Isolation and Identification of Enterococci from Seawater Samples: Assessment of Their Resistance to Antibiotics and Heavy Metals

A hundred Enterococcus strains were isolated from seawater samples collected from coastal areas of Istanbul. Isolates were identified to the species level using standard biochemical tests specified by Facklam and Collins. The species distribution was as follows Enterococcus faecalis (96%), Enterococcus gallinarum (3%) and Enterococcus solitarius (1%). The resistance of bacteria to both heavy metals (zinc [Zn], iron [Fe], cadmium [Cd], chrome [Cr], cobalt [Co]) and antibiotics (ampicillin 10 μg [AP], penicillin G 10 Units [PG], gentamycin 10 μg [GM], streptomycin 10 μg [S], chloramphenicol 10 μg [C], erythromycin 15 μg [E], kanamycin 30 μg [K], amikacin 30 μg [AK], nalidixic acid 30 μg [NA], and vancomycin 30 μg [VA]) was evaluated. None of the strains was resistant to VA. It was found that among the 100 isolates, those that exhibit resistance to antibiotics, particularly NA, S and K, were also resistant all the heavy metals tested. To our knowledge this is the first report focusing on determination of resistance of environmental enterococci found in Istanbul against heavy metals and antibiotics. Thus, combined expressions of antibiotic and heavy metal resistance may help to reinforce ecological and epidemiological studies and to determine the role of these strains in antibiotic and heavy metal resistance dissemination.     

Determining Sources of Fecal Bacteria in Waterways

The microbiological contamination of waterways by pathogenic microbes has been, and is still, a persistent public safety concern in the United States and in most countries of the world. As most enteric pathogens are transmitted through the fecal–oral route, fecal pollution is generally regarded as the major contributor of pathogens to waterways. Fecal pollution of waterways can originate from wastewater treatment facilities, septic tanks, domestic- and wild-animal feces, and pets. Because enteric pathogens are derived from human or animal sources, techniques capable of identifying and apportioning fecal sources have been intensively investigated for use in remediation efforts and to satisfy regulatory concerns. Pollution of human origin is of the most concern, since human feces is more likely to contain human-specific enteric pathogens. Fecal indicator bacteria have been used successfully as the primary tool for microbiologically based risk assessment. However measurement of fecal indicator bacteria does not define what pathogens are present, or define the sources of these bacteria. Microbial source tracking (MST) methods that have the ability to differentiate among sources of fecal pollution are currently under development. These methods will ultimately be useful for risk assessment purposes and to aid regulatory agencies in developing strategies to remediate microbiologically impaired waterways.     

Cu(II), Fe(III) and Mn(II) combinations as environmental stress factors have distinguishing effects on Enterococcus hirae

Pollution by various heavy metals as environmental stress factors might affect bacteria. It was established that iron (Fe(III)), manganese (Mn(II)) and copper (Cu(II)) ion combinations caused effects on Enterococcus hirae that differed from the sum of the effects when the metals were added separately. It was shown that the Cu²⁺-Fe³⁺ combination decreased the growth and ATPase activity of membrane vesicles of wild-type E. hirae ATCC9790 and atpD mutant (with defective F_oF₁-ATPase) MS116. Addition of Mn²⁺-Fe³⁺ combinations within the same concentration range had no effects on growth compared to control (without heavy metals). ATPase activity was increased in the presence of Mn²⁺-Fe³⁺, while together with 0.2 mmol/L N,N'-dicyclohexylcarbodiimide (DCCD), ATPase activity was decreased compared to control (when only 0.2 mmol/L DCCD was present). These results indicate that heavy metals ion combinations probably affect the FOF1-ATPase, leading to conformational changes. Moreover the action may be direct or be mediated by environment redox potential. The effects observed when Fe³⁺ was added separately disappeared in both cases, which might be a result of competing processes between Fe³⁺ and other heavy metals. These findings are novel and improve the understanding of heavy metals ions effects on bacteria, and could be applied for regulation of stress response patterns in the environment.     

Cu(II), Fe(III) and Mn(II) combinations as environmental stress factors have distinguishing effects on Enterococcus hirae

Pollution by various heavy metals as environmental stress factors might affect bacteria. It was established that iron (Fe(III)), manganese (Mn(II)) and copper (Cu(II)) ion combinations caused effects on Enterococcus hirae that differed from the sum of the effects when the metals were added separately. It was shown that the Cu^2 +–Fe^3 + combination decreased the growth and ATPase activity of membrane vesicles of wild-type E. hirae ATCC9790 and atpD mutant (with defective F_oF₁-ATPase) MS116. Addition of Mn^2 +–Fe^3 + combinations within the same concentration range had no effects on growth compared to control (without heavy metals). ATPase activity was increased in the presence of Mn^2 +–Fe^3 +, while together with 0.2 mmol/L N,N′-dicyclohexylcarbodiimide (DCCD), ATPase activity was decreased compared to control (when only 0.2 mmol/L DCCD was present). These results indicate that heavy metals ion combinations probably affect the F_OF₁-ATPase, leading to conformational changes. Moreover the action may be direct or be mediated by environment redox potential. The effects observed when Fe^3 + was added separately disappeared in both cases, which might be a result of competing processes between Fe^3 + and other heavy metals. These findings are novel and improve the understanding of heavy metals ions effects on bacteria, and could be applied for regulation of stress response patterns in the environment.     

Cu(Ⅱ), Fe(Ⅲ) and Mn(Ⅱ) combinations as environmental stress factors have distinguishing effects on Enterococcus hirae

Pollution by various heavy metals as environmental stress factors might affect bacteria. It was established that iron(Fe(Ⅲ)), manganese(Mn(Ⅱ)) and copper(Cu(Ⅱ)) ion combinations caused effects on Enterococcus hirae that differed from the sum of the effects when the metals were added separately. It was shown that the Cu2+–Fe3+combination decreased the growth and ATPase activity of membrane vesicles of wild-type E. hirae ATCC9790 and atp D mutant(with defective FoF1-ATPase) MS116. Addition of Mn2+–Fe3+combinations within the same concentration range had no effects on growth compared to control(without heavy metals). ATPase activity was increased in the presence of Mn2+–Fe3+, while together with0.2 mmol/L N,N′-dicyclohexylcarbodiimide(DCCD), ATPase activity was decreased compared to control(when only 0.2 mmol/L DCCD was present). These results indicate that heavy metals ion combinations probably affect the FOF1-ATPase, leading to conformational changes. Moreover the action may be direct or be mediated by environment redox potential.The effects observed when Fe3+was added separately disappeared in both cases, which might be a result of competing processes between Fe3+and other heavy metals. These findings are novel and improve the understanding of heavy metals ions effects on bacteria,and could be applied for regulation of stress response patterns in the environment.     

Simulating the fate of indigenous antibiotic resistant bacteria in a mild slope wastewater polluted stream

The fate of indigenous surface-water and wastewater antibiotic resistant bacteria in a mild slope stream simulated through a hydraulic channel was investigated in outdoor experiments.The effect of(i) natural(dark) decay,(ii) sunlight,(iii) cloudy cover,(iv) adsorption to the sediment,(v) hydraulic conditions,(vi) discharge of urban wastewater treatment plant(UWTP)effluent and(vii) bacterial species(presumptive Escherichia coli and enterococci) was evaluated.Half-life time(T1/2) of E. coli under sunlight was in the range 6.48–27.7 min(initial bacterial concentration of 10~5 CFU/mL) depending on hydraulic and sunlight conditions. E. coli inactivation was quite similar in sunny and cloudy day experiments in the early 2 hr, despite of the light intensity gradient was in the range of 15–59 W/m~2; but subsequently the inactivation rate decreased in the cloudy day experiment(T1/2= 23.0 min) compared to sunny day(T_(1/2)= 17.4 min). The adsorption of bacterial cells to the sediment(biofilm) increased in the first hour and then was quite stable for the remaining experimental time. Finally, when the discharge of an UWTP effluent in the stream was simulated, the proportion of indigenous antibiotic resistant E. coli and enterococci was found to increase as the exposure time increased, thus showing a higher resistance to solar inactivation compared to the respective total populations.     

Quantitative catchment profiling to apportion faecal indicator organism budgets for the Ribble system, the UK's sentinel drainage basin for Water Framework Directive research

Under the EU Water Framework Directive (WFD) 20/60/EC and the US Federal Water Pollution Control Act 2002 management of water quality within river drainage basins has shifted from traditional point-source control to a holistic approach whereby the overall contribution of point and diffuse sources of pollutants has to be considered. Consequently, there is a requirement to undertake source-apportionment studies of pollutant fluxes within catchments. The inclusion of the Bathing Water Directive (BWD), under the list of 'protected areas' in the WFD places a requirement to control sources of faecal indicator organisms within catchments in order to achieve the objectives of both the BWD (and its revision - 2006/7/EC) and the WFD. This study was therefore initiated to quantify catchment-derived fluxes of faecal indicator compliance parameters originating from both point and diffuse sources. The Ribble drainage basin is the single UK sentinel WFD research catchment and discharges to the south of the Fylde coast, which includes a number of high profile, historically non-compliant, bathing waters. Faecal indicator concentrations (faecal coliform concentrations are reported herein) were measured at 41 riverine locations, the 15 largest wastewater treatment works (WwTWs) and 15 combined sewer overflows (CSOs) across the Ribble basin over a 44-day period during the 2002 bathing season. The sampling programme included targeting rainfall-induced high flow events and sample results were categorised as either base flow or high flow. At the riverine sites, geometric mean faecal coliform concentrations showed statistically significant elevation at high flow compared to base flow. The resultant faecal coliform flux estimates revealed that over 90% of the total organism load to the Ribble Estuary was discharged by sewage related sources during high flow events. These sewage sources were largely related to the urban areas to the south and east of the Ribble basin, with over half the load associated with the relatively small subcatchment of the River Douglas. The majority of this load was attributed to two WwTWs that discharge through a common outfall close to the tidal limit of this catchment. Budgets adjusted to accommodate the impact of proposed UV disinfection of these effluents showed that the load from these sources would be reduced significantly during base flow conditions. However, during high flow events loads would still remain high due to the operation of storm sewage overflows from stormwater retention tanks. The study identified untreated storm sewage spills from urban infrastructure and WwTW stormwater retention tanks as the dominant component of the high flow flux of faecal indicators to receiving waters of the Fylde coast and the associated bathing waters.     

Characteristics and significance of liquid effluent from woodchip corrals in Scotland

Woodchip corrals are increasingly used as cost effective means of over-wintering livestock in temperate regions but there is little information on their potential environmental impact. Four woodchip corrals of varying characteristics were instrumented to capture and quantify the flows reaching the base, where pollutant fluxes may move either vertically to groundwater, or laterally to a water course. Samples for chemical analysis were collected daily by auto-sampler. Samples for bacterial analysis were aseptically hand-sampled. Sampling frequency was increased during high flow events and sampling was conducted over a 12-month period. Microbiological samples were analysed for total coliform (TC), presumptive Escherichia coli (EC) and intestinal enterococci (IE). Leachate was also analysed for total phosphorus, phosphate, total nitrogen, ammonium, total oxidised nitrogen, nitrite and nitrate. Each corral had a recording rain gauge sited within 10 m of the corral surface. Mean total nitrogen concentration in leachate was 339.5 mg l(-1), of which ammoniacal-N comprised approximately 57%. Mean total phosphorus concentration was 94.7 mg l(-1). Geometric mean concentrations of TC, EC and IE were 95,461, 94,983 and 55,552 cfu100 ml(-1), respectively. Significant flows of leachate occurred at the base of the corrals on most days during the 1-year sampling period and flow rate increased with stocking density. Strong positive linear relationships were found between the concentrations of the nutrient parameters and discharge. Strong positive curvilinear relations were found between faecal indicator concentrations and discharge. Different relationships were observed in the stocked and unstocked corrals. The resulting fluxes are sufficient to give concern and to indicate that corral development is worthy of regulatory attention.     

水质标准中指示微生物的发展及现状

饮用水病原微生物污染是公共卫生面临的主要威胁之一,微生物监测在水质监测中的必要性日益受到人们的认可。在实际工作中,一般是通过检测指示微生物间接反映病原微生物的存在。通过调研国内外各组织、机构颁布的水质标准发现,近年来,我国环境质量标准和污染物排放标准中对于指示微生物的选择有从总大肠菌群向粪大肠菌群和大肠埃希氏菌(E.coli)转变的趋势,而美国环保署、欧盟、世界卫生组织、澳大利亚国家健康与医疗研究委员会等根据最新的流行病学证据,强调了大肠埃希氏菌(E.coli)、肠球菌(Enterococci)与粪便污染的相关性更强,可用于替代大肠菌群。建议我国在后续水质标准中对微生物指标进行增补修订时,参考国外经验,形成集成多种指示微生物与多种特定病原体的监测指标体系,以更好地保护环境功能和民众健康。     

Microbiological water quality and its relation to nitrogen and phosphorus at the Pareja limno-reservoir (Guadalajara, Spain)

Bordering on the edge of the Entrepeñas reservoir (Guadalajara, Spain), next to the village of Pareja, a small dam that allows a body of water to develop with a constant level has been built. Initiatives like this (which we have termed “limno-reservoirs”) are innovative in Spain and around the world. Earlier reservoirs such as this one were constructed to create a habitat for birds, but the Pareja limno-reservoir is the first to promote socio-economic development.