Isolation and characterization of filterable marine bacteria

Anderson, J. I. W. (Northeast Shellfish Sanitation Research Center, Narragansett, R.I.), and W. P. Heffernan. Isolation and characterization of filterable marine bacteria. J. Bacteriol 90:1713-1718. 1965.-By a process of double filtration of seawater, first through a membrane filter with a pore diameter of 0.45 mu and then through a membrane filter with a pore diameter of 0.22 mu, it was possible to isolate on the surface of the latter membrane a group of marine organisms not usually encountered by conventional techniques of pour plates or one-stage filtration. Many of the isolates could not be identified, but the largest single group belonged to the genus Spirillum; other isolates were placed in the genera Leucothrix, Flavobacterium, Cytophaga, and Vibrio. A group of four organisms which was not identified was characterized by the formation of large, club-shaped cells, 20 to 30 mu long. Of the 25 strains studied in detail, 22 required seawater for growth and 8 retained their filterable property after cultivation. No filterable bacteria were isolated from terrestrial samples.     

Enzymatic,outer membrane proteins and plasmid alterations of starved <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> cells in seawater

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 μm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.     

Investigation of 0.2 µm filterable bacteria from the Western Mediterranean Sea using a molecular approach: dominance of potential starvation forms

Although the existence of 0.2 μm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 μm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 μm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 μm and the 0.2 μm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 μm filterable and the total bacterial populations within the samples. Additionally the 0.2 μm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 μm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 μm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 μm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.     

Detection and characterization of filterable heterotrophic bacteria from rural groundwater supplies

AIMS: The chemical/physical environment of groundwater may contribute to the existence of a subpopulation of small-sized bacteria (filterable bacteria) that fails to be trapped on conventional 0.45 microm-pore-size membrane filters during routine bacteriological water quality analyses. Efforts were directed to determining an efficient recovery method for detection of such cells. METHODS AND RESULTS: Individual groundwater supplies in a rural setting were examined by a double membrane filtration procedure to determine the presence of heterotrophic plate count (HPC) bacteria capable of escaping detection on conventional pore size (0.45 microm) membrane filters but retained on 0.22 microm-pore-size filters. Since optimum cultural conditions for recovery of filterable bacteria are not well defined, initial efforts focused on evaluation of various media (R2A, m-HPC and NWRI) and incubation temperatures (15, 20, 28 and 35 degrees C) for specific recovery of filterable bacteria. Maximum recovery of small-sized HPC bacteria occurred on low-nutrient concentration R2A agar incubated for 7 d at 28 degrees C. Similarly, identical cultural conditions gave enhanced detection of the general HPC population on 0.45 microm-pore-size filters. A 17-month survey of 10 well water supplies conducted with the cultural conditions described above resulted in detection of filterable bacteria (ranging in density from 9 to 175 cfu ml-1) in six of the groundwater sources. The proportion of filterable bacteria in any single sample never exceeded 10% of the total HPC population. A majority of the colonies appearing on the 0.22 microm membrane filters was pigmented (50-90%), whereas the proportion of colonies demonstrating pigmentation on the larger porosity filters failed to exceed 50% for any of the samples (19-49%). CONCLUSION: A reliable recovery method was developed for the detection of filterable bacteria from groundwater. During a subsequent survey study using this procedure, filterable bacteria were detected in a majority of the groundwater supplies examined; however, the density of filterable bacteria in any single sample never exceeded 10% of the total HPC population. Identification of randomly selected isolates obtained on the 0.22 microm filters indicated that some of these filterable bacteria have been implicated as opportunistic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: We have determined the presence of small-sized HPC bacteria in ground water that may go undetected when using standard porosity membrane filters for water quality analyses. Further study is needed to assess the significance and possible health risk associated with presence of filterable bacteria in drinking water supplies from groundwater sources.     

Survival of the fish pathogen Aeromonas salmonicida in seawater

Survival of Aeromonas salmonicida in natural (non-sterile) seawater, as determined from colony counts on marine agar, was found to be influenced by the presence of potentially inhibitory organisms, i.e., Acinetobacter, Aeromonas hydrophila, Chromobacterium, Escherichia coli, Flavobacterium and Pseudomonas, and their metabolites. Yet, samples, thought to be devoid of culturable A. salmonicida, were found to contain cells, which were filterable through 0.22 and 0.45 microns Millipore Millex porosity filters, and were recoverable on a specialised medium for L-forms, i.e. L-F medium.     

Ultrastructural alterations in branchial chloride cells of Atlantic salmon, Salmo salar, during parr-smolt transformation and early development in sea water

The ultrastructure of the gill primary lamellae of juvenile Atlantic salmon was examined during the parr-smolt transformation and for 42 days after smolts were exposed to sea water. Scanning electron microscopy indicated that primary lamellae were characterized by rough convoluted surfaces that became rougher throughout the experimental period and that crypts did begin to form in freshwater fish. Crypt formation increased in sea water.
Transmission electron microscopy indicated that parr preadapt for life in sea water in part by changes in chloride cells. Chloride cells show an elaboration of rough endoplasmic reticulum in fresh water and a decline of rough endoplasmic reticulum after 42 days of sea water exposure. The tubular membrane system becomes well developed in fresh water, and apical vesicles become abundant only after seawater exposure. Mitochondria are both spherical and elongate through the period and contain well developed cristae. No evidence of mitochondrial rupture was observed. The junctions between chloride cells and adjacent cells were characterized in fresh water by long tight junctions with desmosomes. This type of junction continued in sea water and was the norm between chloride cells and accessory cells after 42 days of seawater exposure. While leaky junctions appeared to be forming, no evidence was found of membrane interdigitation between accessory cells and chloride cells after 42 days of seawater exposure. It also appeared that seawater exposure influenced the number of chloride cells exposed to the external milieu.
Pavement cells showed an elaboration in fresh water of free ribosomes and rough endoplasmic reticulum and these elements became less prominent after seawater exposure.     

Growth of Vibrio cholerae O1 in red tide waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6- micro m-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day(-1) (4.2 day(-1) +/- 3.9) for V. cholerae and 0.1 to 9.7 day(-1) (2.2 +/- 2.8 day(-1)) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10(4) cell ml(-1)) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.     

Morpholino gene knockdown in adult Fundulus heteroclitus: role of SGK1 in seawater acclimation

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism.     

Development of Intracellular Membrane Systems in the Columnar Epithelium of the Urinary Bladder of the Euryhaline Goby Gillichthys mirabilis

The membrane systems in "columnar cells" of the goby urinary bladder were studied after staining with ferrocyanide-reduced osmium tetroxide (K arnovsky ). In addition to the endoplasmic reticulum, two distinct systems of membranes were observed: 1) a vesiculotubular system made up of vesicles and short tubules located between the Golgi area and the apical membrane and 2) well-developed infoldings of the laterobasal plasma membrane which form either complete or fenestrated sheets. Adaptation to 5% seawater or prolactin exposure of seawater fish induces a proliferation of these membrane systems and, in particular, of the complete infoldings of the laterobasal plasma membrane. These observations suggest high activity of these bladder cells in osmoregulatory adjustments to hypotonic environments. The divergence between cytological and physiological indicators of activity is considered.     

Viability and virulençe of Escherichia coli suspended by membrane chamber in semitropical ocean water

Abstract Escherichia coli H10407 was suspended in seawater (38.5‰ salinity) contained in membrane chambers (0.4-μm polycarbonate membrane) incubated in situ at 25°C in Nixon's Harbor, South Bimini, Bahamas. Although colonies of E. coli could not be cultured after 13 h post chamber inoculation, the number of fluorescent-antibody staining cells remained constant. Direct viable counts revealed that viable cells were present, even though the cell suspension was not culturable on the media tested. After exposure to seawater for 112 h, cells were concentrated by centrifugation and introduced into ligated rabbit ileal loops. E. coli H10407 proved viable for recovery from inoculated loops and was confirmed by detection of characteristic plasmid bands. Results indicate that enteric pathogens remain viable in seawater long after they cease to be cultivable on laboratory media.     

Effect of antibiotic action on the submicroscopic structure of Salmonella. The action of monomycin on S. typhi]

The effect of subinhibitory concentrations of monomycin on the submicroscopic structures of 2 strains of S. typhi, 5 and 799 was studied. It was shown that formation of filamentoue forms, separation of the cell wall, thinning out of the cytoplasm granular component, increasing of the size of the matter with low electron optic density of fine granular structure were common in the cell response of both strains. Formation of vacuoli containing the thinned out granular component, filterable elements and complex membrane structures followed by their liberation into the medium and formation of the forms devoid of the cell walls was a characteristic peculiar property of the cell response in strain 799. The cells of strain 5 were characterized by formation of large granular osmiefilic matters and their excretion from the cells.     

Seawater-resistant, non-spherical protoplasts from seagrass leaves

Two distinct types occurred among enzymatically isolated protoplasts from leaves of eelgrasses ( Zostera marina L., Z. japonica Ascherson and Phyllospadix iwatensis Makino). Spherical protoplasts with a smooth cell membrane were obtained only from young leaf tissues at the basal portions of blades protected from seawater by tightly enclosing sheaths. Non-spherical protoplasts had a highly invaginated cell membrane and were obtained from mature leaf blades, where the cells also in situ have this type of membrane. The protoplasts from mature leaves were rather rigid in shape and resistant to wide ranges of osmotic potential and salinity without change in their non-spherical shape, while the spherical protoplasts were rapidly destroyed in seawater. Detergents lysed the spherical protoplasts but not the non-spherical ones, suggesting that the highly invaginated enclosing structures of the non-spherical protoplasts contained detergent-resistant materials. Thus, the seagrass leaf cells develop seawater resistance, and this change alters the nature of the enclosing structures during the growth of the leaf blades. The non-membranous enclosing structures and their characteristic materials in the mature leaf cells remain to be defined.     

Isolation of ultrasmall (filterable) bacteria from patients suffering from ME,and patients and staff of a paediatric hospital

A total of 108 blood samples obtained from 28 male and 80 female patients diagnosed with ME were diluted in sterile, Ringer’s Solution and forced (by suction) through 0.2 µm filters. Of the 28 male samples, 4 yielded filterable bacteria and of the 80 female samples, 18 gave filterable bacteria; as a result, of the total of 124 samples, 22 yielded FB. Filterable (0.4 and 0.2, but not 0.1micron filterable) bacteria were also isolated from the nose throat and skin of paediatric patients and from the throat and skin of staff at an emergency paediatric hospital. The highest percentage of bacterial passage occurred through the largest (0.4 µm) pores. The results show that ultrasmall bacteria occur in ME patients and in paediatric patients and nurses. The potential pathogenic role of such filterable bacteria is briefly discussed.     

Symbiotic Role of the Viable but Nonculturable State of Vibrio fischeri in Hawaiian Coastal Seawater

To achieve functional bioluminescence, the developing light organ of newly hatched juveniles of the Hawaiian squid Euprymna scolopes must become colonized by luminous, symbiosis-competent Vibrio fischeri present in the ambient seawater. This benign infection occurs rapidly in animals placed in seawater from the host's natural habitat. Therefore, it was surprising that colony hybridization studies with a V. fischeri-specific luxA gene probe indicated the presence of only about 2 CFU of V. fischeri per ml of this infective seawater. To examine this paradox, we estimated the total concentration of V. fischeri cells present in seawater from the host's habitat in two additional ways. In the first approach, the total bacterial assemblage in samples of seawater was collected on polycarbonate membrane filters and used as a source of both a crude cell lysate and purified DNA. These preparations were then assayed by quantitative DNA-DNA hybridization with the luxA gene probe. The results suggested the presence of between 200 and 400 cells of V. fischeri per ml of natural seawater, a concentration more than 100 times that revealed by colony hybridization. In the second approach, we amplified V. fischeri-specific luxA sequences from microliter volumes of natural seawater by PCR. Most-probable-number analyses of the frequency of positive PCR results from cell lysates in these small volumes gave an estimate of the concentration of V. fischeri luxA gene targets of between 130 and 1,680 copies per ml. From these measurements, we conclude that in their natural seawater environment, the majority of V. fischeri cells become nonculturable while remaining viable and symbiotically infective. Experimental studies indicated that V. fischeri cells suspended in natural Hawaiian seawater enter such a state within a few days.     

Development of a nondestructive fluorescence-based enzymatic staining of microcolonies for enumerating bacterial contamination in filterable products

Aims: Develop a nondestructive fluorescence‐based staining procedure to rapidly detect and enumerate bacteria in filterable samples. Methods and Results: The study consists in the development of a staining solution and a protocol to fluorescently detect microcolonies on cellulose membranes. After detection, membranes can be re‐incubated on media to yield colonies. Carboxyfluorescein diacetate was selected among other carboxyfluorescein derivatives for its staining efficiency and the absence of background. Several permeabilizers were evaluated for their ability to promote dye uptake into cells without affecting viability. We demonstrated that a combination of n‐Octyl β‐d ‐glucopyranoside, sodium hexametaphosphate, lithium chloride and rubidium chloride significantly increased the staining efficiency of bacteria without affecting their viability. The method developed allowed the detection in <9 h of all tested aerobic bacteria and in 48 h of the anaerobic slow grower Propionibacterium acnes. Conclusions: This method allows the rapid detection of bacteria in filterable samples in at least three to five times faster than traditional microbiological method. Significance and Impact of the Study: The advantage of this nondestructive procedure is to allow contaminants identification after membrane re‐incubation. This method could be easily applied in routine in pharmaceutical, clinical and food and beverage industries to monitor contaminations.     

Decimal reduction times of Pyrodinium bahamense var. compressum and Escherichia coli in chlorine- and ultraviolet-treated seawater

AIMS: Decimal reduction times (D-values) of the vegetative cells of Pyrodinium bahamense var. compressum and Escherichia coli in ultraviolet- and chlorine-treated seawater were established. METHODS AND RESULTS: The cells of the test organisms were exposed to ultraviolet- and chlorine-treated seawater and maintained at 20-35 ppt salinity and 20 to 35 degrees C. The dinoflagellate cells which cause Paralytic Shellfish Poisoning (PSP) were found to be more resilient than the bacterial cells. Ultraviolet treatment was found to be more effective than chlorine to both test organisms. Irreversible morphological changes in the treated dinoflagellate cells were noted, including protoplast discoloration, cellular membrane leakage and damage to the thecal armour. CONCLUSIONS: The vegetative cells of both test organisms in seawater were more sensitive to ultraviolet treatment than to chlorine exposure. Generally, the dinoflagellate cells were less susceptible than bacterial cells to both disinfection treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study may have significant implications in depuration procedures for molluscs and cleaning protocols for ballast waters of ships.     

Prokaryotic ultramicroforms in a <Emphasis Type="Italic">Sphagnum</Emphasis> peat bog of upper Volga catchment

The waters of small lakes located in swampy catchment areas of upper Volga contain considerable amounts of ultrasmall microbial cells that pass through 0.22-μm-pore-size filters. As shown in our previous study [1], most of these cells represent the bacterial genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia of the class Betaproteobacteria, as well as euryarchaea of the uncharacterized clade LDS. The aim of the present study was to investigate the possible effect of the waters draining swampy areas on the composition of the filterable microbial fraction in lakes fed by swampy catchments. To address this question molecular identification was performed of prokaryotic ultramicroforms in the peat of the ombrotrophic Sphagnum bog Obukhovskoe, located, like the lakes studied previously [1], in the Mologa-Sheksna catchment area. The number of filterable microorganisms in 1 g wet peat was 3.8 × 10⁶ cells, or 0.5% of total microbial cell number in the peat. From the DNA of the filterable cell fraction, 100 clones of bacterial and 77 clones of archaeal 16S rRNA genes were obtained. The bacterial clone library contained 16S rRNA gene sequences representing the classes Beta- and Gammaproteobacteria (the genera Janthinobacterium and Pseudomonas, respectively) and the phylum Bacteroidetes (the genera Chryseobacterium and Epilithonimonas) and differed significantly from the clone library of bacterial ultramicroforms of lake water. By contrast, the pools of filterable archaea in bogs and lakes were essentially similar. They were represented by the euryarchaeal clade LDS and methanogens of the orders Methanobacteriales and Methanosarcinales. Additionally, the pool of filterable archaea of the bog included methanogens of the order Methanomicrobiales and representatives of the uncharacterized euryarchaeal clade RC-V (Rice Cluster V) and of the phylum Thaumarchaeota.     

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.     

Growth of Vibrio cholerae O1 in Red Tide Waters off California

Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters. However, its population dynamics in such environments are not well understood. We tested the proliferation of V. cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions. Microcosms containing 100-kDa-filtered seawater were inoculated with V. cholerae or the 0.6-μm-pore-size filterable fraction of seawater assemblages. These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles. Growth rates ranged from 0.3 to 14.3 day⁻¹ (4.2 day⁻¹ ± 3.9) for V. cholerae and 0.1 to 9.7 day⁻¹ (2.2 ± 2.8 day⁻¹) for bacterial assemblage. Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V. cholerae in seawater. Under the conditions tested, free-living V. cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10⁴ cell ml⁻¹) and remained viable under many conditions. If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V. cholerae in disease propagation and prevention during phytoplankton blooms.     

In Vitro Bleaching of Hardwood Kraft Pulp by Extracellular Enzymes Excreted from White Rot Fungi in a Cultivation System Using a Membrane Filter

To clarify the role of excreted extracellular enzymes during long-term incubation in a pulp biobleaching system with white rot fungi, we developed a cultivation system in which a membrane filter is used; this membrane filter can prevent direct contact between hyphae and kraft pulp, but allows extracellular enzymes to attack the kraft pulp. Phanerochaete sordida YK-624 brightened the pulp 21.4 points to 54.0% brightness after a 5-day in vitro treatment; this value was significantly higher than the values obtained with Phanerochaete chrysosporium and Coriolus versicolor after a 7-day treatment. Our results indicate that cell-free, membrane-filtered components from the in vitro bleaching system are capable of delignifying unbleached kraft pulp. Obvious candidates for filterable reagents capable of delignifying and bleaching kraft pulp are peroxidase and phenoloxidase proteins. The level of secreted manganese peroxidase activity in the filterable components was substantial during strain YK-624 in vitro bleaching. A positive correlation between the level of manganese peroxidase and brightening of the pulp was observed.