The effect of sodium azide concentration on the recovery of enterococci from water.

The ability of Slanetz and Bartley medium to recover chlorine-stressed enterococci has been studied. Results showed that chlorine injury significantly affected the ability of Slanetz and Bartley medium to recover enterococci while lower concentrations of sodium azide in the same basal medium allowed their recovery. However, reducing the concentration of sodium azide considerably reduced the specificity making it unsuitable for use in the routine examination of water. A non-azide-containing medium, Enterolert(®)-DW appeared to be able to recover injured and non-injured enterococci with similar efficiency. The data presented here suggest that further work is required to improve the recovery of chlorine-injured enterococci by Slanetz and Bartley medium.

[Role of increased tissue pressure in the pathophysiology of osteoarthritis]. / Rolle des erhöhten Gewebedruckes in der Pathophysiologie der Osteoarthrose.

Osteoarthritis is characterized by increased internal pressure in the subchondral bone tissue as well as in the joint capsule. In analogy to the pathophysiological mechanism of venous compartment syndrome it is demonstrated that the increased pressure is maintained by high protein edema and initiates a vicious circle. Drug-induced resorption of the osmotically active proteins leads to elimination of the high protein edema and is able to interrupt the pathophysiological process.

Understanding the cause of false negative beta-D-glucuronidase reactions in culture media containing fermentable carbohydrate.

AIMS: To explain the basis for false negative beta-glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. METHODS AND RESULTS: Escherichia coli strains were assessed for their reactions in culture media containing a beta-d-glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of beta-D-glucuronidase at pH 5.0 and pH 7.2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme beta-D-glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose-containing media expressed virtually no beta-D-glucuronidase activity at pH 5.0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. CONCLUSIONS: E. coli that failed to produce green colonies on MLGA produced lower levels of beta-D-glucuronidase than did strains that formed green colonies, the difference being greater at pH 5.0 than pH 7.2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme beta-D-glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7.2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).

Identification of coliform genera recovered from water using different technologies.

AIMS: Methods for the detection of coliforms in water have changed significantly in recent years with procedures incorporating substrates for the detection of beta-d-galactosidase becoming more widely used. This study was undertaken to determine the range of coliform genera detected with methods that rely on lactose fermentation and compare them to those recovered using methods based upon beta-d-galactosidase. METHODS AND RESULTS: Coliform isolates were recovered from sewage-polluted water using m-endo, membrane lauryl sulfate broth, tergitol TTC agar, Colilert-18, ChromoCult and ColiScan for primary isolation. Organisms were grouped according to whether they had been isolated based upon lactose fermentation or beta-d-galactosidase production. CONCLUSIONS: A wide range of coliform genera were detected using both types of methods. There was considerable overlap between the two groups, and whilst differences were seen between the genera isolated with the two method types, no clear pattern emerged. Substantial numbers of 'new' coliforms (e.g. Raoutella spp.) were recovered using both types of methods. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here confirm that both methods based on lactose fermentation or detection of beta-d-galactosidase activity recover a range of coliform organisms. Any suggestion that only methods which are based upon fermentation of lactose recover organisms of public health or regulatory significance cannot be substantiated. Furthermore, the higher recovery of coliform organisms from sewage-polluted water using methods utilizing beta-d-galactosidase-based methods does not appear to be because of the recovery of substantially more 'new' coliforms.

Use of the ISO 9308-1 procedure for the detection of E. coil in water utilizing two incubation temperatures and two confirmation procedures and comparison with defined substrate technology.

Disinfected and non-disinfected samples have been used to determine the accuracy of the ISO procedure (ISO 9308-1) for detection of E. coli in drinking water. Samples were analysed using the ISO procedure at both 36 and 44 degrees C and using the defined substrate technology method Colilert-18/Quanti-Tray (Colilert-18). Utilizing the confirmation procedure described in ISO 9308-1, large numbers of false positive E. coli results were obtained using the ISO primary isolation procedure at 36 degrees C. However, when glucuronidase production was used as the confirmation procedure, the 'confirmed' count of E. coli was lowest with ISO 9308-1 performed at 36 degrees C. When TTC medium was incubated at 36 degrees C confirmation using production of indole at 44 degrees C resulted in 29% more 'E. coli' being recovered than when confirmation was performed using production of glucuronidase. When 44 degrees C was used as the primary isolation temperature the difference between the number of 'confirmed' E. coli identified using the two confirmation procedures was less than 1% and was not significant. Identification of isolates which 'confirmed' when using production of indole at 44 degrees C as the test method but which failed to produce beta-D-glucuronidase, showed that the majority of these isolates were Klebsiella oxytoca.

False-negative beta-D-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia coli from water.

AIMS: Testing for beta-D-glucuronidase activity has become the basis of many methods for the detection of Escherichia coli in both food and water. Used in combination with tests for the presence of beta-D-glucuronidase, these tests offer a simple method for simultaneously detecting coliforms and E. coli. The purpose of this study was to determine the effectiveness of several different procedures in detecting beta-D-glucuronidase activity and hence in detecting E. coli. METHODS AND RESULTS: The ability of membrane lactose glucuronide agar (MLGA), Colilert-18, MI agar, Colitag and Chromocult agar to detect beta-D-glucuronidase activity was tested with over 1000 isolates of E. coli recovered from naturally contaminated water samples. Four of the media gave very similar results but MLGA failed to detect glucuronidase activity in 15.6% of the cultures tested. CONCLUSIONS: MLGA had very poor sensitivity for the detection of beta-D-glucuronidase activity in strains of E. coli isolated from naturally contaminated water. This is probably because of the fact that beta-D-glucuronidase activity is pH-sensitive and that acid is formed by E. coli during fermentation of lactose in MLGA. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of E. coli in drinking water is the primary test used to establish faecal contamination. The poor sensitivity of MLGA in detecting beta-D-glucuronidase activity suggests that this medium and others containing high concentrations of fermentable carbohydrate should not be used for the detection of E. coli.

Heterotrophic plate count measurement in drinking water safety management: report of an Expert Meeting Geneva, 24-25 April 2002.

A group of microbiology and public health experts including regulatory and medical expertise was convened in Geneva, Switzerland, 25-26 April 2002 to consider the utility of heterotrophic plate count (HPC) measurements in addressing drinking water quality and safety. The group was convened following the NSF International/World Health Organization Symposium on HPC Bacteria in Drinking Water--Public Health Implications? The Expert Meeting was attended by 31 participants from Australia, Canada, France, Germany, Italy, Japan, the Netherlands, South Africa, Switzerland, UK and USA.

Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.

A comparison of the International Standards Organisation reference method for the detection of coliforms and Escherichia coli in water with a defined substrate procedure.

AIMS: This study investigated the use of the International Standards Organisation (ISO) procedure for the comparison of microbiological methods. Using this procedure the ISO reference procedure for the detection of coliforms and Escherichia coli in water was compared with a defined substrate method (ColilertTM). METHODS AND RESULTS: A total of 20 laboratories from 13 European countries compared the use of Colilert/Quanti-TrayTM, a quantitative defined substrate test (DST) for the presence of coliforms and E. coli with the ISO reference procedure, which utilizes tergitol-TTC medium. Results of the study showed that DST detected significantly more coliforms and E. coli than did the reference procedure. In the case of E. coli the recoveries were also higher when using DST and the difference seen was statistically significant. The confirmation rate obtained when using the DST product suggested that no confirmation of wells positive for E. coli was necessary during routine use. CONCLUSIONS: Colilert is a suitable alternative to the ISO reference procedure for the detection of coliforms and E. coli in water. The methods used during the comparison study indicated that confirmation of all colonies/positive wells led to the most accurate information and it is recommended that for future comparison studies this should become standard practice. Confirmation of a small proportion of colonies led to misleading conclusions and should be avoided when comparing microbiological methods. SIGNIFICANCE AND IMPACT OF STUDY: It has been demonstrated that the ISO reference procedure fails to detect a significant proportion of coliforms and E. coli in drinking water. Colilert/QuantiTrayTM is a more suitable alternative.

Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts.

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 degrees C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.

Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts.

cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD-1 mice using standardized protocols and a well-characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose-response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose-response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.

Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water.

A species-specific Peptide Nucleic Acid (PNA) oligonucleotide probe directed against the V(1)region of the 16S rRNA molecule was synthesized for the detection of Escherichia coli in water. The specificity of the probe was tested in dot blot hybridizations against a number of environmental isolates including those from the genera Escherichia, Klebsiella, Enterobacter and Citrobacter. In situ hybridization experiments were performed with biotinylated PNA oligonucleotide probes with subsequent detection of the biotin label using a combination of Streptavidin-Horseradish Peroxidase and a tyramide signal amplification system. The results obtained enabled the specific detection of E. coli in under 3 h. Hybridizations were also performed on cells which were treated with chlorine (1.5 mg l(-1)) for up to 30 min. Escherichia coli cells were still detected after storage for 14 days at room temperature. No cells were detected by conventional plate count or the <> assay, a method currently used for the routine detection of E. coli and coliforms in the water industry. Cell viability was assessed by the ability of cells to reduce 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to highly fluorescent formazan crystals through bacterial respiration. Only cells that had not been chlorinated were detected. These results confirm that ribosomal RNA exists within the cell long after cell death has occurred and that rRNA cannot be used to assess the viability of individual cells. However rRNA probes in combination with viability markers should enable the specific detection of viable cells in situ. Hybridization experiments were also performed successfully on seeded tap water samples. The number of fluorescent cells detected correlated well with those obtained by plate count analysis. This represents the first reported use of PNA oligonucleotides for in situ detection of micro-organisms and offers a fast efficient alternative to conventional DNA approaches.

Application of laser scanning for the rapid and automated detection of bacteria in water samples.

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using 'viability markers' may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z-test, P < 0.05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.

Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data.

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.

In situ reverse transcription for the specific detection of bacteria and protozoa.

In situ hybridization experiments with oligonucleotide probes directed against the 16S and 18S rRNA molecules have been used successfully to identify specific organisms in mixed microbial populations. However, there are limitations in applying these techniques to environmental samples. In the present study we have examined the possibility of using in situ reverse transcription as an alternative to hybridization methods for the rapid detection of Escherichia coli and the waterborne parasite Cryptosporidium parvum. Following fixation and permeabilization of the cells, extension reactions were performed with species-specific primers, AMV reverse transcriptase and either cy3-AP3-dUTP or fluorescein-11-dUTP at 45 degrees C for 45 min. The cells or oocysts were then filtered onto Costar metallic membrane filters and images captured with a CCD camera. The results have shown that this technique can successfully detect E. coli cells and C. parvum oocysts in under 2 h.

Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7.

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

An evaluation of the Gelman Envirochek capsule for the simultaneous concentration of Cryptosporidium and Giardia from water.

The Gelman Envirochek capsule is a membrane device for the simultaneous concentration of Cryptosporidium oocysts and Giardia cysts from water. Samples are filtered through a Supor polyethersulphone membrane with a 1 micron absolute pore size. (Oo)cysts are mechanically eluted from the membrane fibre using a wrist action shaker and a nonionic detergent and concentrated by centrifugation. The concentrate can be further processed using any separation technique to separate the target organisms from other debris. This method enables multiple samples to be processed within 1 h. Recoveries from seeded tap and source water samples were in excess of 70% for Cryptosporidium and 80% for Giardia.

Immunomagnetic separation of Cryptosporidium parvum from source water samples of various turbidities.

Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts.

A simple method for the comparison of commercially available ATP hygiene-monitoring systems.

The purpose of this study was to evaluate a methodology which could easily be used in any test laboratory in a uniform and consistent way for determining the sensitivity and reproducibility of results obtained with three ATP hygiene-monitoring systems. The test protocol discussed here allows such comparison to be made, thereby establishing a method of benchmarking both new systems and developments of existing systems. The sensitivity of the LUMINOMETER K, PocketSwab (Charm Sciences) was found to be between 0.4 and 4.0 nmol of ATP with poor reproducibility at the 40.0 nmol level (CV, 35%). The sensitivity of the IDEXX LIGHTING system and the Biotrace UNILITE Xcel were both between 0.04 and 0.4 nmol with coefficients of variation (CVs) of between 9% at 0.04 nmol and 10% at 0.4 nmol for the IDEXX system and 17% at 0.04 nmol and 21% at 0.4 nmol for the Biotrace system. The three systems were tested with a range of dilutions of different food residues: orange juice, raw milk, and ground beef slurry. All three test systems allowed detection of orange juice and raw milk at dilutions of 1:1,000, although the CV of results from the Charm system (54 and 74% respectively) was poor at this dilution for both residues. The sensitivity of the test systems was poorer for ground beef slurry than it was for orange juice and raw milk. Both the Biotrace and IDEXX systems were able to detect a 1:100 dilution of beef slurry (with CVs of 17 and 10% respectively), whilst at this dilution results from the Charm system had a CV of 55%. It was possible by using the method described in this paper to rank in order of sensitivity and reproducibility the three single-shot ATP hygiene-monitoring systems investigated, with the IDEXX LIGHTNING being the best, followed by the Biotrace UNILITE Xcel, and then the charm LUMINOMETER K, PocketSwab.