Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples.

Sewage effluent and outfall confluence samples were collected at the Barceloneta Regional Treatment Plant in Barceloneta, Puerto Rico; outfall confluence samples at Ocean City, Md., were also collected. Samples from uncontaminated open ocean areas served as clean-water controls. Bacteria were enriched in marine broth 2216 amended with 1 microgram of one of a set of chemicals selected for study per ml: nitrobenzene, dibutyl phthalate, m-cresol, o-cresol, 4-nitroaniline, bis(tributyltin) oxide, and quinone. MICs of the chemicals were determined individually for all isolates. Bacterial isolates were evaluated for resistance to nine different antibiotics and for the presence of plasmid DNA. Treated sewage was found to contain large numbers of bacteria simultaneously possessing antibiotic resistance, chemical resistance, and multiple bands of plasmid DNA. Bacteria resistant to penicillin, erythromycin, nalidixic acid, ampicillin, m-cresol, quinone, and bis(tributyltin) oxide were detected in nearly all samples, but only sewage outfall confluence samples yielded bacterial isolates that were resistant to streptomycin. Bacteria resistant to a combination of antibiotics, including kanamycin, chloramphenicol, gentamicin, and tetracycline, were isolated only from sewage effluent samples. It is concluded that bacterial isolates derived from toxic chemical wastes more frequently contain plasmid DNA and demonstrate antimicrobial resistance than do bacterial isolates from domestic sewage-impacted waters or from uncontaminated open ocean sites.     

Impact of long-term application of industrial wastewater on the emergence of resistance traits in Azotobacter chroococcum isolated from rhizospheric soil

A total of 57 (36 and 21) Azotobacter chroococcum were isolated from wheat (Triticum aestivum) rhizospheric soil irrigated with industrial wastewater (about a decade) and ground water (uncontaminated) and characterized on the basis of morphological, cultural and biochemical characteristics. Rhizospheric soils were analyzed for metal concentrations by atomic absorption spectrophotometery and the test soil samples were contaminated with Fe, Zn, Cu, Cr, Ni and Pb. All the isolates of A. chroococcum were tested for their resistance against Hg2+, Cd2+, Cu2+, Cr3+, Cr6+, Zn2+, Ni2+ and Pb2+. Among 36 isolates of Azotobacter from soil irrigated with industrial wastewater, 94.4% were resistant to Pb2+ and Hg2+ and 86.1%, 77.5% and 63.8% were resistant to Zn2+, Cr6+ and Cr3+ respectively. The highest minimum inhibitory concentration of 200 microg/ml for Hg2+ and 1600 microg/ml for other metals were observed against these bacteria from soil. The incidences of metal resistance and MICs of metals for A. chroococcum from wastewater irrigated soil were significantly different to those of uncontaminated soil. All A. chroococcum isolates were tested for their resistance against 11 commonly used antibiotics/drugs. 91.6% were found to be resistant against nitrofurantoin while 86.4% and 80.5% were found to be resistant against polymyxin-B and co-trimoxazole respectively. Agarose gel electrophoresis using the miniprep method for plasmid isolation revealed that these isolates harboured plasmids of molecular weights 58.8 and 64.5 kb using EcoRI and HindIII digests of X DNA and undigested X DNA as standard markers.     

Concentrations of nitrifying bacteria in sewages, effluents, and a receiving stream and resistance of these organisms to chlorination.

Estimates of NH4+-and NO2-oxidizers in samples from four activated sludge plants treating mainly domestic sewage were obtained using a most-probable-number (MPN) technique. Ranges of concentrations per milliliter of each, respectively, were 1,010 to 3,880 and 79 to 145 in settled sewages, 32 to 7,420 and 2 to 1,010 in secondary effluents, and less than 0.1 to 622 and 0.1 to 70 in chlorinated secondary effluents. The results of this field study indicated that nitrifiers were more resistant to chlorination than fecal streptococci, which were also enumerated. In laboratory studies the survivals of these bacterial groups in secondary effluents were determined after exposure to chlorine residuals of up to 2 mg/liter for 0 to 60 min. The nitrifiers proved considerably more resistant than fecal streptococci, with NO2-oxidizers showing greater resistance than NH4+-oxidizers. Below the outfall of one of the plants that discharges heavily chlorinated unnitrified effluent, NH4+-oxidizers amounted to approximately 200 X 10(5) per g of slime scraped from stream-bed rocks. Upstream of the outfall this was approximatley 3 X 10(5)/G.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

A method for the enumeration of Aeromonas bacteriophage in aquatic environments

K.P. FLINT. 1996. Bacteriophage were isolated against type strains and environmental isolates of Aeromonas hydrophila, Aeromonas sobria and Aeromonas caviae . A most probable number method for estimating the number of bacteriophage in a water sample was devised and tested using some of these isolates. The maximum number of bacteriophage against all three type strains were found in water from below a sewage effluent outfall. This corresponds to the increased numbers of each species of bacterium also found in this water sample. High numbers of bacteriophage against Aer. hydrophila were also found in the lake sample examined. Bacteriophage against Aer. caviae were rare in water samples other than those contaminated with sewage effluent.     

Multiresistance, beta-lactamase-encoding genes and bacterial diversity in hospital wastewater in Rio de Janeiro, Brazil

Aims: To investigate the bacterial diversity, antimicrobial resistance patterns and types of beta‐lactamase genes in Gram‐negative bacteria isolated from a hospital sewage treatment plant. Methods and Results: Between July and December 2008, we collected samples from influent, clarifier tank effluent and chlorine contact tank effluent from a sewage treatment plant service of a hospital located in the city of Rio de Janeiro, Brazil. Of the 221 isolates identified, 40% were characterized as extended‐spectrum beta‐lactamase (ESBL) producers. Nonpathogenic micro‐organisms and some pathogenic genera were quantified. The most common ESBL‐producing isolates were Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli. The bla_TEM, bla_SHV and bla_CTX‐M genes were detected in 82, 48 and 67% of bacterial isolates, respectively. Conclusions: Results showed that hospital wastewater treatment plant is not suitable systems for the removal of all antibiotic‐resistant micro‐organisms present in hospital wastewaters. Significance and Impact of the Study: This study provides evidence that bacteria resistant to multiple antibiotics and their resistance genes that are usually present in the hospital can reach the environment, even after the use of hospital wastewater treatment plants.     

The microbiology of a pharmaceutical effluent and its public health implications

The effluent of a pharmaceutical company was examined microbiologically. Its bacterial count was 2.15 × 10⁵ c.f.u./ml and there was evidence of faecal contamination with MPN of > 1800. The organisms encountered included Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Serratia marcescens and Pseudomonas aeruginosa. The resistances of the 25 bacterial strains isolated from the effluent to the commonly used antibiotics were studied. About 80% of the isolates were resistant to Amoxycillin, 76% to Nitrofurantoin, 64% to Cotrimoxazole and Augmentin, 60% were resistant to Nalidixic acid, 52% were resistant to Tetracycline and Ofloxacin, while resistance of 12% was obtained for Gentamicin. Among the eight antibiotics tested, seven patterns of drug resistance were obtained and all of them were multiple-drug resistance with the number of antibiotics ranging from 2–8. All the strains of E. coli and S. aureus had high MIC values for Cloxacillin and Amoxycillin. In all, 13 strains of the bacterial isolates had evidence for the production of -lactamases. The potential of the effluent in spreading drug resistance and the public health implications are discussed.     

Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Analysis of the structure of bacteria communities and detection of resistance genes of quinolones from pharmaceutical wastewater

The aim of the present study was to investigate the distribution of bacteria and detect the presence of quinolone resistance gene (qnrA) and integrons (intI1, intI2) in a habitat polluted by pharmaceutical sewage. The bacteria were isolated by nutrient agar and nutrient broth from waste water and sludge collected from the sewage outfall of a pharmaceutical factory. The bacteria were identified by Gram staining and biochemical tests, and the bacterial community diversity was analyzed by Shannon–Wiener diversity index (H), Pielou evenness index (J) and Simpson’s diversity index (D). The occurrence of qnrA and integrons (intI1, intI2) were detected by Real-time PCR assays. The results showed that 90 strains were isolated from water samples and sludge samples including 22 genera and 26 species. Types of bacteria in water samples contained 18 genera and 20 species, while 13 genera and 14 species were detected in sludge samples. Fifty-five Enterobacteriaceae isolates (61.11 %, 55 of 90) were the predominant bacteria in water and sludge samples. Bacterial species richness and evenness in water samples were higher than in sludge samples. The resistance genes of qnrA and integrons (intI1, intI2) with the total DNA and single isolate plasmid DNA were detected. There were a variety of bacterial species and the presence of qnrA and integrons (intI1, intI2) genes in pharmaceutical wastewater habitats, in which Enterobacteriaceae strains were the dominant bacteria. These results suggested that pharmaceutical wastewater had potential risks to public health.     

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。     

Antimicrobial susceptibility of non-O1 Vibrio cholerae isolated from wastewater stabilization ponds in Marrakesh,Morocco

The 365 strains of Vibrio cholerae, isolated in Marrakesh from raw sewage and stabilization pond effluent, were all identified as non-O1 Vibrio cholerae. When tested for their susceptibilities to ampicillin, amoxicillin, cephalothin, streptomycin, novobiocin, chloramphenicol, nalidixic acid and trimethoprim-sulphamethoxazole, 13% of the strains from raw sewage and 20% of those from stabilization pond effluent were found to be resistant to one or more of the antibiotics. There were no significant differences, in terms of drug resistance, between isolates from the new sewage and those from the ponds' effluent.The authors are with the Université Cadi Ayyad, Faculté des Sciences Semialia, Département de Biologie, Laboratoire de Microbiologie, BP S/15, Marrakesh, Morocco     

Effect of chlorination on antibiotic resistance profiles of sewage-related bacteria

A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage influent and chlorinated sewage effluent from a sewage treatment plant, as well as from chlorinated and neutralized dilute sewage, before and after a 24-h regrowth period in the laboratory. Of these isolates, 84% were resistant to one or more antibiotics. Chlorination of influent resulted in an increase in the proportion of bacteria resistant to ampicillin and cephalothin, the increase being most marked after regrowth occurred following chlorination. Of the other nine antibiotics tested, chlorination resulted in an increased proportion of bacteria resistant to some, but a decrease in the proportion resistant to the remainder. Multiple resistance was found for up to nine antibiotics, especially in regrowth populations. Identification of about 5% of the isolates showed that the highest proportion of Escherichia coli fell in untreated sewage. Some rare and potentially pathogenic species were isolated from chlorinated and regrowth samples, including Yersinia enterocolitica, Yersinia pestis, Pasteurella multocida, and Hafnia alvei. Our results indicate that chlorination, while initially lowering the total number of bacteria in sewage, may substantially increase the proportions of antibiotic-resistant, potentially pathogenic organisms.     

Effect of chlorination on antibiotic resistance profiles of sewage-related bacteria.

A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage influent and chlorinated sewage effluent from a sewage treatment plant, as well as from chlorinated and neutralized dilute sewage, before and after a 24-h regrowth period in the laboratory. Of these isolates, 84% were resistant to one or more antibiotics. Chlorination of influent resulted in an increase in the proportion of bacteria resistant to ampicillin and cephalothin, the increase being most marked after regrowth occurred following chlorination. Of the other nine antibiotics tested, chlorination resulted in an increased proportion of bacteria resistant to some, but a decrease in the proportion resistant to the remainder. Multiple resistance was found for up to nine antibiotics, especially in regrowth populations. Identification of about 5% of the isolates showed that the highest proportion of Escherichia coli fell in untreated sewage. Some rare and potentially pathogenic species were isolated from chlorinated and regrowth samples, including Yersinia enterocolitica, Yersinia pestis, Pasteurella multocida, and Hafnia alvei. Our results indicate that chlorination, while initially lowering the total number of bacteria in sewage, may substantially increase the proportions of antibiotic-resistant, potentially pathogenic organisms.     

Determination of mercury and organomercurial resistance in obligate anaerobic bacteria

A methodology for determining the minimum inhibitory concentration of inorganic and organomercurial compounds for obligate anaerobic bacteria is described. A wide variation in the susceptibility of anaerobic clinical and sewage isolates was observed. Isolates of Bacteroides ruminicola and Clostridium perfringens resistant to mercury were examined for their plasmid content and ability to demonstrate inducible resistance. None of the resistant anaerobes contained any plasmids, while resistant facultative isolates from the same source contained several plasmids. In 24 h, resistant strains of clostridia and Bacteroides volatilized 20 and 43% of the 203Hg2+ added to cultures, while Escherichia coli R100 and a sewage isolate of Enterobacter cloacae volatilized 63 and 27%, respectively, of the added 203Hg2+. Attempts to induce mercury resistance in the aerobic isolates were successful, but no induction was seen in the anaerobes. Thus, mercury resistance in these anaerobic isolates was neither inducible nor plasmid mediated.     

Identification of sources of Escherichia coli in South Carolina estuaries using antibiotic resistance analysis

Fecal pollution from nonhuman (pets, livestock or wildlife) and human sources is often one of the major factors associated with urbanization that contribute to the degradation of water quality. Methods to differentiate animal from human sources of fecal coliform contamination could assist resource managers in developing strategies to protect shellfish harvesting areas and recreational waters. In this study, surface water samples were collected from both a developed and an undeveloped watershed in coastal South Carolina. Influent and effluent samples from several wastewater treatment plants (WWTPs) in the same area were also collected. Most Probable Numbers (MPNs) of fecal coliforms were determined for all samples. Escherichia coli isolates were analyzed for antibiotic resistance (AR) to 10 antibiotics. Then, AR indices (no. of resistant/total no. of antibiotics tested), were calculated for each isolate and site. Results indicated that MPNs from the WWTP samples were significantly higher than those from the developed watershed which were significantly higher than those from the undeveloped watershed (p<0.0001). The AR analyses suggested that there was a trend toward increased antibiotic resistance in samples for the urbanized Broad Creek (BC) watershed. In the Okatee River (OR), E. coli isolates from three sites (20%) showed resistance to a single antibiotic (penicillin) but in BC, isolates from seven sites (47%) were resistant to multiple antibiotics, and the predominant resistance pattern was chlortetracycline-oxytetracycline-tetracycline. Raw sewage isolates from most WWTPs contained E. coli that exhibited resistance to multiple antibiotics. Cluster analysis indicated that all resistant OR sites had antibiotic resistant isolates that matched AR patterns found in isolates from WWTPs. Similarly, six of the seven sites in BC had AR patterns that matched with resistance patterns in WWTPs. These results suggest that AR testing may be a useful tool for differentiating E. coli from human and wildlife sources. Further testing of bacterial isolates from known animal sources is necessary to better assess the utility of this approach.     

Effects of Sewage Dumping on Macrobenthic Invertebrates in the Jordan Gulf of Aqaba,Red Sea

There are two sewage outfalls along the Jordanian coastline in the Gulf of Aqaba. During 1982 and 1983 a total of 328 core samples (0.01 m² to a depth of 15 cm) were collected from the two outfalls, two control stations which resemble the outfalls in depth and sediment texture, and from two stations 100 m on both sides of each outfall. Faunal analysis revealed that the total number of individuals, number of species, species richness, and faunal similarities of macrobenthic invertebrates were lower at the sewage outfall near the phosphate loading port than the control station during both collections. At the 100 m stations, the numbers of individuals were generally higher than the sewage and control stations. The number of species, however, was highest at the control station and lowest at the sewage outfall. At the other sewage outfall, where the sewage effluent is discharged sporadically, no measurable effects on macrobenthic invertebrates were found.     

Characterization of plasmids from antibiotic-resistant Shigella isolates by agarose gell electrophoresis.

Gel electrophoresis of DNA from 95 clinical isolates of Shigella sonnei and Shigella flexneri resistant to antibiotics revealed a heterogeneous plasmid population. Most of the plasmids were smaller than 6 megadaltons (Mdal). Six S. sonnei isolates with the most common antibiotic resistance pattern were characterized. They had two plasmids in common: one was a self-transmissible Fi+ plasmid of 46 Mdal encoding resistance to streptomycin and sulphafurazole. In addition, several cryptic plasmids ranging in size from 1.0 to 24.5 Mdal were present. Mobilization of the 5.5 Mdal SuSm plasmid and a 1.0 Mdal cryptic plasmid was demonstrated with all six S. sonnei isolates during conjugation. This mobilization was mediated by the 46 Mdal self-transmissible Fi+ R plasmid and also by a 24.5 Mdal Fi- plasmid carrying no known drug resistance determinants.     

Plasmid Incidence in Bacteria from Deep Subsurface Sediments

Bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of South Carolina. A total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid DNA content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. MICs of Cu²⁺, Cr³⁺, and Hg²⁺ for each isolate were also determined. The overall frequency of plasmid occurrence in the subsurface bacteria was 33%. Resistance was most frequent to penicillin (70% of all isolates), ampicillin (49%), and carbenicillin (32%) and was concluded to be related to the concentrations of the individual antibiotics in the disks used for assaying resistance and to the production of low levels of β-lactamase. The frequencies of resistance to penicillin and ampicillin were significantly greater for isolates bearing plasmids than for plasmidless isolates; however, resistance was not transferable to penicillin-sensitive Escherichia coli. Hybridization of subsurface bacterial plasmids and chromosomal DNA with a whole-TOL-plasmid (pWWO) probe revealed some homology of subsurface bacterial plasmid and chromosomal DNAs, indicating a potential for those bacteria to harbor catabolic genes on plasmids or chromosomes. The incidences of antibiotic resistance and MICs of metals for subsurface bacteria were significantly different from those for drill mud bacteria, ruling out the possibility that bacteria from sediments were derived from drill muds.     

Multiple antibiotic resistances of <Emphasis Type="Italic">Enterococcus</Emphasis> isolates from raw or sand-filtered sewage

Fifty antibiotic-resistant Enterococcus strains were isolated from raw sewage of a wastewater treatment plant and from the same sewage after trickling through a 25-cm sand column, which retained >99% of the initial population. All 50 Enterococcus isolates were resistant against triple sulfa and trimethoprim/sulfamethoxazole and none were resistant against vancomycin. Most of the isolates from raw sewage were resistant to more antibiotics than the isolates from sand column effluent. One Enterococcus isolate from raw sewage (no. 61) and one Enterococcus isolate from sand column effluent (no. 95) had ten antibiotic resistances each. Isolate no. 95 maintained its resistances in the absence of antibiotics during the whole study. It was compared with isolate no. 70, which was one of the isolates, being resistant only against the two sulfonamides. Phenotypically and biochemically, the two organisms were strains of Enterococcus faecalis. Sequence analysis of partical 16S rDNA allowed alignment of isolate no. 95 as a strain of Enterococcus faecium and of isolate no. 70 as a strain of E. faecalis. E. faecium strain no. 95 carried at least six different plasmids, whereas for E. faecalis strain no. 70, no discrete plasmid band was seen on the gels.