failed to yield the typical bluish colonies, while E. coli were counted in the same samples using the Colilert method. The data indicate that the m-FC agar culture method is inadequate for the enumeration of fecal coliforms in subtropical water samples. Significant correlations were observed between the total number of bacteria and various indicator bacteria in river water samples, but no such correlations were found for groundwater and spring water. This finding suggested that the river water was polluted by anthropogenic sources. The counts of total coliforms, E. coli, and other indicator bacteria were significantly correlated in all river water samples, while in groundwater and spring water, significant (p<0.01) correlation was only observed with enterococcal counts. The presence of total coliforms/E. coli generally implies the presence of fecal pollution possibly including pathogenic enteric bacteria. However, no Salmonella spp. were detected in any of the 107 water samples analyzed. The results of this study suggest that the use of these commonly employed microbial indicators for assessing subtropical water quality, especially in a pristine body of water (ie, mountain spring and groundwater), is highly questionable.

Prevalence of sulfonamide-resistant bacteria, resistance genes and integron-associated horizontal gene transfer in natural water bodies and soils adjacent to a swine feedlot in northern Taiwan.

Antibiotics are commonly used in swine feed to treat and prevent disease, as well as to promote growth. Antibiotics released into the environment via wastewater could accelerate the emergence of antibiotic-resistant bacteria and resistance genes in the surrounding environment. In this study, we quantified the occurrence of sulfonamides, sulfonamide-resistant microorganisms and resistance genes in the wastewater from a swine farm in northern Taiwan and its surrounding natural water bodies and soils. Sulfonamide levels were similar in the receiving downstream and upstream river water. However, the prevalence of sulfonamide-resistant bacteria and resistance genes, as analyzed by cultivation-dependent and -independent molecular approaches, was significantly greater in the downstream compared to the upstream river water samples. Barcoded-pyrosequencing revealed a highly diverse bacterial community structure in each sample. However, the sequence identity of the sulfonamide resistance gene sul1 in the wastewater and downstream environment samples was nearly identical (99-100%). The sul1 gene, which is genetically linked to class 1 integrons, was dominant in the downstream water bodies and soils. In conclusion, the increased prevalence of sulfonamide resistance genes in the wastewater from a swine farm, independent of the persistent presence of sulfonamides, could be a potential source of resistant gene pools in the surrounding environment.

An investigation of total bacterial communities, culturable antibiotic-resistant bacterial communities and integrons in the river water environments of Taipei city.

The intensive use of antibiotics may accelerate the development of antibiotic-resistant bacteria (ARB). The global geographical distribution of environmental ARB has been indicated by many studies. However, the ARB in the water environments of Taiwan has not been extensively investigated. The objective of this study was to investigate the communities of ARB in Huanghsi Stream, which presents a natural acidic (pH 4) water environment. Waishuanghsi Stream provides a neutral (pH 7) water environment and was thus also monitored to allow comparison. The plate counts of culturable bacteria in eight antibiotics indicate that the numbers of culturable carbenicillin- and vancomycin-resistant bacteria in both Huanghsi and Waishuanghsi Streams are greater than the numbers of culturable bacteria resistant to the other antibiotics tested. Using a 16S rDNA sequencing approach, both the antibiotic-resistant bacterial communities (culture-based) and the total bacterial communities (metagenome-based) in Waishuanghsi Stream exhibit a higher diversity than those in Huanghsi Stream were observed. Of the three classes of integron, only class I integrons were identified in Waishuanghsi Stream. Our results suggest that an acidic (pH 4) water environment may not only affect the community composition of antibiotic-resistant bacteria but also the horizontal gene transfer mediated by integrons.

[Isolation and identification of four DBP-degrading strains and molecular cloning of the degradation genes].

Four di-butyl-phthalate(DBP)-degrading bacterial strains, JDC-1, JDC-8, JDC-9 and JDC-12, were isolated from soil. The strains were gram positive. The 16S rRNA sequence analysis revealed that the four strains had similarities of 99% with Arthrobacter sp.. According to the morphologic, physiobiochemical characteristics and the analysis of their 16S rRNA, all the four strains were identified as Arthrobacter sp.. A 900 bp DNA fragment was obtained from the four strains by PCR amplified and clone. When compared with the large subunit of phthalate dioxygenase gene (phtA) of Arthrobacter keyseri, more than 96% similarities were evident in the nucleotide sequences. The optimal growth conditions and degradation rates of DBP were tested and the result indicated that the optimal growth conditions of the four bacteria strains were pH 7.0-8.5 and 30-35 degrees C. All the four bacteria strains performed efficiently for DBP degrading capabilities under optimal conditions. The most efficient strain JDC-1 degraded 500 mg/L DBP completely within 28 h whereas the least efficient strain JDC-8 degraded 500 mg/L DBP completely within 40 h. This study is helpful to the investigation of DBP-degrading mechanisms and the development of microbial resources.

Evaluation of Colilert-18 for detection of coliforms and Eschericha coli in subtropical freshwater.

The accuracy of Colilert-18 as a test for coliforms and Escherichia coli in subtropical freshwater was evaluated by using API 20E strips and fatty acid methyl ester analysis. The false-positive and -negative rates of detection were 7.4 and 3.5%, respectively, for E. coli and 9.6 and 6.3%, respectively, for coliforms.