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.     

Persistence of Escherichia coli O157:H7 on the rhizosphere and phyllosphere of lettuce

Aims:  The major objective of this study was to determine the effects of low levels of Escherichia coli O157:H7 contamination on plant by monitoring the survival of the pathogen on the rhizosphere and leaf surfaces of lettuce during the growth process.
Methods and Results:  Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the rhizosphere and leaf surfaces after planting. Real-time PCR assays were designed to amplify the stx 1, stx 2 and the eae genes of E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real-time PCR was 2·4 × 10³ CFU g⁻¹ of starting DNA in rhizosphere and phyllosphere samples and about 10² CFU g⁻¹ by plate count. The time for pathogens to reach detection limits on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere. However, real-time PCR continued to detect stx 1, stx 2 and the eae genes throughout the experimental period.
Conclusion:  Escherichia coli O157:H7 survived throughout the growth period as was determined by real-time PCR and by subsequent enrichment and immunomagnetic separation of edible part of plants.
Significance and impact of the Study:  The potential presence of human pathogens in vegetables grown in soils contaminated with E. coli O157:H7 is a serious problem to our national food supply as the pathogen may survive on the leaf surface as they come in contact with contaminated soil during germination.     

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Aims:  The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae .
Methods and Results:  A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 10³ CFU ml⁻¹ or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 10⁴ CFU g⁻¹ or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
Conclusion:  The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
Significant and Impact of the study:  The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae . This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.     

Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures

Aim:  Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen.
Methods and Results:  A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20°C and pH 5·3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15°C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15°C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through 'acetoclastic' pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15°C. Addition of 3 ml of PXYL1 at the rate of 12 × 10² CFU ml⁻¹ increased the biogas 1·7-fold (33 l per kg cowdung) when compared to control (19·3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l⁻¹ when compared to 1140 mg l⁻¹ in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6·8 ± 10² CFU ml⁻¹ in addition to PXYL1 served to further improve the biogas yields to 46 l kg⁻¹ as well as significantly brought down the VFA levels to 1350 mg l⁻¹.
Conclusions:  Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens.
Significance and Impact of the Study:  Stimulation of biomethanation at low temperature by coculture.     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

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.     

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.     

Detection of genogroup IV norovirus in wastewater and river water in Japan

Aims:  To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV).
Methods and Results:  Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption–elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6·9 × 10⁴ and 1·5 × 10⁴ copies l⁻¹, respectively.
Conclusions:  Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks.
Significance and Impact of the Study:  This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

Effect of selected antimicrobial compounds on the radiosensitization of Salmonella Typhi in ground beef

Aims:  In this study, we extended our previous work to determine the efficiency of antimicrobial compounds in increase of relative radiosensitivity of Salmonella Typhi in medium fat ground beef (23% fat) by testing 41 different essential oils (EOs), oleoresins and food sauces.
Methods and Results:  Ground beef samples inoculated with Salmonella Typhi (10⁶ CFU g⁻¹ ) were treated with each antimicrobial compound at a concentration of 0·5% (w/w). Then, the samples (25 g each) were packaged under air and irradiated in a ⁶⁰Co irradiator at doses from 0 to 1·75 kGy. Radiosensitivity was evaluated by calculating relative radiation sensitivity, defined as the ratio of radiation D ₁₀ value in the absence/presence of antimicrobial compound.
Conclusions:  Depending on the compound tested, the addition of antimicrobial compound decreased the D ₁₀ value of Salmonella Typhi, resulting in an increase of the radiation sensitivity up to more than four times. Among these antimicrobial compounds, Chinese cinnamon EO, clove EO and trans -cinnamaldehyde were most effective to increase the radiosensitivity of Salmonella Typhi in ground beef.
Significance and Impact of the Study:  These observations demonstrate that some active compounds can function as radiosensitizers of Salmonella Typhi.     

Legionella pollution in cooling tower water of air-conditioning systems in Shanghai, China

Aims:  To determine Legionella pollution prevalence, describe the amount of Legionellae with respect to temperature in Shanghai cooling tower water (CTWs) in various types of public sites.
Methods and Results:  Six urban districts were selected as the study fields, adopting multiple-phase sampling methods. Routine culture was used to identify Legionellae. Of the samples, 58·9% (189/321) were observed to be positive, 19·9% were isolated over 100 CFU ml⁻¹. Legionella pneumophila serogroup 1 was the most frequently isolated species (155/189, 82·0%), followed by Leg. micdadei that was at the second place (44/189, 23·3%). The mean CFU ml⁻¹ of Legionellae in CTWs reached its peak from July to September. Over all 15·4% of the samples exceeding 100 CFU ml⁻¹ were observed in a hospital setting.
Conclusions:  The prevalence of Legionella pollution in CTWs, especially in CTWs of subway stations and hospitals, is worrying, and the positive rate and CFU ml⁻¹ of Legionellae in CTWs have a close relationship with air temperature.
Significance and Impact of the Study:  The study demonstrates pollution prevalence rates in different types of sites and various seasons, and provides a proportion of different serogroups of Legionellae . It illuminates an urgent need for dealing with the potential risk of legionellosis in Shanghai, through improved control and prevention strategies.     

The anti-inflammatory drug Diclofenac retains anti-listerial activity in vivo

Aims:  The interactions between nonsteroidal anti-inflammatory drugs (NSAID) and Listeria monocytogenes have not been sufficiently documented to date. The aim of this study was to investigate the possible effects of Diclofenac (Dc) in a murine listerial infection model.
Methods and Results:  Dc was administered orally at 2·5 μg g⁻¹ to female albino strain of laboratory mouse (BALB/c) thrice postinfection (1 × 10⁸ CFU ml⁻¹ oral challenge with L. monocytogenes ATCC 51774), which resulted in significantly ( P  < 0·01) reduced bacterial counts in liver and spleen, decreased (10-fold, P  < 0·05) hepatic colonization and necrosis, and caused up-regulation of the expression of inflammatory cytokines (interferon-γ, interleukin-1β, tumour necrosis factor-α), compared with drug-free control.
Conclusions:  Dc may be useful as a promising adjuvant to the existing therapies in controlling systemic listerial infection. Further, quantitative structure–activity relationship studies might contribute in manipulating it as a lead compound for the synthesis of new, more effective nonantibiotics, perhaps, devoid of side-effects that could be recommended as a compassionate therapy for listeriosis.
Significance and Impact of the study:  This is the first in vivo study designed to evaluate the antilisterial effect of the NSAID Dc with special emphasis on the immunological mechanism of action of the drug.     

Effect of heat treatment on Cronobacter spp. in reconstituted, dried infant formula: preparation guidelines for manufacturers

Aim:  To explore safe guidelines for manufacturers and consumers to prepare, handle and store dry infant formula (DIF) to protect infants against Cronobacter spp.
Methods and Results:  Selected strains (2.45, FSM 293, ATCC-12868, FSM-271) screened from 68 strains of Cronobacter spp . were used to study growth and survival in commercial DIF. Prototype growth patterns in Enterobacteriaceae enrichment broth (EEB) containing a cocktail comprised of ATCC 12868, ATCC 29004, ATCC 29544 and ATCC 51329 showed a rapid increase in cell count (2·0 log₁₀ to 6·2 log₁₀ CFU ml⁻¹). Infant formula provided a better protective environment for the cells of Cronobacter strains than did buffered peptone water . Experiments on survival in inoculated (10⁴–10⁶ CFU ml⁻¹) reconstituted infant formula (RIF), preparation temperature, the effect of preparation volume (one-serving or two-serving) and effect of storage at room temperature for up to 10 h provided information to develop consumer guidelines for DIF preparation and handling.
Conclusions:  Reconstituted DIF in water at >70°C in larger volumes, minimizing storage time before feeding and storing unused reconstituted formulate at <4°C, may reduce the risk of Cronobacter infection in infants.
Significance and Impact of the Study:  Meningitis, necrotizing enterocolitis and bacteremia in premature babies has been linked to contaminated milk powder and DIF; better handling practices may improve the safety of these foods for neonates.     

Synbiotics growth optimization of Bifidobacterium pseudocatenulatum G4 with prebiotics using a statistical methodology

Aims:  This study demonstrated the optimum growth of Bifidobacterium pseudocatenulatum G4 with prebiotics via statistical model.
Methods and Results:  Commercial prebiotics [inulin and fructooligosaccharide (FOS)], together with sorbitol, arabinan and inoculum rate, were tested by fractional factorial design to determine their impact on growth of Bif. pseudocatenulatum G4 in skim milk. At 48 h incubation, bacterial growth was mainly influenced by FOS and inoculum rate. Growth reduction was observed in all samples incubated for 72 h. Central composite design (CCD) was adopted using FOS and inoculum rate at 48 h incubation to develop the statistical model for optimization. The model predicted that 2·461 log CFU ml⁻¹ produced the optimum growth increase of Bif. pseudocatenulatum G4. The combination that produced the optimum point was 2·86% FOS (g/v) and 0·67% inoculum rate (v/v).
Conclusion:  At optimum combination of inoculum rate and FOS, validation experiments recorded 2·40 ± 10·02 log CFU ml⁻¹. The application in 1-l bioreactor for 24 h showed higher growth increase of 2·95 log CFU ml⁻¹.
Significant and Impact of the Study:  Response surface methodology approach is useful to develop optimum synbiotics combination for strain G4 with FOS.     

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 .     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

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.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.