Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China

AIMS: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. METHODS AND RESULTS: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3.7% and 9.9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. CONCLUSIONS: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world.     

海口市污水处理厂活性污泥中细菌的分离鉴定及耐药性分析

为了解海口市白沙门污水处理厂活性污泥中细菌抗生素耐性情况,采用平板分离技术分离、纯化细菌,并通过BIOLOG微生物鉴定系统对筛选到的细菌进行鉴定,同时采用Kirby-Bauer纸片琼脂扩散法进行药敏试验并进行抗生素耐性分析。本研究共分离到18株细菌,分属8个属,14个种,其中G+和G-均为9株。抗生素药敏性试验结果表明,所有菌株均耐药,菌株单重耐药率、双重耐药性及多重耐药性分别为50%、38.9%、和11.1%。菌株对9种常用抗生素:头孢他啶、环丙沙星、庆大霉素、链霉素、氨苄西林、红霉素、氯霉素、四环素、卡那霉素的耐药率分别为61.1%、0%、5.6%、16.7%、50%、16.7%、11.1%、0%、5.6%。综上所述,白沙门污水处理厂活性污泥中的细菌耐药性比较严重,存在潜在的环境生态和人畜健康风险。本研究揭示了当前白沙门污水处理厂活性污泥中细菌对常见抗生素耐药的严重现状,为建议污水处理厂加强出水及污泥中抗生素耐药性及耐药基因的检测并评估其生态影响提供基础,避免出水及污泥中的抗性菌和耐药基因可能带来的风险问题。     

Screening of bacteria with antiviral activity from fresh water salmonid hatcheries

Bacteria isolated from two salmonid hatcheries were screened for antiviral activity against infectious hematopoietic necrosis virus (IHNV) to ascertain the presence of bacteria with anti-IHNV activity in the aquatic environment. Out of 710 bacterial isolates from the water and sediment samples, 190 strains showed anti-IHNV activities of more than 50% plaque reduction. These antiviral activities were detected predominantly in Pseudomonas, Aeromonas/Vibrio, and coryneforms. In one hatchery, the bacteria with antiviral activities were more prevalent in sediment samples than in water samples. Seventy-seven percent of the isolates with higher antiviral activities (greater than 90% plaque reduction) belonged to Pseudomonas.     

Protozoan Predation Is Differentially Affected by Motility of Enteric Pathogens in Water vs. Sediments

Survival of enteric bacteria in aquatic habitats varies depending upon species, strain, and environmental pressures, but the mechanisms governing their fate are poorly understood. Although predation by protozoa is a known, top-down control mechanism on bacterial populations, its influence on the survival of fecal-derived pathogens has not been systematically studied. We hypothesized that motility, a variable trait among pathogens, can influence predation rates and bacterial survival. We compared the survival of two motile pathogens of fecal origin by culturing Escherichia coli O157 and Salmonella enterica Typhimurium. Each species had a motile and non-motile counterpart and was cultured in outdoor microcosms with protozoan predators (Tetrahymena pyriformis) present or absent. Motility had a significant, positive effect on S. enterica levels in water and sediment in the presence or absence of predators. In contrast, motility had a significant negative effect on E. coli O157 levels in sediment, but did not affect water column levels. The presence/absence of protozoa consistently accounted for a greater proportion of the variability in bacterial levels (>95 %) than in bacterial motility (<4 %) in the water column. In sediments, however, motility was more important than predation for both bacteria. Calculations of total CFU/microcosm showed decreasing bacterial concentrations over time under all conditions except for S. enterica in the absence of predation, which increased ～0.5–1.0 log over 5 days. These findings underscore the complexity of predicting the survival of enteric microorganisms in aquatic habitats, which has implications for the accuracy of risk assessment and modeling of water quality.     

Bacteriological Investigation of Alberta Meat-Packing Plant Wastes with Emphasis on Salmonella Isolation

The waste treatment facilities and final effluents of 11 meat-packing plants in the Province of Alberta were investigated primarily to determine the numbers of indicator bacteria and the presence of Salmonella. This was done to discover the efficiency of the treatment systems presently in operation in reducing bacterial numbers and to establish the need for disinfection and for bacterial standards for these effluents. Data obtained showed that the final effluents were of very poor quality bacteriologically, with numbers of indicator organisms comparable to those found in raw sewage. Primary treatment facilities were ineffective in reducing the numbers of these bacteria. The secondary treatment facility investigated achieved greater than a 99% reduction of indicator bacteria. Salmonella were isolated from the final effluents of 78% of the plants, including the plant using secondary treatment. In total, 21 Salmonella serotypes were isolated. Salmonella isolates were not antibiotic resistant, but certain coliform and fecal coliform isolates demonstrated resistance to chloramphenicol, tetracycline, and ampicillin.     

Antibiotic resistance patterns of gram-negative bacteria isolated from environmental sources.

A total of 2,445 gram-negative bacteria belonging to fecal coliform, Pseudomonas, Moraxella, Acinetobacter, and Flavobacterium-Cytophaga groups were isolated from the rivers and bay of Tillamook, Oregon, and their resistances to chloramphenicol (25 microgram/ml), streptomycin (10 microgram/ml), ampicillin (10 microgram/ml), tetracycline (25 microgram/ml), chlortetracycline (25 microgram/ml), oxytetracycline (25 microgram/ml), neomycin (50 microgram/ml), nitrofurazone (12.5 microgram/ml), nalidixic acid (25 microgram/ml), kanamycin (25 microgram/ml), and penicillin G (10 IU/ml) were determined. Among fecal coliforms the bay isolates showed greater resistance to antibiotics than those from tributaries or surface runoff. No such well-defined difference was found among other bacterial groups. The antibiotic resistance patterns of gram-negative bacteria from different sources correlated well, perhaps indicating their common origin. The antibiotic resistance patterns of gram-negative bacteria of different general also correlated well, perhaps indicating that bacteria which share a common environment also share a common mode for developing antibiotic resistance.     

Dominant chloramphenicol-resistant bacteria and resistance genes in coastal marine waters of Jiaozhou Bay,China

Studies of abundance, diversity and distribution of antibiotic-resistant bacteria and their resistance determinants are necessary for effective prevention and control of antibiotic resistance and its dissemination, critically important for public health and environment management. In order to gain an understanding of the persistence of resistance in the absence of a specific antibiotic selective pressure, microbiological surveys were carried out to investigate chloramphenicol-resistant bacteria and the chloramphenicol acetyltransferase resistance genes in Jiaozhou Bay after chloramphenicol was banned since 1999 in China. About 0.15–6.70% cultivable bacteria were chloramphenicol resistant, and the highest abundances occurred mainly in the areas near river mouths or sewage processing plants. For the dominant resistant isolates, 14 genera and 25 species were identified, mostly being indigenous estuarine or marine bacteria. Antibiotic-resistant potential human or marine animal pathogens, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Shewanella algae, were also identified. For the molecular resistance determinants, the cat I and cat III genes could be detected in some of the resistant strains, and they might have the same origins as those from clinical strains as determined via gene sequence analysis. Further investigation about the biological, environmental and anthropogenic mechanisms and their interactions that may contribute to the persistence of antibiotic-resistance in coastal marine waters in the absence of specific antibiotic selective pressure is necessary for tackling this complicated environmental issue.     

Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia

AIMS: To carry out a preliminary assessment of the occurrence of resistance to antimicrobials in bacteria that has been isolated from a variety of aquaculture species and environments in Australia. METHOD AND RESULTS: A total of 100 Gram-negative (Vibrio spp. and Aeromonas spp. predominantly) and four Gram-positive bacteria isolated from farmed fish, crustaceans and water from crab larval rearing tanks were obtained from diagnostic laboratories from different parts of Australia. All the isolates were tested for sensitivity to 19 antibiotics and Minimal Inhibitory Concentrations were determined by the agar dilution method. Plasmid DNA was isolated by the alkali lysis method. Resistance to ampicillin, amoxycillin, cephalexin and erythromycin was widespread; resistance to oxytetracycline, tetracycline, nalidixic acid and sulfonamides was common but resistance to chloramphenicol, florfenicol, ceftiofur, cephalothin, cefoperazone, oxolinic acid, gentamicin, kanamycin and trimethoprim was less common. All strains were susceptible to ciprofloxacin. Multiple resistance was also observed and 74.4% of resistant isolates had between one and ten plasmids with sizes ranging 2-51 kbp. CONCLUSIONS: No antibiotics are registered for use in aquaculture in Australia but these results suggest that there has been significant off-label use. SIGNIFICANCE AND IMPACT OF STUDY: Transfer of antibiotic resistant bacteria to humans via the food chain is a significant health concern. In comparison with studies on terrestrial food producing animals, there are fewer studies on antibiotic resistance in bacteria from aquaculture enterprises and this study provides further support to the view that there is the risk of transfer of resistant bacteria to humans from consumption of aquaculture products. From the Australian perspective, although there are no products registered for use in aquaculture, antimicrobial resistance is present in isolates from aquaculture and aquaculture environments.     

Antibiotic resistance in surface water ecosystems: Presence in the aquatic environment,prevention strategies,and risk assessment

Antibiotic-resistant bacteria (ARB) have gained increased notoriety due to their continued detection in environmental media and consequently their threat to human and animal health. The continuing spread of antibiotic resistance throughout the environment is of growing environmental and public health concern, making it difficult to treat harmful resistant diseases. This paper examines the presence of antibiotics, ARB, and antibiotic-resistant genes (ARGs) in aquatic environments; the effectiveness of current water treatment strategies to remove them; and risk assessment methods available that can be used to evaluate the risk from antibiotic resistance. Antibiotics, ARB, and ARGs have been reported at varying levels in wastewater treatment plants, hospital wastewater, irrigation water, recreational water, and drinking water. There are many different water treatments capable of reducing antibiotic resistance (including chlorination, UV, and ozone); however, no one method can fully eliminate it with much variation in the reported effects. Risk assessment models can be used for interpreting field data into the risk to human health from antibiotic resistance. Currently, there is no gold standard risk assessment method for evaluating antibiotic resistance. Methods in this area need further development to reflect evolving risk assessment methodologies and dynamic data as it emerges.     

Phenotypic characteristics of coliform and noncoliform bacteria from a public water supply compared with regional and national clinical species

During the summer and fall of 1984, elevated total coliform counts were observed in the distribution system of a public water supply serving 350,000 people in south central Connecticut. As part of an investigation of possible health risks associated with the presence of bacteria in the water supply, bacterial isolates from the distribution system were compared with bacterial isolates of the same species obtained from a large regional teaching hospital and from a national compendium of clinical isolates. Characteristics analyzed included phenotypic metabolic activity, antimicrobial susceptibilities to clinically utilized antibiotics, temperature tolerance at 44.5 degrees C, and beta-glucuronidase activity in single-test form and on a selective medium. Environmental isolates lacked known plasmid-mediated characteristics, with the exception of one Escherichia coli isolate which showed some antibiotic resistance. Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter agglomerans from all sources were temperature tolerant and yielded positive fecal coliform tests. Only E. coli showed beta-glucuronidase activity (both in a single biochemical test and on a selective medium). No single characteristic analyzed was sufficient to establish an organism as either environmental or clinical in origin.     

Phenotypic characteristics of coliform and noncoliform bacteria from a public water supply compared with regional and national clinical species.

During the summer and fall of 1984, elevated total coliform counts were observed in the distribution system of a public water supply serving 350,000 people in south central Connecticut. As part of an investigation of possible health risks associated with the presence of bacteria in the water supply, bacterial isolates from the distribution system were compared with bacterial isolates of the same species obtained from a large regional teaching hospital and from a national compendium of clinical isolates. Characteristics analyzed included phenotypic metabolic activity, antimicrobial susceptibilities to clinically utilized antibiotics, temperature tolerance at 44.5 degrees C, and beta-glucuronidase activity in single-test form and on a selective medium. Environmental isolates lacked known plasmid-mediated characteristics, with the exception of one Escherichia coli isolate which showed some antibiotic resistance. Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter agglomerans from all sources were temperature tolerant and yielded positive fecal coliform tests. Only E. coli showed beta-glucuronidase activity (both in a single biochemical test and on a selective medium). No single characteristic analyzed was sufficient to establish an organism as either environmental or clinical in origin.     

Association of metal tolerance with multiple antibiotic resistance of bacteria isolated from drinking water

Bacterial isolates from the drinking water system of an Oregon coastal community were examined to assess the association of metal tolerance with multiple antibiotic resistance. Positive correlations between tolerance to high levels of Cu2+, Pb2+, and Zn2+ and multiple antibiotic resistance were noted among bacteria from distribution waters but not among bacteria from raw waters. Tolerances to higher levels of Al3+ and Sn2+ were demonstrated more often by raw water isolates which were not typically multiple antibiotic resistant. A similar incidence of tolerance to Cd2+ was demonstrated by isolates of both water types and was not associated with multiple antibiotic resistance. These results suggest that simultaneous selection phenomena occurred in distribution water for bacteria which exhibited unique patterns of tolerance to Cu2+, Pb2+, and Zn2+ and antibiotic resistance.     

Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat samples and 18.0% of shellfish samples were contaminated with Salmonella spp., and more than 90% of all food sources contained Escherichia coli. The isolates were screened for antibiotic resistance against 15 antibiotics, and 50.5% of Salmonella isolates and 83.8% of E. coli isolates were resistant to at least one antibiotic. Isolates were examined for the presence of mobile genetic elements conferring antibiotic resistance. Fifty-seven percent of E. coli and 13% of Salmonella isolates were found to contain integrons, and some isolates contained two integrons. Sequencing results revealed that the integrons harbored various gene cassettes, including aadA1, aadA2, and aadA5 (resistance to streptomycin and spectinomycin), aacA4 (resistance to aminoglycosides), the dihydrofolate reductase gene cassettes dhfrXII, dfrA1, and dhfrA17 (trimethoprim resistance), the beta-lactamase gene bla_PSE1 (ampicillin resistance), and catB3 (chloramphenicol resistance). Plasmids were also detected in all 23 antibiotic-resistant Salmonella isolates and in 33 E. coli isolates. Thirty-five percent of the Salmonella isolates and 76% of the E. coli isolates contained plasmids of more than 95 kb, and some of the isolates contained two large plasmids. Conjugation experiments showed the successful transfer of all or part of the antibiotic resistance phenotypes among the Salmonella and E. coli food isolates. Our results show that enteric bacteria in raw food samples from Vietnam contain a pool of mobile genetic elements and that the transfer of antibiotic resistance can readily occur between similar bacteria.     

Antibiotic and heavy metal resistance in Gram-negative bacteria isolated from the Seyhan Dam Lake and Seyhan River in Turkey

This study aimed to determine the pattern of antibiotic and heavy metal resistance in Gram-negative bacteria isolated from five different sites in the Seyhan Dam Lake and Seyhan River in Adana, Turkey. The susceptibility of 268 isolates to 16 different antibiotics and five heavy metals was investigated by agar diffusion and dilution methods, respectively. The most common species isolated from the samples were Aeromonas hydrophila (17.5 %), Aeromonas caviae (8.9 %) and Citrobacter freundii (8.9 %). There was a high incidence of resistance to ampicillin (80.2 %), streptomycin (71.6 %) and cefazolin (60.4 %). Multiple antibiotic resistance indices ranged from 0.2 to 0.81, suggesting exposure to antibiotic contamination. The isolates showed tolerance to different concentrations of heavy metals. These results indicate that antibiotic and heavy metal resistance among the Seyhan Dam Lake and Seyhan River bacteria may pose a risk to the fish population and public health. At the same time, the finding in the aquatic environments of different combinations of resistance genes suggests their involvement in the spread of multidrug-resistant strains.     

Spatial Analysis of Antibiotic Resistance Along Metal Contaminated Streams

The spatial pattern of antibiotic resistance in culturable sediment bacteria from four freshwater streams was examined. Previous research suggests that the prevalence of antibiotic resistance may increase in populations via indirect or coselection from heavy metal contamination. Sample bacteria from each stream were grown in media containing one of four antibiotics—tetracycline, chloramphenicol, kanamycin, and streptomycin—at concentrations greater than the minimum inhibitory concentration, plus a control. Bacteria showed high susceptibilities to the former two antibiotics. We summarized the latter two more prevalent (aminoglycoside) resistance responses and ten metals concentrations per sediment sample, by Principal Components Analysis. Respectively, 63 and 58% of the variability was explained in the first principal component of each variable set. We used these multivariate summary metrics [i.e., first principal component (PC) scores] as input measures for exploring the spatial correlation between antibiotic resistance and metal concentration for each stream sampled. Results show a significant and negative correlation between metals PC scores versus aminoglycoside resistance scores and suggest that selection for metal tolerance among sediment bacteria may influence selection for antibiotic resistance differently in sediments than in the water column. Our most important finding comes from geostatistical cross-variogram analysis, which shows that increasing metal concentration scores are spatially associated with decreasing aminoglycoside resistance scores—a negative correlation, but holds for contaminated streams only. We suspect our field results are influenced by metal bioavailability in the sediments and by a contaminant promoted interaction or “cocktail effect” from complex combinations of pollution mediated selection agents.     

Antibiotic resistant bacteria in Windermere and two remote upland tarns in the English Lake District

The incidence of antibiotic resistance was determined in over 2000 bacteria which were divided into the following groups: faecal streptococci, coliforms (excluding Escherichia coli ), E. coli, Pseudomonas spp. and aquatic bacteria (i.e. bacteria predominant in the lake water which were excluded from the previous four categories). The isolates were obtained from the water of Windermere (English Lake District) and from a sewage effluent which entered the lake. With the exception of the faecal streptococci, the incidence of antibiotic resistance was higher in the bacteria isolated from the lake water than in those from the effluent, and ranked according to groups Pseudomonas spp. > E. coli > aquatic bacteria > coliforms > faecal streptococci. The highest incidence of multiple resistance was found among the pseudomonads. When corrected for the relative size of each population the pool of antibiotic resistance in the aquatic bacteria was by far the largest. The incidence of antibiotic resistance in aquatic bacteria isolated from Windermere was, however, lower than in those isolated from two remote upland tarns. This finding may have been due to differences in the species composition of the three sites except that the same results were obtained when only fluorescent pseudomonads were tested. The upland tarns were not totally isolated from man and other animals but did not receive any sewage or other effluents and therefore the results were surprising. Possible explanations include a lack of susceptibility in aquatic bacteria and increased resistance associated with growth in nutrient poor environments.     

Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four danish rainbow trout farms

Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark. Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88 Flavobacterium psychrophilum isolates and 134 Yersinia ruckeri isolates) and 313 motile Aeromonas isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples. MICs were obtained applying a standardized agar dilution method. A markedly decreased susceptibility of F. psychrophilum isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected Y. ruckeri isolates remained largely sensitive to all therapeutic substances. Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads. High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.     

Incidence of multiple antibiotic resistant Escherichia coli in the Bhavani River

Of 107 Escherichia coli strains isolated from the water, sediment and fish of the Bhavani River, all of which are considered potential causes of human enteric disease, 62% were resistant to more than four antibiotics. Levels of resistance to bacitracin, penicillin, and novobiocin were generally high whereas those to polymyxin-B and chloramphenicol were much lower. A high incidence of multiple antibiotic resistant E. coli was noted in all samples and the multiple antibiotic resistance index of the strains showed that 95% of the strains originated either from man or cattle.     

Selection of antibiotic-resistant standard plate count bacteria during water treatment.

Standard plate count (SPC) bacteria were isolated from a drinking-water treatment facility and from the river supplying the facility. All isolates were identified and tested for their resistance to six antibiotics to determine if drug-resistant bacteria were selected for as a consequence of water treatment. Among the isolates surviving our test procedures, there was a significant selection (P less than 0.05) of gram-negative SPC organisms resistant to two or more of the test antibiotics. These bacteria were isolated from the flash mix tank, where chlorine, alum, and lime are added to the water. Streptomycin resistance in particular was more frequent in this population as compared with bacteria in the untreated river water (P less than 0.01). SPC bacteria from the clear well, which is a tank holding the finished drinking water at the treatment facility, were also more frequently antibiotic resistant than were the respective river water populations. When 15.8 and 18.2% of the river water bacteria were multiply antibiotic resistant, 57.1 and 43.5%, respectively, of the SPC bacteria in the clear well were multiply antibiotic resistant. Selection for bacteria exhibiting resistance to streptomycin was achieved by chlorinating river water in the laboratory. We concluded that the selective factors operating in the aquatic environment of a water treatment facility can act to increase the proportion of antibiotic-resistant members of the SPC bacterial population in treated drinking water.