In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus.

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. The bacterium occurs naturally in molluscan shellfish, and ingestion of raw oysters is typically the source of human infection. V. vulnificus is also known to enter a viable but nonculturable (VBNC) state, wherein the cells are no longer culturable on routine plating media but can be shown to remain viable. Whether or not this human pathogen remains virulent when entering the VBNC state has not been definitively demonstrated. In this study, the VBNC state was induced through a temperature downshift to 5 degrees C, with cells becoming nonculturable (< 0.1 CFU/ml) within 7 days. As they became nonculturable, virulence was determined by employing an iron overload mouse model. At the point of nonculturability (7 days), injections of the diluted microcosm population resulted in death when < 0.04 CFU was inoculated, although > 10(5) cells in the VBNC state were present in the inoculum. Culturable cells of V. vulnificus, with identification confirmed through PCR, were recovered from the blood and peritoneal cavities of mice which had died from injections of cells present in the VBNC state for at least 3 days. Thus, our data suggest that cells of V. vulnificus remain virulent, at least for some time, when present in the VBNC state and are capable of causing fatal infections following in vivo resuscitation. Our studies also indicate, however, that virulence decreases significantly as cells enter the VBNC state, which may account, at least to some extent, for the decrease in infections caused by this bacterium during winter months.     

In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state

Isolation of Vibrio vulnificus during winter months is difficult due to the entrance of these cells into the viable but nonculturable (VBNC) state. While several studies have investigated in vitro gene expression upon entrance into and persistence within the VBNC state, to our knowledge, no in situ studies have been reported. We incubated clinical and environmental isolates of V. vulnificus in estuarine waters during winter months to monitor the expression of several genes during the VBNC state and compared these to results from in vitro studies. katG (periplasmic catalase) was down-regulated during the VBNC state in vitro and in situ compared to the constitutively expressed gene tufA. Our results indicate that the loss of catalase activity we previously reported is a direct result of katG repression, which likely accounts for the VBNC response of this pathogen. While expression of vvhA (hemolysin) was detectable in environmental strains during in situ incubation, it ceased in all cases by ca. 1 h. These results suggest that the natural role of hemolysin in V. vulnificus may be in osmoprotection and/or the cold shock response. Differences in expression of the capsular genes wza and wzb were observed in the two recently reported genotypes of this species. Expression of rpoS, encoding the stress sigma factor RpoS, was continuous upon entry into the VBNC state during both in situ and in vitro studies. We found the half-life of mRNA to be less than 60 minutes, confirming that mRNA detection in these VBNC cells is a result of de novo RNA synthesis.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus.

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Oleic acid is elevated in cell membranes during rapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis

The integrity of cellular membranes is critical to the survival of insects at low temperatures, thus an advantage is conferred to insects that can adjust their composition of membrane fatty acids (FAs). Such changes contribute to homeoviscous adaption, a process that allows cellular membranes to maintain a liquid-crystalline state at temperatures that are potentially low enough to cause the membrane to enter the gel state and thereby lose its ability to maintain homeostasis. Flesh flies (Sarcophaga crassipalpis) were subjected to two experimental conditions that elicit low temperature tolerance: rapid cold-hardening and diapause. FAs were isolated and analyzed using gas chromatography-mass spectrometry. FAs changed in response to both rapid cold-hardening and diapause. In response to rapid cold-hardening (8 h at 4 degrees C), the proportion of oleic acid (18:1n-9) in pharate adults increased from 30% to 47% of the total FA pool. The proportion of almost every other FA was reduced. By entering diapause, pupae experienced an even greater increase in oleic acid proportion, to 58% of the total FA pool. Oleic acid not only promotes membrane fluidity at low temperature but also allows the cell membrane to maintain a liquid crystalline state if temperatures increase.     

Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.     

Differential effects of temperature and starvation on induction of the viable-but-nonculturable state in the coral pathogens Vibrio shiloi and Vibrio tasmaniensis

We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20 degrees C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17 degrees C), the VBNC state was not induced by incubation at 4 degrees C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30 degrees C), was induced into the VBNC state by incubation at 4 degrees C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20 degrees C, and V. shiloi became VBNC after incubation for 126 days at 20 degrees C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.     

Characterization of Aspergillus species based on fatty acid profiles

Cellular fatty acid (FA) composition was utilized as a taxonomic tool to discriminate between different Aspergillus species. Several of the tested species had the same FA composition and different relative FA concentrations. The most important FAs were palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which represented 95% of Aspergillus FAs. Multivariate data analysis demonstrated that FA analysis is a useful tool for differentiating species belonging to genus Aspergillus. All the species analyzed showed significantly FA acid profiles (p < 0.001). Furthermore, it will be possible to distinguish among Aspergillus spp. in the Flavi Section. FA composition can serve as a useful tool for the identification of filamentous fungi.     

Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State

Like many other gram-negative bacteria, the human pathogen Vibrio vulnificus is induced into a viable but nonculturable (VBNC) state by incubation at low temperatures. The ability of any bacterium to resuscitate from this dormant state would appear to be essential if the VBNC state is truly a survival strategy. The question as to whether the culturable cells which appear following removal of the inducing stress are a result of true resuscitation or of regrowth of a few residual culturable cells has long been debated. V. vulnificus was examined for its ability to resuscitate from this state following a temperature upshift. Several lines of investigation, including dilution studies, determination of the time necessary for appearance of a culturable population, and the effects of nutrient on recovery, all indicated that, at least for V. vulnificus, true resuscitation does occur. Our studies further suggest that nutrient is in some way inhibitory to the resuscitation of cells in the VBNC state and that studies which add nutrient in an attempt to detect resuscitation are able to detect only residual culturable cells which might be present and which were not inhibited by the added nutrient.     

Unravelling the significance of cellular fatty acid-binding proteins

Cellular long-chain fatty acid (FA) transport and metabolism are believed to be regulated by membrane-associated and soluble proteins that bind and transport FAs. Several different classes of membrane proteins have been proposed as FA acceptors or transmembrane FA transporters. New evidence from in-vitro and whole-animal studies supports the existence of protein-mediated transmembrane transport of FAs, which is likely to coexist with passive diffusional uptake. The trafficking of FAs by intracellular fatty acid-binding proteins may involve their interaction with specific membrane or protein targets. Evidence is also emerging for concerted actions between the membrane and cytoplasmic fatty acid-binding proteins that allow for efficient regulation of FA transport and metabolism.     

Bioengineered emulsans from Acinetobacter calcoaceticusRAG-1 transposon mutants

Transposon mutants of Acinetobacter calcoaceticus strain RAG-1 were studied in an effort to control fatty acid (FA) substitution patterns of emulsan, a bioemulsifier secreted by the organism. The disrupted genes, involved in the biosynthetic pathways of biotin, histidine, cysteine or purines, influenced the level and types of FAs incorporated into emulsan. The structural variants of emulsan generated by the transposon mutants were characterized for yield, FA content, molecular weight, and emulsification behavior when grown on a series of FAs of different chain lengths from C11 to C18. Yields of emulsan from the transposon mutants were found to be lower than the parent strain and depended on the type of FA used to supplement the growth medium. Mutants 13D (His-) and 52D (Cys-) grown on LB plus C16 or C14, respectively, exhibited enhanced emulsifying activity compared to A. calcoaceticus RAG-1. The presence and composition of long chain FAs on the polysaccharide backbone influenced emulsification behavior: particularly a high mole percentage of C16 (48%) and C18 (42%). The results provide important insight into the bioengineering of bioemulsifier-producing microorganisms and provide a path towards highly tailored novel amphipathic structures to utilize as biodegradable in environmental, biomedical, and personal care applications.     

Changes in the content and composition of lipid fatty acids in tobacco leaves and roots at low-temperature hardening

Changes in the fatty acid (FA) composition of leaf and root lipids of heat-loving tobacco (Nicotiana tabacum L., cv. Samsun) plants during low-temperature hardening (8°C for 6 days) were studied. Hardening could improve leaf but not root cold tolerance. As this took place, the relative content of polyunsaturated (18:2n-6 and 18:3n-3) FAs increased and the proportion of saturated and monounsaturated FAs decreased. In contrast, in the roots hardening slightly increased the concentration of saturated FAs (16:0 and 18:0) and reduced the level of unsaturated FAs (18:1n-9, 18:2n-6, and 18:3n-3). At the same time, root lipids contained much C_20–24 FAs, and their content increased during hardening. It was suggested that an increased FA saturation and elevated proportion of C_20–24 FAs in the root lipids resulting in the lower membrane fluidity could be a reason for incapability of heat-loving tobacco plant roots of hardening and plant death at the lowtemperature stress.     

Cellular fatty acid composition from Sarcobium lyticum (Legionella lytica comb. nov.)--an intracellular bacterial pathogen of amoebae

Legionella lytica comb. nov. an intracellular bacterial pathogen of small free-living amoebae was subjected to cellular fatty acid (FA) analysis employing base and acid catalyzed cleavage, gas-liquid chromatography and mass spectrometry. Both unbranched and branched (iso and anteiso) FA of chains ranging from 14 to 30 carbon atoms occurred. The presence of two long-chain FA: 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid, characteristic for legionellae, was found. Nine amide-linked 3-hydroxy-FA were revealed. The main 3-hydroxy-fatty acids comprise: 3-OH-14:0, 3-OH-16:0, 3-OH-18:0, 3-OH-i18:0, 3-OH-15:OH, 3-OH-i16:0 amd 3-OH-i17:0. The profile of hydroxy FAs permits allocation of L. lytica to group 3 of legionellae which comprise blue-white fluorescent species.     

Induction of viable but nonculturable state in Vibrio parahaemolyticus and its susceptibility to environmental stresses

AIMS: This work analysed factors that influence the induction of viable but nonculturable (VBNC) state in the common enteric pathogen, Vibrio parahaemolyticus. The susceptibility of the VBNC cells to environmental stresses was investigated. METHODS AND RESULTS: Bacterium was cultured in tryptic soy broth-3% NaCl medium, shifted to a nutrient-free Morita mineral salt-0.5% NaCl medium (pH 7.8) and further incubated at 4 degrees C in a static state to induce the VBNC state in 28-35 days. The culturability and viability of the cells were monitored by the plate count method and the Bac Light viable count method, respectively. Cells grown at the optimum growth temperature and in the exponential phase better induced the VBNC state than those grown at low temperature and in the stationary phase. Low salinity of the medium crucially and markedly shortened the induction period. The VBNC cells were highly resistant to thermal (42, 47 degrees C), low salinity (0% NaCl), or acid (pH 4.0) inactivation. CONCLUSIONS: Optimal conditions for inducing VBNC V. parahaemolyticus were reported. The increase in resistance of VBNC V. parahaemolyticus to thermal, low salinity and acidic inactivation verified that this state is entered as part of a survival strategy in an adverse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods for inducing VBNC V. parahaemolyticus in a markedly short time will facilitate further physiological and pathological study. The enhanced stress resistance of the VBNC cells should attract attention to the increased risk presented by this pathogen in food.     

Comparison of fatty acid compositions of azooxanthellate Dendronephthya and zooxanthellate soft coral species

Ten zooxanthellae-free Dendronephthya species , twelve zooxanthellate soft coral species of the genera Sarcophyton, Lobophytum, Cladiella, Lytophyton, Cespitularia, and Clavularia, and the hermatypic coral Caulastrea tumida were examined for the first time to elucidate the fatty acid (FA) composition of total lipids. In Dendronephthya species, the main FAs were 20:4n-6, 24:5n-6, 16:0, 18:0, 7-Me-16:1n-10, and 24:6n-3 which amounted on the average to 26.0, 12.7, 12.1, 6.0, 4.8, and 4.0% of the total FA contents, respectively. For zooxanthellate soft corals, the main FAs were 16:0 (25.7%), 20:4n-6 (18.2%), 24:5n-6 (6.2%), and 18:4n-3 (5.6%), as well as 16:2n-7, which amounted up to 11.8% in Sarcophyton aff. crassum. Corals with zooxanthellae had low contents of 24:6n-3. The significant difference (p<0.01) between azooxanthellate and zooxanthellate soft corals was indicated only for 12 of 46 FAs determined. The principal components analysis confirmed that 7-Me-16:1n-10, 17:0, 18:4n-3, 18:1n-7, 20:4n-6, 22:5n-6, 24:5n-6, and 24:6n-3 are useful for chemotaxonomy of Dendronephthya. The azooxanthellate soft corals studied were distinguished by the absence of significant depth-dependent and species-specific variations of FA composition, low content of 16:2n-7, an increased proportion of bacterial FAs, predominance of n-6 FAs connected with active preying, and a high ability for biosynthesis of tetracosapolyenoic FAs.     

Effect of temperature and salinity on Vibrio (Beneckea) vulnificus occurrence in a Gulf Coast environment.

Vibrio (Beneckea) vulnificus is a recently recognized halophilic organism that may cause serious human infections. Patients infected with V. vulnificus often have a history of exposure to the sea, suggesting that the organism may be a common inhabitant of marine environments. Twenty-one inshore sites around Galveston Island in the Gulf of Mexico were cultured for V. vulnificus over a 12-month period. The organism was recovered from all but one of the sites at some time during the study. It was frequently isolated during the summer and fall from environments of relatively low salinity (7 to 16%). V. vulnificus was rarely isolated from any of the sites during the winter months, when water temperatures dropped below 20 degrees C. In vitro growth characteristics of environmental isolates of V. vulnificus demonstrated salinity optima of 1.0 to 2.0% NaCl and a temperature optimum of 37 degrees C. These growth characteristics may account for the seasonal and geographical variations in occurrence of the organism. Overall, the results of these studies indicate that V. vulnificus is commonly found in Gulf Coast environments and that the occurrence of the organism is favored by warm temperatures and relatively low salinity.     

Temporal fatty acid dynamics of the octocoral Veretillum cynomorium

The objectives of the present work were to investigate the temporal variation in the fatty acid (FA) composition of the octocoral Veretillum cynomorium, examine the effects of reproduction and environmental factors on FA variation, and establish a chemotaxonomic identification for this species. Mean oocyte size-frequency distributions showed that the majority of the oocytes had an intermediate size (Group II) before spawning (April and June). The late-vitellogenic oocytes (Group III) became absent in August and October and, during this post-spawning period, oocytes were primarily of small size (Group I). Most of the major FA, 16:0, 18:0, 20:4n-6, 20:5n-3, and the tetracosapolyenoic fatty acid (TPA), 24:6n-3, varied significantly throughout the year (p < 0.01), with two peaks in August/October and February. The boost in early oogenesis, also associated with warmer temperatures, seemed to be responsible for the observed increase in FA content between June and August. The highest values of FA content were observed in February when intermediate oogenesis (Group II) was at its peak and there were considerable levels of available food in the environment. Also, the increase in food availability seemed to trigger the final stages of gametogenesis. The high quantity of 18:1n-7, odd-numbered and branched FAs, suggested the presence of a dynamic bacterial community in V. cynomorium, probably as an adaptive response to the lack of symbiotic microalgae. Although the presence of TPAs is the main feature distinguishing octocorals from other coral species, here we showed that there was no single FA clearly dominating the FA composition of V. cynomorium throughout the year. Instead, four main FAs share similar concentrations: 16:0, 20:4n-6, 20:5n-3 and 24:6n-3. The predominance of these four FAs combined with the higher amount of 24:6n-3 when compared to 24:5n-6 may serve as a chemotaxonomic feature to distinguish this octocoral species (or genus).     

Fatty acid composition of the parasitic mite Varroa destructor and its host the worker prepupae of Apis mellifera

The present study analyzes the fatty acid (FA) profile of lipids isolated from Varroa destructor Anderson & Trueman, a parasitic mite of the honey bee (Apis mellifera L.), uninfected and infected worker prepupae of the Carnolian subspecies Apis mellifera carnica Pollmann, and bee bread fed to the worker brood. Significant differences are observed in the FA profiles of lipids isolated from parasites, hosts and bee bread. Parasitism by V. destructor (henceforth, varroosis) induces visible changes in the lipid profile of worker prepupae. In infected prepupae, the percentage of total saturated FAs is lower and the percentage of unsaturated FAs is higher than in uninfected insects. These differences result from significant changes in the percentages of FAs that are most abundant in the evaluated groups (i.e. C16:0, C18:1 9c, C18:2n‐6 and C18:3n‐3 FAs). In mites and in uninfected and infected prepupae, the predominant FAs are oleic acid (41.07 ± 2.26%, 42.79 ± 1.21% and 45 ± 0.20%, respectively) and palmitic acid (22.62 ± 0.87%, 39.48 ± 0.43% and 36.84 ± 0.22%, respectively). Highly significant differences in FA composition are noted between bee bread and worker brood. The results suggest specific mechanisms of FA uptake, accumulation and metabolism in the food chain of this parasitic association, beginning from the food processed by nurse bees for larval feeding, through host organisms (worker brood) to V. destructor mites.     

Effect of age on the fatty acid content of Blumeria graminis conidia

Blumeria (=Erysiphe) graminis f.sp. tritici (Bgt), the causal agent of wheat powdery mildew, is responsible for an important disease leading to considerable yield reductions in wheat worldwide. Conidia of the obligate plant pathogen Bgt were analysed for their total fatty acid (FA) composition as a function of their ontogeny. A total of 17 FAs were detected (C(12)-C(24) saturated and unsaturated ones), including the presence of unusual long-chain monoenoic FAs. In young conidia, the major FAs were C(18:2) (23%), C(16:0) (16%), C(18:0) (15.2%) and C(18:1) (14.3%). In old conidia, the main FAs were C(24:1) (20.7%), C(22:0) (15%), C(22:1) (13.5%) and C(24:0) (9.7%). The amount of total FA was about 39 microg.mg of dry weight(-1) in young conidia and decreased clearly to 18 microg.mg of dry weight(-1) in older conidia. For the first time, we have demonstrated that the FA composition of conidia changes greatly with age. Medium-chain FAs (C(12)-C(18)) are predominant in very young conidia (75%), whereas long-chain FAs (C(22)-C(24)) are the major compounds in old conidia (74%). This study showed a significant elongation of FAs and a drastic decrease in the total FA amount during the ontogeny of conidia.