Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.     

Isolation of Pectolytic clostridia from Potatoes

S ummary : A method for selective counting and isolation of pectolytic clostridia in the presence of Erwinia carotovora is described; using this method pectolytic clostridia have been found in numbers of 8 × 10⁵–1 × 10⁸/g of rotting potato tissue in the presence of 1–4 × 10⁸ E. carotovora /g.     

Attempts to control cabbage root flies Delia radicum L. and Delia floralis (Fall.) (Dipt., Anthomyiidae) with entomopathogenic fungi: laboratory and greenhouse tests

One laboratory and three greenhouse experiments were conducted to study the pathogenicity and efficacy of Finnish isolates of entomopathogenic hyphemycetous fungi against cabbage root flies. In Petri dishes, exposure to 1.5 × 10¹⁰ spores of Metarhizium anisopliae per dish caused 40–50% mortality of undifferentiated second- and third-stage larvae of Delia floralis , and 1 × 10⁷ spores per dish caused 40–50% mortality of Delia radicum larvae. In one greenhouse test, 1 × 10⁸ and 1 × 10⁹ spores of M. anisopliae and Paecilomyces fumosoroseus reduced the root damage of head cabbage by 20–70% compared with untreated controls, although this was not accompanied by significant reductions in the number of pupae. Only M. anisopliae consistently grew out of larvae and pupae of D. floralis during incubation that followed their recovery from the soil at the end of an experiment testing different formulations of M. anisopliae and Beauveria bassiana , but the frequency of the latent infections of the pest by M. anisopliae was not associated with reduced severity of damage to seedlings of head cabbage.     

Evaluation of a commercial fluorochromic system for the rapid detection and estimation of wine lactic acid bacteria by DEFT

A commercial fluorochromic system was evaluated for the rapid detection of lactic acid bacteria in fortified wines by the epifluorescent filter technique (DEFT). The viability test used, employing the fluorescence dyes SYTO 9 and propidium iodide, was able to detect and clearly differentiate viable from non-viable cells (killed with a 50% v/v ethanol solution). A good overall agreement ( r ₌ 0·92) was obtained between the DEFT count and the plate count in the range studied (5 × 10²–4 × 10⁹ cells ml⁻¹). Wine components which might otherwise interfere with the method could be removed by including simple wash steps in the protocol. This measure proved critical to the success of the procedure. For practical purposes, the rapid method studied seems to be a good alternative to the traditional cultural methods as part of quality control programmes in wine making. It may also be useful when studying the efficacy of certain treatments in the elimination of wine bacterial contaminants.     

DIRECT DETECTION OF ESCHERICHIA COLI 0157:H7 IN UNPASTEURIZED APPLE JUICE WITH AN EVANESCENT WAVE BIOSENSOR

An evanescent wave biosensor was used to detect Escherichia coli O157:H7 in unpasteurized apple juice. Light is launched from a 635 nm laser diode into silica or polystyrene optical waveguides, generating an evanescent field which extends from the waveguide surface. Fluorescent molecules within the evanescent field are excited resulting in an emission signal that the biosensor then detects and quantifies. A sandwich immunoassay was performed on the waveguides using cyanine 5 dye-labeled anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. The lower limit of detection was between 6.0 × 10² and 6.0 × 10⁴ CFU/mL with silica waveguides and between 3.2 × 10⁴ and 3.2 × 10⁴ CFU/mL using polystyrene waveguides. One-hundred percent correct identification of true positive samples occurred at 6.0 × 10⁴ and 3.2 × 10⁴ CFU/mL for silica and polystyrene waveguides, respectively. Signals from a variety of non-E. coli O157 bacteria, including closely related enterotoxigenic strains of E. coli at concentrations of ˜ 10⁶ CFU/mL, were below the limits of detection. Assays were conducted in near real-time with results obtained within 15 min of sample processing.     

Monitoring the fate of genetically engineered bacteria sprayed on the phylloplane of bush beans and grass

Abstract The fate of a Bacillus amyloliquefaciens with the recombinant plasmid pSB20 sprayed on the phyllosphere of grass, and of a Tn 5 marked Pseudomonas syringae sprayed on the phyllosphere of bush beans was studied in planted soil microcosms. B. amyloliquefaciens showed a decline from 1.5×10⁸ to 3.1×10² cfu g⁻¹ on the phylloplane of grass in the course of the experiment. B. amyloliquefaciens was easy to follow by selective cultivation due to the complete absence of bacterial background growth. Southern blot hybridization of Hin dIII digested genomic DNA showed plasmid restriction patterns identical with pSB20 indicating high plasmid stability. In total DNA extracts from phyllosphere bacteria the recombinant plasmid was detectable by Southern blot hybridization up to 6×10⁴ cfu g⁻¹ (wet weight). Counts of hybridizing colonies showed that P. syringae established on the phyllosphere of bush beans at between 5×10³ and 4×10⁶ cfu g⁻¹ fresh weight. During senescence of the bean plants the strain was no longer detectable by selective cultivation and subsequent colony hybridization. In contrast, Tn5 marked DNA was detected after PCR amplification over the whole period of the experiment.     

Association of bacteria with the fungal fermentation of soybean tempe

Bacteria grew to viable populations of 10⁸–10⁹ cfu/g during the fermentation of soybeans into tempe with the fungus, Rhizopus oligosporus. Bacillus pumilus and B. brevis were the predominant bacterial species, reaching populations of approximately 10⁸ cfu/g during the 48 h fermentation. Species of Streptococcus faecium, Lactobacillus casei, Klebsiella pneumoniae and Enterobacter cloacae also contributed to the fermentation and achieved populations of 10⁶–10⁷ cfu/g. and accepted 25 May 1989     

Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

Aflatoxin content and number of fungi in poultry feedstuffs from Indonesia

The content of aflatoxin and associated fungi was determined in 56 samples, including 34 of corn, 10 of soybean meal, nine of rice bran and three of broken rice, collected from different poultry farms and poultry feedmills situated around Jakarta-Bogor, Indonesia.
Ninety-one per cent of the corn samples contained aflatoxins and the total concentration ranged from 22 to 6171 μ g/kg. With rice bran, 100% of the samples were positive for aflatoxin B₁, ranging from 36 to 71 μ g/kg. No aflatoxin was detected in samples of soybean meal or broken rice. All the samples were contaminated by several fungi (8 times 10³–5 times 10⁶cfu/g) and further identification was limited to Aspergillus flavus and A. parasiticus. The dominant species was A. flavus (2 times 10³–4 times 10⁶cfu/g in corn samples, 1·0 times 10³–1·0 times 10⁵cfu/g in soybean meal, 2 times 10⁴–4·4 times 10⁵cfu/g in rice bran and 2 times 10⁴–6 times 10⁴cfu/g in broken rice). Some of the corn samples also contained A. parasiticus (2 times 10³–9·5 times 10⁴cfu/g).     

Regulation of Fusarium oxysporum population introduced into soil: the amoebal predation hypothesis

Abstract Inoculation of fungi into soil has been suggested for biological control of plant diseases. The aim of our work was to test the ability of protozoa to reduce the density of introduced fungal populations. The survival of Fusarium oxysporum in non-sterile soil was studied after introduction at densities of: 1 × 10⁴, 1 × 10⁶ and 5 × 10⁷ cfu/g soil. The dynamics of protozoa were also followed. The fungal populations remained close to the initial inoculation densities and did not induce the growth of indigenous protozoa. A bacterial population ( Enterobacter aerogenes ) was used to promote and stimulate the predatory activity of amoebae. Then, after simultaneous inoculation with bacteria and fungi, the density of protozoa increased but this had no effect on the fungal population, although some amoebae are able to feed on small fungal propagules such as conidia. The physiological state of Fusarium in soil and intraspecific competition seem to be more important in regulating introduced fungal populations than amoebal predation. We conclude that the regulation of bacterial and fungal populations in soil depend on different mechanisms.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.