Addressing the Joint Impact of Temperature and pH on Vibrio harveyi Adaptation in the Time of Climate Change.

Global warming and acidification of the global ocean are two important manifestations of the ongoing climate change. To characterize their joint impact on Vibrio adaptation and fitness, we analyzed the temperature-dependent adaptation of Vibrio harveyi at different pHs (7.0, 7.5, 8.0, 8.3 and 8.5) that mimic the pH of the world ocean in the past, present and future. Comparison of V. harveyi growth at 20, 25 and 30 °C show that higher temperature per se facilitates the logarithmic growth of V. harveyi in nutrient-rich environments in a pH-dependent manner. Further survival tests carried out in artificial seawater for 35 days revealed that cell culturability declined significantly upon incubation at 25 °C and 30 °C but not at 20 °C. Moreover, although acidification displayed a negative impact on cell culturability at 25 °C, it appeared to play a minor role at 30 °C, suggesting that elevated temperature, rather than pH, was the key player in the observed reduction of cell culturability. In addition, analyses of the stressed cell morphology and size distribution by epifluorescent microscopy indicates that V. harveyi likely exploits different adaptation strategies (e.g., acquisition of coccoid-like morphology) whose roles might differ depending on the temperature-pH combination.

Efficiency and specificity of CARD-FISH probes in detection of marine vibrios.

Vibrio is a bacterial genus widely distributed in natural aquatic systems. Some Vibrio species can cause severe diseases in both marine organisms and humans. Previous studies revealed a link between the current climate change and increased incidence of the Vibrio-associated diseases recently causing sanitary, economic and/or ecological problems worldwide. The conventional culture-based methods (e.g. selection on TCBS agar) used to monitor the presence of Vibrio spp. in environmental samples are not always straightforward and can underestimate the number of cells, especially in microbial populations containing a fraction of 'dormant' cells (e.g. cells in the Viable but Non Culturable [VBNC] state). This problem can be overcome by using alternative culture-free approaches such as Catalysed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH). To select an efficient CARD-FISH probe for detection of Vibrio spp. in environmental samples, we have assessed the most promising probes described in the literature by using both computer-assisted and experimental approaches. Our results demonstrate that the use of the optimized protocol along with a very specific probe, ViB572a, can offer the high sensitivity and selectivity of CARD-FISH detection of marine vibrios in natural seawater.

The Effect of Visible Light on Cell Envelope Subproteome during Vibrio harveyi Survival at 20 °C in Seawater.

A number of Vibrio spp. belong to the well-studied model organisms used to understand the strategies developed by marine bacteria to cope with adverse conditions (starvation, suboptimal temperature, solar radiation, etc.) in their natural environments. Temperature and nutrient availability are considered to be the key factors that influence Vibrio harveyi physiology, morphology, and persistence in aquatic systems. In contrast to the well-studied effects of temperature and starvation on Vibrio survival, little is known about the impact of visible light able to cause photooxidative stress. Here we employ V. harveyi ATCC 14126T as a model organism to analyze and compare the survival patterns and changes in the protein composition of its cell envelope during the long-term permanence of this bacterium in seawater microcosm at 20 °C in the presence and absence of illumination with visible light. We found that V. harveyi exposure to visible light reduces cell culturability likely inducing the entry into the Viable but Non Culturable state (VBNC), whereas populations maintained in darkness remained culturable for at least 21 days. Despite these differences, the starved cells in both populations underwent morphological changes by reducing their size. Moreover, further proteomic analysis revealed a number of changes in the composition of cell envelope potentially accountable for the different adaptation pattern manifested in the absence and presence of visible light.

Analysis of Vibrio harveyi adaptation in sea water microcosms at elevated temperature provides insights into the putative mechanisms of its persistence and spread in the time of global warming.

Discovering the means to control the increasing dissemination of pathogenic vibrios driven by recent climate change is challenged by the limited knowledge of the mechanisms in charge of Vibrio spp. persistence and spread in the time of global warming. To learn about physiological and gene expression patterns associated with the long-term persistence of V. harveyi at elevated temperatures, we studied adaptation of this marine bacterium in seawater microcosms at 30 °C which closely mimicked the upper limit of sea surface temperatures around the globe. We found that nearly 90% of cells lost their culturability and became partly damaged after two weeks, thus suggesting a negative impact of the combined action of elevated temperature and shortage of carbon on V. harveyi survival. Moreover, further gene expression analysis revealed that major adaptive mechanisms were poorly coordinated and apparently could not sustain cell fitness. On the other hand, elevated temperature and starvation promoted expression of many virulence genes, thus potentially reinforcing the pathogenicity of this organism. These findings suggest that the increase in disease outbreaks caused by V. harveyi under rising sea surface temperatures may not reflect higher cell fitness, but rather an increase in virulence enabling V. harveyi to escape from adverse environments to nutrient rich, host-pathogen associations.

Changes in the Vibrio harveyi Cell Envelope Subproteome During Permanence in Cold Seawater.

Previous work demonstrated that physiological, morphological, and gene expression changes as well as the time-dependent entry into the viable but not culturable (VBNC) state are used by Vibrio species to survive and cope with diverse stress conditions including seasonal temperature downshifts and starvation. To learn more about the nature and specific contribution of membrane proteins to cell adaptation and survival, we analyzed variations in the protein composition of cell envelope and related them to morphological and physiological changes that were taking place during the long-term permanence of Vibrio harveyi in seawater microcosm at 4 °C. We found that after 21 days of permanence, nearly all population (ca. 99 %) of V. harveyi acquired the VBNC phenotype. Although the size of V. harveyi cells gradually decreased during the incubation time, we found that this morphological change was not directly related to their entry into the VBNC state. Our proteomic study revealed that the level of membrane proteins playing key roles in cellular transport, maintenance of cell structure, and in bioenergetics processes remained unchanged along starvation at low temperature, thus suggesting that V. harveyi might need these proteins for the long-term survival and/or for the resuscitation process. On a contrary, the level of two proteins, elongation factor Tu (EF-TU) and bacterioferritin, greatly increased reaching the maximal values by the end of the incubation period. We further discuss the above data with respect to the putative roles likely exerted by membrane proteins during transition to and maintaining of the VBNC state.

Unveiling the Metabolic Pathways Associated with the Adaptive Reduction of Cell Size During Vibrio harveyi Persistence in Seawater Microcosms.

Owing to their ubiquitous presence and ability to act as primary or opportunistic pathogens, Vibrio species greatly contribute to the diversity and evolution of marine ecosystems. This study was aimed at unveiling the cellular strategies enabling the marine gammaproteobacterium Vibrio harveyi to adapt and persist in natural aquatic systems. We found that, although V. harveyi incubation in seawater microcosm at 20 °C for 2 weeks did not change cell viability and culturability, it led to a progressive reduction in the average cell size. Microarray analysis revealed that this morphological change was accompanied by a profound decrease in gene expression affecting the central carbon metabolism, major biosynthetic pathways, and energy production. In contrast, V. harveyi elevated expression of genes closely linked to the composition and function of cell envelope. In addition to triggering lipid degradation via the ß-oxidation pathway and apparently promoting the use of endogenous fatty acids as a major energy and carbon source, V. harveyi upregulated genes involved in ancillary mechanisms important for sustaining iron homeostasis, cell resistance to the toxic effect of reactive oxygen species, and recycling of amino acids. The above adaptation mechanisms and morphological changes appear to represent the major hallmarks of the initial V. harveyi response to starvation.

Mechanisms involved in Escherichia coli and Serratia marcescens removal during activated sludge wastewater treatment.

Wastewater treatment reduces environmental contamination by removing gross solids and mitigating the effects of pollution. Treatment also reduces the number of indicator organisms and pathogens. In this work, the fates of two coliform bacteria, Escherichia coli and Serratia marcescens, were analyzed in an activated sludge process to determine the main mechanisms involved in the reduction of pathogenic microorganisms during wastewater treatment. These bacteria, modified to express green fluorescent protein, were inoculated in an activated sludge unit and in batch systems containing wastewater. The results suggested that, among the different biological factors implied in bacterial removal, bacterivorous protozoa play a key role. Moreover, a representative number of bacteria persisted in the system as free-living or embedded cells, but their distribution into liquid or solid fractions varied depending on the bacterium tested, questioning the real value of bacterial indicators for the control of wastewater treatment process. Additionally, viable but nonculturable cells constituted an important part of the bacterial population adhered to solid fractions, what can be derived from the competition relationships with native bacteria, present in high densities in this environment. These facts, taken together, emphasize the need for reliable quantitative and qualitative analysis tools for the evaluation of pathogenic microbial composition in sludge, which could represent an undefined risk to public health and ecosystem functions when considering its recycling.

Reprogramming of Vibrio harveyi gene expression during adaptation in cold seawater.

The life and survival of the marine bacterium Vibrio harveyi during its adaptation in natural aquatic systems is highly influenced by the availability of nutrients and temperature. To learn about adaptation strategies evolved by this bacterium to cope with drastic temperature downshifts and nutrients depletion, we have studied the phenotypical and gene expression changes occurring in V. harveyi during its adaptation to cold seawater. We found that incubation in cold seawater up to 12 h did not cause any significant morphological changes in V. harveyi and had no effect on the number of viable and culturable cells. Microarray analysis revealed that the V. harveyi response to cold seawater leads to up- and downregulation of numerous genes controlling the central carbon metabolism, nucleotide and amino acid biosynthesis as well as DNA repair. In addition, expression of some genes controlling biosynthesis of lipids, molecular transport, and energy production was altered to likely affect the composition and properties of the V. harveyi cell envelope, thus implying the putative role of this compartment in adaptation to stress. Here, we discuss these results with regard to the putative adaptive responses likely triggered by V. harveyi to cope with environmental challenges in natural aquatic systems.

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli.

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T90 values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state.

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations?

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.

Inability of Escherichia coli to resuscitate from the viable but nonculturable state.

After induction of the viable but nonculturable (VBNC) state in Escherichia coli populations, we analysed abiotic and biotic factors suggested to promote the resuscitation process. The response to the stressing conditions implied the formation of three subpopulations, culturable, VBNC and nonviable. In most adverse situations studied, the VBNC subpopulation did not represent the dominant fraction, decreasing with time. This suggests that, in most cases, the VBNC is not a successful phenotype. Combining methods of dilution and inhibition of remaining culturable cells, we designed a working protocol in order to distinguish unequivocally between regrowth and resuscitation. Reversion of abiotic factors inducing nonculturability as well as prevention of additional oxidative stress did not provoke resuscitation. Participation of biotic factors was studied by addition of supernatants from different origin without positive results. These results indicate that the E. coli strain used is not able to resuscitate from the VBNC state. VBNC cells release into the surrounding medium, and could thus aid in the survival of persisting culturable cells. The formation of a VBNC subpopulation could thus be considered as an adaptive process, designed for the benefit of the population as a whole.