Adaptative responses of E. coli to marine environmental stresses: a modelling approach based on viability and dormancy concepts

In an attempt to synthesize conceptual and experimental information on the behaviour of enteric bacteria in seawater, a mathematical model has been developed. This model is based on changes in intracellular metabolisable components depending on physiological responses of the cell when subjected to nutrient starvation, salinity stress and solar radiation. Following a strategy which takes into account short- and mid-term physiological adaptation and reversal processes, the cells can develop in different states: culturable (B₁), viable and definitively nonculturable (B₂), and reversibly dormant (B₃) cells. Model parameters were deduced either from original microcosm experiments, from literature data or calibration procedures. Validation of the model was performed through specific data on the patterns of change in culturable cell abundances when Escherichia coli populations were subjected to separate or simultaneous experimental stresses. It was shown that model simulations fit these data satisfactorily. Simulation results clearly show the effectiveness of the antistress response and the importance of dormancy which confers higher resistance properties. The model also makes it possible to test the effect of the different stresses, and the role of pre-adaptation with regard to the dynamics of the different cellular states.     

Some factors affecting the survival of coliform bacteria in seawater

Some experiments were carried out to explain thein situ phenomena that the number of coliform organisms decreased rapidly from estuaries to offshore, and also at deeper layer, and that the appearance of coliform types varied.In natural seawater, experimental results did not show thatEscherichia coli was extinct by self-purification or anti-biosis action of seawater, but it showed that this organism decreased mainly because of their starvation caused by lack of nourishment. Although the decreasing rate of bacterial density was delayed in enriched seawater, addition of nutrient even at the time of bacterial extinction promoted the appearance of variated form of this bacteria with floc formation.Flocculation of bacterial cells was influenced by quality and quantity of added nutrients. Temperature was shown to have an effect on the floc formation, but appearance of variated form in flocculated cells ofE. coli was not affected by temperature. Flocculated particles of coliform bacteria were adsorbed on suspended particles in seawater and precipitated rapidly. This phenomenon seems to be a cause of the rapid disappearance of coliform bacteria in coastal waters.In bottom deposits the coliform bacteria probably survive longer as physiologically variated forms when suitable nutrients were supplied.     

Effects of dechlorinated seawater on fish behavior

Behavioral responses to chemically dechlorinated seawater were studied using two species of marine fish associated with power plant discharge areas off Southern California. Opaleye (Girella nigricans) neither avoided nor were attracted to chlorinated seawater containing 0·1 mg/liter total residual oxidants (TRO) which had been dechlorinated using equimolar or excess sodium thiosulfate. Blue-banded goby (Lythrypnus dalli) did not avoid dechlorinated seawater initially containing 0·1 or 0·9–1·0 mg/liter TRO. Previously it has been shown that opaleye avoided chlorinated seawater with oxidant concentrations of 0·1 mg/liter and blue-banded gobies avoided 0·9–1·0 mg/liter TRO. Elimination of chlorine-produced oxidants by thiosulphate dechlorination resulted in 100% survival in both species. The toxicological implications of exposure to dechlorinated seawater are discussed.     

Survie et maintien de la virulence de Salmonella Typhimurium VNC exposée simultanément à trois facteurs stressants expérimentaux

The release of enteric pathogenic bacteria in aquatic environments poses the problem of the fate of these bacteria under the effects of environmental factors (solar radiation, salt concentration, temperature, nutrient level, pH, competition). Frequently, these bacterial cells, potentially pathogenic, enter into a non-culturable state on routine bacteriological plating media. However, the use of direct detection methods DAPI stained cells allows the visualization of these Viable but Non Culturable cells VNC. But, beyond the characterization of the viability of the cells electron transport activity, metabolic activity, membrane integrity, structure and/or quantity of DNA, what happens with the virulence of these cells? This problem was experimentally investigated according to the bacterial model Salmonella Typhimurium. The virulence of this strain, which is the agent of the murine typhoid, was evaluated on a mouse model. Experimentally, the effects of some environmental factors on the survival and on the maintenance of virulence of Salmonella Typhimurium were measured in microcosms exposed to UV radiation (four germicidal lamps 8 mW s⁻¹ cm⁻², wave length: 254 nm, salt concentration (Sea Salt Sigma, 37, nutrient starvation. The microcosms were simultaneously submitted to these three factors, with variable exposure times. For each of those times, the viability of the nonculturable cells which became nonculturable because of the exposure to the three factors was measured through different physiological states notable in the cells, after using different fluorescent dyes. The stained cells were observed by epifluorescence microscopy and analysed by image cytometry. So, the cellular populations are characterised by enumeration of respiring bacteria CTC, [39], metabolising bacteria YEK, [22] modified, bacteria owning an undamaged cytoplasmic membrane LD, Live/Dead BacLight Viability Kit. Molecular Probes Inc.; we also determined the quantity and/or structure of DNA of the cells fluorescence level of DAPI stained cells), After exposure to the three factors for one hour 13.56 J cm⁻², while the plate count cell density rapidly decreased from #10⁷ cells mL⁻¹ to0.1 cell mL⁻¹, physiological states of these viable but non-culturable cells are similar to those of non-exposed cells. On the other hand, after exposure for three hours, only 10 % of the cells deposit a CTC formazan-crystal and 20 % are substrate responsive cells enlarged cells in presence of Yeast Extract and Cephalexin: YEC. Half of the cellular population presents an undamaged cytoplasmic membrane and the level of fluorecence of DAPI stained cells is close to 85 %, which shows that the DNA of these cells is weakly damaged. After exposure to the three experimental factors for24 hours 315 J cm⁻², weak replies to the physiological tests used in this study to characterize the viability of the non-culturable cellular population are observed CTC: 4 %; YEC: 2 %; LD: 11.8 % while the fluorescence level of DAPI stained cells remains firm at 80 %. At the same time, the virulence expression of VNC cells of Salmonella Typhimurium, evaluated by intraperitoneal injection to the mouse route which excludes uncontrolled parameters, unlike the per os route does not seem to be correlated with the cellular viability such as it has been evaluated in this study. A 30 min exposure (6.73 J cm⁻²) to the three environmental factors, leading to the non-culturability of almost the entire exposed cell population 0.08 culturable cell mL⁻¹ whereas the level of viability of those culturable cells is closed to the one of non-exposed cells. The injection of 1000 of those cells 0.001 culturable cells in 100 μL inoculated into the mouse a group of ten mice does not cause any mortality four weeks post-inoculation, whereas the injection of the same dose of non-exposed cells leads to the death of all mice in the group one week post-inoculation. According to our preliminary experiments on Salmonella Typhimurium, the loss of the state of culturability and the loss of virulence towards mice by intra-peritoneal route, because of the exposure to associated effects of UV irradiation 254 nm, salinity 37 and nutrient starvation, seem to be concomitant.     

Metagenomic characterization of viral communities in Goseong Bay,Korea

In this study, seawater samples were collected from Goseong Bay, Korea in March 2014 and viral populations were examined by metagenomics assembly. Enrichment of marine viral particles using FeCl₃ followed by next-generation sequencing produced numerous sequences. De novo assembly and BLAST search showed that most of the obtained contigs were unknown sequences and only 0.74% of sequences were associated with known viruses. As a result, 138 viruses, including bacteriophages (87%), viruses infecting algae and others (13%) were identified. The identified 138 viruses were divided into 11 orders, 14 families, 34 genera, and 133 species. The dominant viruses were Pelagibacter phage HTVC010P and Roseobacter phage SIO1. The viruses infecting algae, including the Ostreococcus species, accounted for 9.4% of total identified viruses. In addition, we identified pathogenic herpes viruses infecting fishes and giant viruses infecting parasitic acanthamoeba species. This is a comprehensive study to reveal the viral populations in the Goseong Bay using metagenomics. The information associated with the marine viral community in Goseong Bay, Korea will be useful for comparative analysis in other marine viral communities.     

The respiratory response of juvenile mud crabs, Rhithropanopeus harrisii to variations in salinity and following short-term exposure to halowax 1099®, a polychlorinated naphthalene (PCN)

Juvenile mud crabs Rhithropanopeus harrisii were exposed for five days to Halowax 1099, a polychlorinated naphthalene (approximately 1:1 ratio of tri- and tetrachlorinated isomers). Exposure concentrations were 0, 20, 50 or 100 μg/litre. The exposure salinity was 15%. At the end of the exposure period, respiratory rates of the crabs were determined at 15% S. The respiratory rates were also determined for control and exposed animals which were osmotically shocked by a move from 15% S to either 5 or 25% S immediately before respirometry. Exposure to PCNs always caused the mean respiratory rates of juvenile crabs to increase over control values. The respiratory response of crabs to PCN exposure was similar at the steady-state salinity (15% S) and after hyperosmotic shock (25% S). However, the mean rate for animals exposed to PCNs and given a hyposmotic shock was significantly higher than that for controls and for PCN-exposed animals at the other two salinities. Since hyperosmotic regulation of body fluids is most active at low salinities in this species, we suggest that the PCNs interfere with hyperosmotic regulation and reduce the efficiency of metabolic compensation for hyposmotic shock. The higher energetic cost of acclimation to transient reductions in salinity would be potentially detrimental to wild populations in impacted areas.     

Devenir des virus entériques en mer et influence des facteurs environnementaux

To improve the knowledge of the survival of enteric viruses in a marine environment, the influence of physico-chemical parameters (temperature, UV, salinity) on the survival of infectious poliovirus 1 and hepatitis A virus (HAV) in seawater was first studied, the influence of suspended solids (SS) on poliovirus adsorption and survival in seawater was then evaluated and the detection of rotavirus genome in environmental samples (shellfish, river water, treated wastewater) was finally investigated. The results show that temperature has a major impact on virus survival in seawater as the time necessary to inactivate 90 % of the virus (T₉₀) is 671 days at 4 °C and only 25 days at 25 °C. Ultraviolet light (42 mW s cm⁻²) rapidly inactivates viruses but HAV is more resistant (T₉₀ = 2.6 min) than the poliovirus 1 (T₉₀ = 1.3 min). By contrast, seawater salinity has no effect on virus survival. In presence of SS, 90 % to 99.9 % of the viruses were adsorbed. This adsorption does not provide any protection for viruses with low SS concentrations (3 and 15 mg L⁻¹) but a slight increase in virus survival was observed with a high SS concentration (500 mg L⁻¹). Finally environmental sample analysis indicated that 20 % shellfish, about 40 % river water and 40 % treated wastewater tested positive for the rotavirus genome.     

海绵共栖细菌抗菌活性及其群体感应信号的研究

利用平板涂布法从南海海绵生物样品中分离海绵共栖细菌,以枯草芽孢杆菌、金黄色葡萄球菌、大肠杆菌及酿酒酵母作为实验用指示菌对海绵共栖细菌进行了抗菌活性筛选。利用气相质谱联用仪(GC-MS)建立了群体感应信号分子酰基高丝氨酸内酯类化合物(AHLs)的检测方法,分析了活性菌株粗提液中信号分子的产生及种类。同时采用16S rDNA同源性和系统发育分析对活性菌株进行种属鉴定和系统发生学研究。结果表明,在分离到的94株海绵共栖细菌中18株(占19.1%)细菌具有抗菌活性。通过GC-MS检测确定大多数活性菌株都含有AHLs,因此提出了海绵共栖细菌抗菌活性与群体感应之间可能存在必然联系的推测。通过细菌分类鉴定,结果显示具有抗菌活性的细菌大部分属于芽孢杆菌属(Bacillus)。     

Characterization of bacterial communities associated with seasonal water masses from Tongyoung in South Sea of Korea

Understanding the patterns of microbial diversity and their composition in seawater is necessary to assess the impacts of microbes on marine ecosystem. Although the potential roles of microbes in the ocean have been studied, their diversity and relationship with environmental factors remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with two different water masses from Tongyoung in the South Sea of Korea. Our results revealed highly diverse bacterial communities up to 900 Operational Taxonomic Units (OTUs) estimated from each seawater which was collected in the month of March and May, when the environmental conditions including temperature differed significantly: 7.2°C and 17.6°C in March and May, respectively. Altogether, 13 bacterial phyla were recovered from the seawater, of which Proteobacteria was the most dominant group. In addition, the value of the Shannon index, which measures the evenness of the distribution of individuals among OTUs, in May is higher than that in March, indicating that it displays a wider diversity of bacteria. Interestingly, the proportion of pathogenic bacteria was significantly increased in the month of May compared to March, suggesting that pathogenic bacteria were increasingly emerging in May. In particular, Pseudoalteromonas and Vibrio spp. were determined as major pathogenic bacteria from both water masses, of which Vibrio spp. were dominant.     

海绵共栖细菌抗菌活性及其群体感应信号的研究

利用平板涂布法从南海海绵生物样品中分离海绵共栖细菌,以枯草芽孢杆菌、金黄色葡萄球菌、大肠杆菌及酿酒酵母作为实验用指示菌对海绵共栖细菌进行了抗菌活性筛选。利用气相质谱联用仪(GC-MS)建立了群体感应信号分子酰基高丝氨酸内酯类化合物(AHLs)的检测方法,分析了活性菌株粗提液中信号分子的产生及种类。同时采用16S rDNA同源性和系统发育分析对活性菌株进行种属鉴定和系统发生学研究。结果表明,在分离到的94株海绵共栖细菌中18株(占19.1%)细菌具有抗菌活性。通过GC-MS检测确定大多数活性菌株都含有AHLs,因此提出了海绵共栖细菌抗菌活性与群体感应之间可能存在必然联系的推测。通过细菌分类鉴定,结果显示具有抗菌活性的细菌大部分属于芽孢杆菌属(Bacillus)。     

近海沉积物与水体中天然大分子聚合物降解菌的原位富集与多样性分析

本研究选取5种海洋动植物大分子高聚物或其天然组织,包括虾壳、鱼鳞、海带叶片、几丁质和壳聚糖,分别在海水表层和沉积物环境中进行富集,定期取样,通过高通量测序分析菌群多样性。结果发现,不同有机物原位富集的细菌多样性存在较大的差异,而且同种底物在海水表层与沉积物中的降解菌菌群差异较大。从物种多样性看,在海水表层环境中富集的鱼鳞样品种群最丰富,而沉积物环境中富集的海带叶片样品菌群多样性最低,除其优势菌群为热袍菌门外,其他所有富集物中优势菌均为变形菌门。其中脱硫杆菌科、黄杆菌科、脱硫弧菌科和弧菌科占有较大比例,脱硫杆菌科在所有样品中优势较大,黄杆菌科在海水表层环境样品中为优势菌群,弧菌科在沉积物样品几丁质和壳聚糖样品中占比较高。本研究通过对同种海洋环境中不同富集样品之间以及同种底物不同环境中富集菌群之间的比较,分析结果得到了原位条件下参与大分子聚合物降解的菌群种类,但有待于在更多不同的海域富集物中进行验证。     

繁茂膜海绵清除大肠杆菌的过程

通过分析海绵清除大肠杆菌的过程,研究海绵净化细菌的机理。作者利用荧光显微镜和激光共聚焦显微镜观测等手段,监测和分析了绿色荧光大肠杆菌(Escherichia coli)在繁茂膜海绵(Hymeniacidon perlevis)体内、体外水环境中数量变化过程。在1 L含有3×107个/m L绿色荧光大肠杆菌的海水中放入鲜重(1.02±0.11)g的繁茂膜海绵24块,处理7 h,海水中的荧光大肠杆菌数量逐渐降低;而海绵体内荧光大肠杆菌数量在2 h时内逐渐增多,之后的2 h趋于稳定,4 h以后开始逐渐减少。水体中大肠杆菌不仅进入海绵体内,而且进入海绵细胞内。含有荧光大肠杆菌的海绵块转入无菌海水中后,海绵体内及细胞中大肠杆菌逐渐消失,而且大肠杆菌没有被释放到环境海水中。分析表明,繁茂膜海绵能够以摄食的方式净化水环境中的大肠杆菌。     

Acute toxicity of heavy metals to two species of marine nematodes

The acute toxicity of seawater solutions of copper, zinc, lead, cadmium and mercury to two species of free-living marine nematodes was studied. Enoplus brevis (Bastian) is a euryhaline species inhabitating intertidal estuarinemud. The closely related Enoplus communis (Bastian) is stenohaline and may be found as a commensal in the byssus threads of seed mussels. Specimens of both species were collected from two sites on the Northeast coast of England showing different levels of pollution by heavy metals. Both species were more susceptible to copper, lead and mercury than to zinc and cadmium, but the order of toxicity of the metals changed with exposure concentration. The euryhaline species survived better than the strictly marine species and there is a suggestion that animals from a polluted site were more tolerant of elevated of heavy metals.     

Numerical modelling of blue mussel (Mytilus edulis) bacterial contamination

Bivalve shellfish such as oysters and mussels can concentrate human pathogens when grown in areas impacted by municipal wastewater. Under EU regulation this risk to consumers is controlled by determining the sanitary quality of bivalve shellfish production areas based on the concentration of Escherichia coli present in shellfish flesh. The authors present a modelling approach to simulate an uptake of E. coli from seawater and subsequent depuration by Mytilus edulis. The model that dynamically predicts E. coli concentration in the mussel tissue is embedded within a 3-D numerical modelling system comprising hydrodynamic, biogeochemical, shellfish ecophysiological and the newly proposed microbial modules. The microbial module has two state variables, namely, the concentrations of E. coli in water and in the mussel tissue. Novel formulations to calculate the filtration rates by mussels and the resulting uptake of bacteria are proposed; these rates are updated at every computational time step. Concentrations of E. coli in seawater are also updated accordingly taking into account the amounts ingested by mussels. The model has been applied to Bantry Bay in the south-west of Ireland. The results indicate that the model is capable of reproducing the official classification of shellfish waters in the bay based on monthly sampling at several stations. The predicted filtration rates and ratios of E. coli in water and mussels also compare well with the literature. The model thus forms a tool that may be used to assist in the classification of shellfish waters at much greater spatial and temporal detail than that offered by a field monitoring programme. Moreover, it can also aid in designing an efficient monitoring programme. The model can also be utilised to determine the contribution of individual point sources of pollution on the microbial loading in mussels and, when incorporated into an operational framework, it can provide a short-term forecasting of microbial contamination in a shellfishery. Also, the model can be easily extended to include other shellfish and pathogen species.     

中国重点海水浴场中粪肠球菌和主要肠道病毒的调查研究

本文调查了中国十大典型海水浴场,旨在监测人类肠道病毒在海水浴场的发生情况,并且找出表面海水中肠道病毒的存在与传统的粪便污染指示物一粪大肠菌群是否存在关系,如果存在,是什么关系。分别采用常规的平板计数法和RT-PCR技术对粪大肠菌群和人类致病性的主要肠道病毒（甲肝病毒、轮状病毒、脊髓灰质炎病毒）进行了检测。结果显示,在2...     

An investigation into the effects of silver nanoparticles on antibiotic resistance of naturally occurring bacteria in an estuarine sediment

The aim of this study was to test whether silver nanoparticles (Ag-NPs) released into estuarine environments result in increased antibiotic resistance amongst the natural bacterial population in estuarine sediments. A 50-day microcosm exposure experiment was carried out to investigate the effects of Ag-NPs (50 nm average diameter) on the antibiotic resistance of bacteria in sediments from an estuary in southwest England. Experimental microcosms were constructed using 3.5 kg sediment cores with 20 l of overlaying seawater treated with (final) Ag-NPs concentrations of 0, 50 or 2000 μg l⁻¹ (n = 3). Sediment samples were screened at the end of the exposure period for the presence of bacteria resistant to eight different antibiotics. Multivariate statistical analyses showed that there was no increase in antibiotic resistance amongst the bacterial population in the sediment due to the dosing of the microcosms with Ag-NPs. This study indicates that, under the tested conditions, Ag-NPs released into the coastal marine environment do not increase antibiotic resistance among naturally occurring bacteria in estuarine sediments. These results contrast previous findings where antimicrobial effects of Ag-NPs on key bacterial species in laboratory experiments have been demonstrated, and reasons for this are discussed. The negligible effects demonstrated on bacterial populations under the selected estuarine conditions, provide important information on no observed effect concentrations (NOECs) for environmental regulation.     

不同长牡蛎群体对海水酸化的生理响应差异分析

海水酸化暴露可对海洋生物产生多层面的影响。本研究以潮间带野生与潮下带养殖长牡蛎(不同生境背景)的不同组织(鳃、外套膜及消化腺)为研究对象,分析在室内调控p CO2模拟海水酸化暴露条件下,其基础代谢活动、能量代谢以及氧化应激相关指标的变化情况。结果显示:海水酸化暴露后,两种长牡蛎(Crassostrea gigas)的基础代谢过程均受到了一定抑制作用且受影响程度差异明显。潮间带野生与潮下带养殖长牡蛎的关键生理过程(能量代谢及氧化应激)对海水酸化暴露存在不同的响应变化,表明两种长牡蛎应对海水酸化的调节机制可能存在差异。依据PLS-DA分析结果显示,在所有生理指标中,对样本的差异贡献较高(VIP值1)的指标为:SDH、AST、ATPase、ATP含量、糖原含量、CAT、GST及SOD,表明海水酸化暴露后,在两种长牡蛎的3种组织中上述指标的响应变化程度更大。综合评价分析多个生理指标的整体变化揭示:在海水酸化暴露条件下,潮间带野生长牡蛎比潮下带养殖长牡蛎对海水酸化的生理响应更为剧烈;相比于鳃及消化腺组织,长牡蛎外套膜组织可能受影响更大。     

Differential response to ocean acidification in physiological traits of Concholepas concholepas populations

Phenotypic adaptation to environmental fluctuations frequently occurs by preexisting plasticity and its role as a major component of variation in physiological diversity is being widely recognized. Few studies have considered the change in phenotypic flexibility among geographic populations in marine calcifiers to ocean acidification projections, despite the fact that this type of study provides understanding about how the organism may respond to this chemical change in the ocean. We examined the geographic variation in CO₂ seawater concentrations in the phenotype and in the reaction norm of physiological traits using a laboratory mesocosm approach with short-term acclimation in two contrasting populations (Antofagasta and Calfuco) of the intertidal snail Concholepas concholepas. Our results show that elevated pCO₂ conditions increase standard metabolic rates in both populations of the snail juveniles, likely due to the higher energy cost of homeostasis. Juveniles of C. concholepas in the Calfuco (southern) population showed a lower increment of metabolic rate in high-pCO₂ environments concordant with a lesser gene expression of a heat shock protein with respect to the Antofagasta (northern) population. Combined these results indicate a negative effect of ocean acidification on whole-organism functioning of C. concholepas. Finally, the significant Population × pCO₂ level interaction in both studied traits indicates that there is variation between populations in response to high-pCO₂ conditions.     

Sub-Lethal Effects of Elevated Concentration of CO<Subscript>2</Subscript> on Planktonic Copepods and Sea Urchins

Data concerning the effects of high CO₂ concentrations on marine organisms are essential for both predicting future impacts of the increasing atmospheric CO₂ concentration and assessing the effects of deep-sea CO₂sequestration. Here we review our recent studies evaluating the effects of elevated CO₂ concentrations in seawater on the mortality and egg production of the marine planktonic copepod, Acartia steueri, and on the fertilization rate and larval morphology of sea urchin embryos, Hemicentrotus pulcherrimus and Echinometra mathaei. Under conditions of +10,000 ppm CO₂ in seawater (pH 6.8), the egg production rates of copepods decreased significantly. The survival rates of adult copepods were not affected when reared under increased CO₂ for 8 days, however longer exposure times could have revealed toxic effects of elevated CO₂ concentrations. The fertilization rate of sea urchin eggs of both species decreased with increasing CO₂ concentration. Furthermore, the size of pluteus larvae decreased with increasing CO₂ concentration and malformed skeletogenesis was observed in both larvae. This suggests that calcification is affected by elevated CO₂ in the seawater. From these results, we conclude that increased CO₂ concentration in seawater will chronically affect several marine organisms and we discuss the effects of increased CO₂ on the marine carbon cycle and marine ecosystem. This revised version was published online in July 2006 with corrections to the Cover Date.