Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters.

Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters. Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars). Pseudomonads were the most frequently identified group of filterable bacteria detected. Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified. Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar. In addition, none of the isolates identified from nonselective media were coliforms. Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.     

Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45-micron-pore-size membrane filters.

Rural drinking water systems supplied by untreated groundwater were examined to determine whether coliform or heterotrophic plate count bacteria are capable of escaping entrapment on standard porosity (0.45-micron-pore-size) membrane filters. Filterable bacteria were present in 42% of the 24 groundwater sources examined by using nonselective media (R2A, full strength m-HPC, and 0.1x m-HPC agars). Pseudomonads were the most frequently identified group of filterable bacteria detected. Flavobacterium, Alcaligenes, Acinetobacter, and Achromobacter isolates were also identified. Total coliforms were not recovered from any of the 24 groundwater samples following filtration through 0.45-micron-pore-size membrane filters by using selective M-Endo LES agar or mT7 agar. In addition, none of the isolates identified from nonselective media were coliforms. Similarly, neither total coliforms nor specifically Escherichia coli were detected in these filtrates when Colilert P/A medium was used.     

Evolution of Pseudomonas aeruginosa cells towards a filterable stage in seawater

In sterile nutrient-free seawater, the number of Pseudomonas aeruginosa culturable cells decreased progressively over time and the bacteria developed cells capable of passing through a 0.45 micron pore membrane. This development was more rapid in non-autoclaved, stirred seawater and the recovery of filterable cells varied depending on the membrane type used. Minicells were observed under an electron microscope. They yielded normal cells in bacteriological media with analogous colonies and an unchanged antibiotic resistance profile. Some biochemical characters, such as gelatinase or urease activity, were however modified in the filterable cells.     

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.     

Membrane Filtration of the Reiter Treponeme

A membrane filtration technique with commercially available membrane filters (Millipore Corp.) was effective for the removal of Reiter treponemes from liquids such as fluorescent-antibody conjugates, to which the organisms are added for adsorption. Reiter treponemes from an 8-day culture were not microscopically detectable in filtrates through membranes with a pore diameter of 0.45 μm, but treponemes were demonstrated in the filtrate by cultural methods. No organisms of the 8-day culture passed through a membrane filter having a pore size of 0.22 μm, as determined by microscopy and culture. Culture data indicated that a filter with a pore size of 0.1 μm was necessary to prevent passage of treponemes from 4-day cultures. It is recommended that a membrane filter with a pore size of 0.22 μm or smaller be used for the removal of Reiter treponemes from suspensions and that the age of the culture be considered in choosing filter pore size.     

Applicability of the Reverse-Flow Filter Technique to Marine Microbial Studies

The use of the reverse-flow filtration technique to quantitatively concentrate marine bacteria was evaluated using both a pure culture and seawater samples. Our data indicate that the cells are altered during the filtration procedure and that a significant and inconsistent number of cells are lost on the membrane filter. The results obtained indicate that data on marine bacteria concentrated in this manner should be interpreted with caution.     

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.     

Passage of Treponema pallidum Through Membrane Filters of Various Pore Sizes

The passage of Treponema pallidum through commercially available Millipore membrane filters of various pore sizes was examined. No microscopically detectable organisms passed through a filter with a pore diameter of 0.22 mum. As pore size was increased, progressively more organisms passed through. Motile organisms passed through filters to a greater extent than nonmotile ones; however, 22% of the motile and 50% of the nonmotile T. pallidum organisms did not pass through the largest pore diameter tested (14.0 mum). Filtration of T. pallidum suspensions through membrane filters may offer a way of separating the organisms from larger particles of debris which accompany their extraction from rabbit testicular syphilomas.     

Composition and variability of biofouling organisms in seawater reverse osmosis desalination plants

Seawater reverse osmosis (SWRO) membrane biofouling remains a common challenge in the desalination industry, but the marine bacterial community that causes membrane fouling is poorly understood. Microbial communities at different stages of treatment processes (intake, cartridge filtration, and SWRO) of a desalination pilot plant were examined by both culture-based and culture-independent approaches. Bacterial isolates were identified to match the genera Shewanella, Alteromonas, Vibrio, and Cellulophaga based on 16S rRNA gene sequencing analysis. The 16S rRNA gene clone library of the SWRO membrane biofilm showed that a filamentous bacterium, Leucothrix mucor, which belongs to the gammaproteobacteria, accounted for nearly 30% of the clone library, while the rest of the microorganisms (61.2% of the total clones) were related to the alphaproteobacteria. 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that bacteria colonizing the SWRO membrane represented a subportion of microbes in the source seawater; however, they were quite different from those colonizing the cartridge filter. The examination of five SWRO membranes from desalination plants located in different parts of the world showed that although the bacterial communities from the membranes were not identical to each other, some dominant bacteria were commonly observed. In contrast, bacterial communities in source seawater were significantly different based on location and season. Microbial profiles from 14 cartridge filters collected from different plants also revealed spatial trends.     

Clustering of Marine Bacteria in Seawater Enrichments

Seawater enrichments of marine bacteria clustered in 20- to 50-(mu)m-wide bands near air-water interfaces. The cells within the band travelled at up to 212 (mu)m s(sup-1) and at an average speed of 163 (mu)m s(sup-1). Mean cell speeds peaked mid-run at 187 (mu)m s(sup-1). At the end of the run, bacteria reversed direction rather than randomly reorienting. The duration of the stops during reversal was estimated at 18 ms, six to seven times shorter than that found in enteric bacteria. Cells hundreds of micrometers from the band travelled at half the speed of the bacteria in the band. The fastest isolate from the seawater enrichment was identified as Shewanella putrefaciens and had an average speed of 100 (mu)m s(sup-1) in culture. Air-water interfaces produced no clustering or speed changes in isolates derived from enrichments. Salinity and pH, however, both influenced speed. The speed and reversal times of the seawater enrichments indicate that the bacteria in them are better adapted for clustering around small point sources of nutrients than are either enteric or cultured marine bacteria.     

Proteobacteria and Bacteroidetes are major phyla of filterable bacteria passing through 0.22 μm pore size membrane filter,in Lake Sanaru,Hamamatsu, Japan

141 filterable bacteria that passed through a 0.22 μm pore size filter were isolated from Lake Sanaru in Hamamatsu, Japan. These belonged to Proteobacteria, Bacteroidetes, Firmicutes, or Actinobacteria among which the first two phyla comprised the majority of the isolates. 48 isolates (12 taxa) are candidates assignable to new bacterial species or genera of Proteobacteria or Bacteroidetes.     

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.     

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

For indirect DNA extraction for metagenomics studies, bacterial cells can be effectively separated from sample debris by using a simple size exclusion technique, such as filtration, and thereafter lysed. The requirement for the optimal recovery of cells in filtrates is critical to achieve sufficient DNA yield and a representative population. Particles smaller than the filter pore size are expected to be found in the filtrate, whereas particles larger than the filter pore sizes are excluded. However, this is not always the case. It is established that the membrane pore size influences filtration efficiency to some degree. In addition the physicochemical characteristics of the filter suspension and characteristics of the microbial cells being filtered influence the exclusion property of a membrane. This review provides an overview of membrane filtration techniques and the factors that affect filterability of bacteria cells through a filter membrane. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:853–866, 2015     

Purification of Chlamydia trachomatis by a simple and rapid filtration method

A simple method for filter purification of Chlamydia trachomatis from cell culture is described. Crude homogenates of chlamydiae-infected cells were passed through a glass prefilter and a 0.6 microns pore diameter polycarbonate filter. The filtrate was then passed through a 0.2 microns pore diameter filter on which the chlamydiae were trapped. This filter was then back-washed to collect the organisms. These procedures removed cell debris and soluble protein, and yielded particles with a narrow size distribution. The mean yield of viable chlamydiae purified by filtration was 64% when the filters were washed at each stage of the process.     

Radiotracer Experiments on Biological Volatilization of Organic Iodine from Coastal Seawaters

Biological volatilization of iodine from seawaters was studied using a radiotracer technique. Seawater samples were incubated aerobically in serum bottles with radioactive iodide tracer (¹²⁵I), and volatile organic and inorganic iodine were collected with activated charcoal and silver wool trap, respectively. Iodine was volatilized mainly as organic iodine, and inorganic iodine volatilization was not observed. Influence of light intensity on the volatilization was determined, but no significant differences were observed under light (70,000 lux) and dark conditions. The effect of the chemical form of iodine on the volatilization was determined, and the results suggested that volatilization preferentially occurs from iodide (I^?) but not from iodate (IO₃ ^?). Volatilization did not occur when the samples were autoclaved or filtered through a 0.22-μm pore size membrane filter. Incubation of the samples with antibiotics caused decreased volatilization. Conversely, enhanced volatilization was observed when the samples were incubated with yeast extract. Fifty-nine marine bacterial strains were then randomly isolated from marine environments, and their iodine-volatilizing capacities were determined. Among these, 19 strains exhibited significant capacities for volatilizing iodine. 16S ribosomal RNA gene comparisons indicated that these bacteria are members of Proteobacteria (α and γ subdivisions) and Cytophaga-Flexibacter-Bacteroides group. One of the strains, strain C-19, volatilized 1 to 2% of total iodine during cultivation, and the gaseous organic iodine was identified as methyl iodide (CH₃I). These results suggest that organic iodine volatilization from seawaters occurs biologically, and that marine bacteria participate in the process. Considering that volatile organic iodine emitted from the oceans causes atmospheric ozone destruction, biological iodine volatilization from seawater is of great importance. Our results also contribute to prediction of movement and diffusion of long-lived radioactive iodine (¹²⁹I) in the environment.     

USE OF POROUS CELLULOSE ESTER MEMBRANES IN THE PRIMARY ISOLATION AND SIZE DETERMINATION OF PLEUROPNEUMONIA-LIKE ORGANISMS

Morowitz, Harold J. (Yale University, New Haven, Conn.), Mark E. Tourtellotte, and Mary E. Pollack. Use of porous cellulose ester membranes in the primary isolation and size determination of pleuropneumonia-like organisms. J. Bacteriol. 85:134-136. 1963.-Millipore filters were used in the primary isolation and sizing of a number of strains of pleuropneumonia-like organisms (PPLO). Maximal pore diameters were checked by filtration of viruses of known sizes. All the PPLO strains studied produced cells capable of passing through a 0.22-mu filter. A number of strains produced forms which could be titered after filtration through a 0.1-mu filter. A number of primary isolations were made from filtrates.     

Rapid separation and quantitation of mixed microorganisms by filtration and bioluminescence

A membrane filtration/bioluminescence system was developed for the differentiation and quantitation of mixed populations of microorganisms. Samples containing microorganisms were filtered through two membrane filters of descending pore size. The microorganisms retained on the filter contain ATP that can be extracted and measured on the filter via the firefly luciferase-luciferin bioluminescence assay. Results, obtained in less than 20 min, show a good correlation (r greater than or equal to 0.95) between the light produced and the number of organisms in the sample. Using these techniques, Escherichia coli can be separated from yeast or mold and measured in samples containing both microorganisms. When lysostaphin is used to selectively lyse Staphylococci on the filter, the specific quantification of these bacteria among other microorganisms can also be accomplished. The filtration/bioluminescence technique offers the potential of being a rapid and sensitive method to differentiate and detect microorganisms, by selective sizing or lysing, in a variety of samples.     

Factors Influencing the Activity of Sterile Filtered and Heat-Sterilized Trypsin Solutions

The mode of sterilization (filtration or heat) was found to significantly affect the activity of trypsin solutions. Trypsin activity was substantially reduced in the initial fractions of filtrate passed through asbestos filter pads; heat-sterilized trypsin was satisfactory for transfer of cell cultures grown on glass. Heat-sterilized trypsin may be useful when elimination of filterable organisms is required.     

抗菌和细胞毒活性海洋细菌的筛选及其次生代谢基因证据

从不同海域的海水、海泥和海洋生物中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选,并对具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析和多聚酮合酶(PKSⅠ型)、非核糖体肽合成酶(NRPS)的筛选。结果显示,在分离到的346株海洋细菌中,42株细菌具有抗菌活性,12株具有细胞毒活性。对12株具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析,它们分别属于Agrobacterium,Pseudoalteromons,Bacillus,Paracoccus,Rheinheimera,Aerococcus,Exiguobacterium和Alteromonas8个属。对这12株具有细胞毒活性的细菌菌株进行进一步的PKS和NRPS筛选,得到了4株含有PKSⅠ型的KS结构域或NPRS的A结构域的海洋细菌,为从聚酮和非核糖体肽等生物合成途径去深入研究其次生代谢产物提供了基因学的证据。     

Optimum Membrane Structures for Growth of Coliform and Fecal Coliform Organisms

The purpose of this study was to determine the optimum membrane filter structure and characteristics for recovery of coliform organisms. Additionally, other factors such as sterilization method and membrane composition were examined. Fecal coliform growth tests with varied samples indicated that the most critical factor in recovery was surface pore morphology and not other factors previously suspected. Fecal coliform counts showed a dramatic increase, with increasing surface opening sizes. Membrane structures with surface openings large enough to surround the entrapped bacteria are required for optimum growth of fecal coliform organisms. Maximum fecal coliform recoveries are obtained using membranes composed of mixed esters of cellulose exhibiting a surface opening diameter of 2.4 μm and a retention pore size of 0.7 μm.