Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discushannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibriosplendidus and Vibriotasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes.     

Isolates of Staphylococcus aureus and saprophyticus resistant to antimicrobials isolated from the Lebanese aquatic environment

The indiscriminate use of antimicrobials especially in developing countries has evoked serious bacterial resistance and led to the emergence of new and highly resistant strains of bacteria to commonly used antimicrobials. In Lebanon, pollution levels and bacterial infections are increasing at a high rate as a result of inadequate control measures to limit untreated effluent discharges into the sea or freshwater resources. The aim of this study was to isolate and molecularly characterize various Staphylococcus strains isolated from sea water, fresh water, sediments, and crab samples collected from representative communities along the coast of Lebanon. The results on the antimicrobial resistance indicated that the level of resistance of Staphylococcus aureus varied with various antimicrobials tested. The resistance patterns ranged between 45% in freshwater isolates and 54.8% in seawater ones. Fifty one percent of the tested isolates have shown resistance to at least one of the five tested antimicrobials; with seawater isolates exhibiting the highest rates of antimicrobial resistance.     

Antibiotic Resistance and Plasmid Profiles of Vibrio Isolates from Cultured Silver Sea Bream, Sparus sarba

A total of 51 potential pathogenic vibrios were isolated from moribund silver sea bream (Sparus sarba) collected from fish farms in Hong Kong. Using the API 20E system and the scheme of Alsina and Blanch (1994), 7 species were identified from all isolates. These species were Vibrio alginolyticus (24 strains), Vibrio vulnificus (12 strains), Vibrio parahaemolyticus (7 strains), Vibrio logei (4 strains), Vibrio pelagius II (2 strains), Vibrio fluvialis (1 strain) and Vibrio meditterranei (1 strain). The three dominant species (V. alginolyticus, V. vulnificus and V. parahaemolyticus) were confirmed to be virulent to sea bream by experimental challenge.

All isolates were screened for plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 16 antibiotics by the agar dilution method. Of the 51 isolates examined, all strains were sensitive to ceftriaxone, streptomycin, nalidixic acid and rifampicin, and almost all were sensitive to ceftazidime, netilimicin, chloramphenicol and sulfamethoxazole. Most isolates were resistant to ampicillin (60.8%), cefuroxime (66.7%), amikacin (55%), kanamycin (58.8%) and trimethoprim (76.5%). Fifteen of the 51 isolates harboured 1–4 plasmids, with sizes ranging from 9 to 123 kb. Both the plasmids and the associated antibiotic resistance (ampicillin, cefuroxime and trimethoprim) of 9 isolates could be transferred to a recipient by single-step conjugation. However, the frequencies were very low, ranging from 10⁻¹¹ to 10⁻⁹. The present results indicate that resistance to these antibiotics is chromosomal.     

Embryotoxicity of the antifouling biocide zinc pyrithione to sea urchin (Paracentrotus lividus) and mussel (Mytilus edulis)

The effects of the new antifouling compound zinc pyrithione (Zpt) on the embryonic development of sea urchin (Paracentrotus lividus) and mussel (Mytilus edulis) were investigated in laboratory toxicity tests. The median effective concentrations (EC50) were 7.7 nM for sea urchin embryos and 8 nM for mussel embryos. Toxic effects of Zpt on the larval growth of the sea urchin were detected at 0.5 nM. Predicted environmental concentrations of Zpt in pleasure craft harbours are higher than the predicted no effect concentrations for sea urchin and mussel embryos, indicating that Zpt may pose a threat to those species from exposure in the field.     

Isolation and characterization of crude-oil-degrading bacteria from the Persian Gulf and the Caspian Sea

Twenty-five crude-oil-degrading bacteria were isolated from oil-contaminated sites in the Persian Gulf and the Caspian Sea. Based on a high growth rate on crude oil and on hydrocarbon degradation ability, 11 strains were selected from the 25 isolated strains for further study. Determination of the nucleotide sequence of the 16S rRNA gene showed that these isolated strains belonged to genera Acinetobacter, Pseudomonas, Gordonia, Rhodococcus, Cobetia, Halomonas, Alcanivorax, Marinobacter and Microbacterium. Among the 11 isolates, strains BS (Acinetobacter calcoaceticus, 98%) and PG-12 (Alcanivorax dieselolei, 98%) were the most effective in degrading crude oil. Rate of crude-oil degradation of 82% (isolate BS) and 71% (isolate PG-12) were observed after 1 week of cultivation in mineral medium. These strains had high emulsification activity and biosurfactant production. GC-MS analysis showed that A. dieselolei PG-12 can degrade different alkanes in crude oil. Screening of the distribution of the alkane hydroxylase gene in 25 isolates in relation to the source of isolation indicated that the group (II) alkane hydroxylase is prevalent in the Caspian Sea, but in the Persian Gulf, the frequency of the group (III) alkane hydroxylase gene is greater than that of the group (II) alkane hydroxylase gene.     

Echinoderms from Azores islands: An unexpected source of antibiotic resistant Enterococcus spp. and Escherichia coli isolates

The prevalence of antibiotic resistance and the implicated mechanisms of resistance were evaluated in Enterococcus spp. and Escherichia coli, isolated from a total of 250 faecal samples of echinoderms collected from Azorean waters (Portugal). A total of 144 enterococci (120 Enterococcus faecium, 14 E. hirae, 8 E. faecalis, 2 E. gallinarum) and 10 E. coli were recovered. High percentages of resistance in enterococci were found for erythromycin, ampicillin, tetracyclin and ciprofloxacin. The erm(A) or erm(B), tet(M) and/or tet(L), vat(D), aac(6′)-aph(2″) and aph(3′)-IIIa genes were found in isolates resistant to erythromycin, tetracycline, quinupristin/dalfopristin, high-level gentamicin and high-level kanamycin, respectively. Resistance in E. coli isolates was detected for streptomycin, amikacin, tetracycline and tobramycin. The aadA gene was found in streptomycin-resistant isolates and tet(A) + tet(B) genes in tetracycline-resistant isolates. The data recovered are essential to improve knowledge about the dissemination of resistant strains through marine ecosystems and the possible implications involved in transferring these resistances either to other animals or to humans.     

Environmental management of marine fish culture in Hong Kong

Marine fish farming is an important commercial practice in Hong Kong. Marine fish farms located in eutrophic coastal waters often face the threat of severe dissolved oxygen depletion associated with algal blooms and red tides. On the other hand, mariculture activities also contribute to pollution. The sustainable management of mariculture requires proper siting of the fish farms and stocking density control. Both of these are related to the carrying capacity of the water body concerned, which is mainly governed by its flushing characteristics. A simple method to determine the carrying capacity of a fish farm has been developed by using three-dimensional (3D) hydrodynamic modelling and its effective coupling with a diagenetic water quality model. A systematic methodology using numerical tracer experiments has been developed to compute the tidal flushing in a fish farm. The flushing time is determined from the results of a numerical tracer experiment using robust 3D hydrodynamic and mass transport models. A unit tracer concentration is initially prescribed inside the region of interest and zero elsewhere; the subsequent mass transport and the mass removal process are then tracked. The fish farms are usually situated in well-sheltered shallow embayments and may not connect directly to the open water. It is found that it is necessary to define both "local" and "system-wide" flushing times to represent the effectiveness of the mass exchange with the surrounding water body and the open sea respectively. A diagenetic water quality model simulating the sediment-water-pollutant interaction is employed to address the response of the water column and the benthic layer to pollution discharges. With the flushing rate reliably computed, the carrying capacity of the fish farm can be determined in terms of key water quality parameters: chlorophyll-a, dissolved oxygen, organic nitrogen and potential lowest dissolved oxygen level on a day of negligible photosynthetic production. The predictions are well-supported by field data.     

Analysis of community structure of a microbial consortium capable of degrading benzo(a)pyrene by DGGE

A microbial consortium was obtained by enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of a mixture of polycyclic aromatic hydrocarbons enrichment strategy. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial composition and community dynamic changes based on PCR amplification of 16S rRNA genes during batch culture enrichment. Using the spray-plate method, three bacteria, designated as BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequence analysis showed that BL01, BL02 and BL03 were phylogenetically close to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens, respectively. The degradation of benzo(a)pyrene (BaP), a model high-molecular-weight polycyclic aromatic hydrocarbon (HMW PAH) compound was investigated using individual isolates, a mixture of the three isolates, and the microbial consortium (BL) originally isolated from the oil port sea water. Results showed that the order of degradative ability was BL > the mixture of the three isolates > individual isolates. BL degraded 44.07% of the 10 ppm BaP after 14 days incubation, which showed the highest capability for HMW PAH compound degradation.Our results revealed that this high selective pressure strategy was feasible and effective in enriching the HMW PAH-degraders from the original sea water samples.     

Antibiotic Resistance of Gram Negatives isolates from loggerhead sea turtles (Caretta caretta) in the central Mediterranean Sea

Previous studies on fish and marine mammals support the hypothesis that marine species harbor antibiotic resistance and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to assess the resistance to antimicrobial agents of Gram negative strains isolated from loggerhead sea turtles (Caretta caretta). Oral and cloacal swabs from 19 live-stranded loggerhead sea turtles, with hooks fixed into the gut, were analyzed. The antimicrobial resistance of the isolates to 31 antibiotics was assessed using the disk-diffusion method. Conventional biochemical tests identified Citrobacter spp., Proteus spp., Enterobacter spp., Escherichia spp., Providencia spp., Morganella spp., Pantoea spp., Pseudomonas spp. and Shewanella spp. Highest prevalences of resistance was detected to carbenicillin (100%), cephalothin (92.6%), oxytetracycline (81.3%) and amoxicillin (77.8%). The isolates showing resistance to the widest range of antibiotics were identified as Citrobacter freundii, Proteus vulgaris, Providencia rettgeri and Pseudomonas aeruginosa. In this study, antibiotic resistant bacteria reflect marine contamination by polluted effluents and C. caretta is considered a bioindicator which can be used as a monitor for pollution.     

A toxicity identification evaluation of silty marine harbor sediments to characterize persistent and non-persistent constituents

Sediment toxicity in silty marine harbor sediments is frequently dominated by ammonia or sulfide, leaving the adverse effects of persistent toxic substances unnoticed. To investigate the latter, we subjected interstitial water from three contaminated silty sediments to toxicity identification evaluation (TIE) phase I manipulations and tested for toxicity with four bioassays: the amphipod Corophium volutator (survival as an endpoint), the sea urchin Psammechinus miliaris (fertilization, embryo development) and the bacterium Vibrio fischeri (bioluminescence inhibition).The graduated pH manipulations identified the prominent toxicity of ammonia in the amphipod and sea urchin embryo tests, and also sulfide toxicity in the bacterium test. In two of the three samples tested with the amphipods, sea urchin embryos and bacteria, a small but significant reduction in interstitial water toxicity was achieved by removing persistent compounds through C(18) solid phase extraction. EDTA chelation resulted in a slight detoxification of the interstitial water for the amphipods and sea urchin embryos, but this was not related to any measured trace metals. Despite the presence of toxic levels of ammonia and sulfide in the harbor sediments, we established the adverse biological effects of persistent constituents by means of the TIE manipulations and in vivo interstitial water bioassays.     

Gilthead seabream (Sparus aurata) carrying antibiotic resistant enterococci. A potential bioindicator of marine contamination?

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin-dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M), aac(6′)-aph(2″) and aph(3′)-IIIa genes were shown in isolates resistant to erythromycin, tetracycline, high-level gentamicin and high-level kanamycin, respectively. Antibiotic resistance in natural microbiota is becoming a concern of human and environmental health.     

Prevalence and genetic profiles of Escherichia coli from mangroves and mangrove associated foods off Goa,India

A total of 120 samples comprising of water (45), sediment (45) and mangrove originated food (30) collected from mangrove ecosystems of Goa were screened for Escherichia coli employing ISO-16654 method. Seventy-one (59.16%) samples were positive for E. coli. The E. coli isolates were further characterized by serotyping, virulence gene profiling and pulsed field gel electrophoresis (PFGE). Water and sediment samples were analyzed for physico-chemical parameters. The serotypes reported were O1, O10, O13, O17, O36, O41, O50, O68, O105, O116, O141, O148, O159, O162 and rough types while, 23 strains could not be typed. The stx1 and stx2 genes were detected in 33(46.47%) and 16(22.53%) isolates, respectively. The XbaI restriction digestion patterns of the stx positive strains were diverse. Interestingly, few strains isolated from diarrheal patients and from water, sediment and food from mangrove sources were genetically similar. The study showed that the mangrove ecosystem could be a potential reservoir for pathogenic E. coli.     

Assessment of the Comparative Toxicity of Sewage Effluent from 10 Sewage Treatment Plants in the Area of Sydney, Australia using an Amphipod and Two Sea Urchin Bioassays

In 1995, Sydney Water Corporation undertook an ecological and human health risk assessment for 10 sewage treatment plants (STPs) that discharge primary (6 STPs) and secondary (4 STPs) treated effluents into coastal waters in the Sydney and Illawarra regions, NSW, Australia. A program of toxicity testing of effluent from the 10 STPs was undertaken to determine the toxic effects of the effluents and as a weight-of-evidence study for the risk assessment.

Three types of bioassays were used to test multiple samples of effluent from each STP. Tests used were the sea urchin (Heliocidaris tuberculata) fertilization and larval development bioassays and the amphipod (Allorchestes compressa) survival bioassay.

Comparing between STPs, primary treated effluent was slightly more toxic than secondary treated effluent for all three tests. The highest toxicity was recorded for those STPs that use primary treatment and discharge to the shoreline. Comparing between tests, the sea urchin fertilization bioassay was slightly more sensitive than the sea urchin larval development bioassay, with the amphipod survival bioassay being the least sensitive.

This paper will describe the results of this toxicity testing program and discuss the application of the study results as weight-of-evidence for the risk assessment study.     

Impact of milk fish farming in the tropics on potentially pathogenic vibrios

Ratios of sucrose-negative to sucrose-positive vibrios on TCBS agar (suc−/suc+) indicate the abundance of potential human pathogenic non-cholera vibrios in coastal mariculture environments of the Lingayen Gulf (Philippines. In guts of adult maricultured milkfish (Chanos chanos) of suc− vibrios reached extreme peak values ranging between 2 and 545 million per g wet weight. Suc− vibrios outnumbered suc+ vibrios in anoxic sediments, too, and were rarely predominant in coastal waters or in oxidized sediments. Suc−/suc+ ratios in sediments increased toward the mariculture areas with distance from the open sea at decreasing redox potentials. There is circumstantial evidence that suc− vibrios can be dispersed from mariculture areas to adjacent environments including coral reefs. An immediate human health risk by pathogenic Vibrio species is discounted, since milkfish guts contained mainly members of the Enterovibrio group. A representative isolate of these contained proteolytic and other virulence factors, but no genes encoding toxins characteristic of clinical Vibrio species.     

The environmental impact of marine fish culture: Towards a sustainable future

The environmental impact of marine fish-farming depends very much on species, culture method, stocking density, feed type, hydrography of the site and husbandry practices. In general, some 85% of phosphorus, 80–88% of carbon and 52–95% of nitrogen input into a marine fish culture system as feed may be lost into the environment through feed wastage, fish excretion, faeces production and respiration. Cleaning of fouled cages may also add an organic loading to the water, albeit periodically. Problems caused by high organic and nutrient loadings conflict with other uses of the coastal zone. The use of chemicals (therapeutants, vitamins and antifoulants) and the introduction of pathogens and new genetic strains have also raised environmental concerns.

Despite the high pollution loadings, results from various studies show that some 23% of C, 21% of N and 53% of P of feed input into the culture system is being accumulated in the bottom sediments and the significant impact is normally confined to within 1 km of the farm. The major impact is on the sea bottom, where high sediment oxygen demand, anoxic sediments, production of toxic gases and a decrease in benthic diversity may result. Decreases in dissolved oxygen and increases in nutrient levels in the water are also evident but are normally confined to the vicinity of the farm. Tributyltin (TBT) contamination and the development of antibiotic-resistant bacteria have been reported near fish farms. The stimulating effects of vitamins/fish wastes on growth of red tide species have been demonstrated in a number of laboratory studies. Nevertheless, there is no evidence to support the suggestion that the present use of therapeutants, vitamins and antibiotics and the introduction of pathogens and new genetic strains would pose a significant threat to the environment.

Marine fish culture can be a sustainable development, provided pollution loadings generated by fish farms are kept well below the carrying capacity of the water body. Effects can be significantly reduced by careful site selection, control of stock density, improved feed formulation and integrated culture (with macroalgae, filter-feeders and deposit-feeders). An example of the application of computer modelling in mariculture management is demonstrated. Environmental impact assessment and monitoring should also be carried out to ensure culture activities are environmentally sustainable.     

Carbon sequestration processes and mechanisms in coastal mariculture environments in China

China is the global leader in mariculture production. Increasing sequestered marine carbon (also known as blue carbon) via mariculture activities is a promising approach for mitigating climate change and promoting the development of a low-carbon economy. Mariculture blue carbon is also considered an important component of China’s “sea granary”. In addition to shellfish and macroalgae yields, which represent carbon removed from mariculture environment, blue carbon also includes other important components, which have been largely neglected in the past, such as the carbon transformed by microbes, dissolved organic carbon (mainly referred to as recalcitrant dissolved organic carbon), and sedimentary particulate carbon. Hence, from different aspects, a comprehensive study on the formation processes and mechanisms of carbon sequestration is of great significance for comprehensively unveiling the carbon sequestration capability in coastal mariculture environment, which will contribute to the sustainable development of the fishery economy and construction of an ecological civilization. Moreover, it may add significant economic benefits to the future carbon-trading market.     

Seasonal variation in the toxicity of sediment-associated contaminants in Corpus Christi Bay, TX

Seasonal variation in pore water and sediment-water interface (SWI) toxicity at two sites of suspected contamination was investigated using sea urchin embryological development (Arbacia punctulata) and copepod hatching success (Schizopera knabeni). Site S1, located inside a marina, was fine-grained and S2, located near a neighboring stormwater outfall, was sandy. Both sites were cored in summer and winter, along with reference sites of equivalent grain sizes. Whereas the muddy contaminated site displayed a slight decrease in sea urchin toxicity from summer to winter, pore water from the sandy contaminated site exhibited an increase in sea urchin toxicity. The sandy sites displayed slight toxicity increase to copepod hatching success in winter, which occurred in the SWI of S2 and in the pore water from the reference site, R2, where it coincided with sedimentation. Cu was the most prevalent metal in the sediment samples. Although Cu concentration increased in the whole sediments and pore waters from summer to winter, its concentration in overlying SWI exposure decreased from summer to winter. Organic compounds exhibited opposite behavior, with smaller variety identified in the winter pore water samples, but more chemicals detected in the SWI samples in winter.     

Bioremediation and fodder potentials of two Sargassum spp. in coastal waters of Shenzhen,South China

In this study, the bioremediation potentials of two seaweeds (Sargassum hemiphyllum and S. henslowianum) against pollution in a coastal mariculture area of Shenzhen, South China, were investigated by comparing the growth, nutrient bioaccumulation capacity of plants from the seaweed bed (control site) with plants from the fish farm. Results indicated that both species are potential candidates for bioremediation in the fish farm areas in terms of their high growth rates and high bioaccumulation capacities on inorganic nutrients. Both Sargassum spp. contain high levels of crude protein (11.7–14.0%) and crude fat (2.2–2.7%), suggesting high nutritional values. The S. hemiphyllum may serve as a good aquaculture fodder with high nutritional compositions and low heavy metal contents. However, heavy metals (Cr, Pb and Cd) of S. henslowianum exceed the maximum allowable concentrations as aquatic feed, which restricts its fodder application. In general, the results of this study may contribute to the marine pollution bioremediation in the coastal areas of South China, especially in mariculture zones.     

Antibiotic resistant pathogens versus human impacts: a study from three eco-regions of the Chennai coast, southern India

We assessed the occurrence of pollution indicators and antibiotic resistant bacterial isolates from water and sediment samples of three different eco-regions of the Chennai coast between March - May of 2010. Total of 960 bacterial strains belonging to four genera were isolated which show the highest frequencies of resistance to vancomycin (53.6%) and penicillin (52.6%) (except Enterococcus sp., which is highly resistant to erythromycin) and lowest frequencies of resistance to chloramphenicol (3.43%), ciprofloxacin (3.95%), gentamicin (4.68%), and tetracycline (6.97%). The E. coli, Vibrio sp., Salmonella sp. and Enterococcus sp. show high frequency of resistance to 2-5 antibacterials of 60.4%, 45.83%, 69.16% and 46.6%, respectively. High pollution indices (PI - 6.66-14.06) and antibiotic resistance indices (ARI - 0.29-0.343) indicate that the coastal environment is highly exposed to antibiotic sources that suggesting to avoid direct contact.     

Effect of sea cucumber (Australostichopus mollis) grazing on coastal sediments impacted by mussel farm deposition

Deposit-feeding holothurians are important processors of surface sediments in many coastal marine systems. The present study examined the effect of grazing by the sea cucumber Australostichopus mollis on sediment impacted by green-lipped mussel biodeposits (faeces and pseudofaeces) from coastal aquaculture activities. Grazing effects were investigated in a series of tank-based feeding experiments conducted over 1, 2, 4 and 8 week periods. Sediment quality indicators routinely applied to determine the impacts of coastal aquaculture were used to evaluate sediment health from grazed and ungrazed sediments. Sea cucumber grazing resulted in reductions in total organic carbon, chlorophyll a and phaeopigment, as well as chlorophyll a/phaeopigment ratio of impacted sediments. These results demonstrate that sea cucumber grazing significantly reduces the accumulation of both organic carbon and phytopigments associated with biodeposition from mussel farms. Sea cucumber grazing offers a means of constraining or reversing the pollutive impacts of coastal bivalve aquaculture.