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.

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.

Water-borne cryptosporidiosis: detection methods and treatment options.

Since the infamous outbreak in Milwaukee, WI, USA, of water-borne cryptosporidiosis affecting over 400,000 people, there have been at least 20 smaller outbreaks associated with this parasite in the UK and North America. These events have led to an explosion of interest in and research on the nature of cryptosporidiosis as a dangerous water-borne pathogen, particularly patients with acquired immune deficiency syndrome (AIDS). In addition, several major environmental laws and proposed regulations specifically address the control of this parasite. The possible ramifications of these laws include billions of dollars of modifications to water-treatment facilities in the USA. Unfortunately, the methods used to gather the information on which these laws are based have serious deficiencies that could lead to gross underestimation of the magnitude of this problem. The present review considers gaps in our understanding of water-borne cryptosporidiosis, new methods under investigation that could improve our ability to monitor water for the presence of this organism, and treatment and control strategies to limit the threat to our water supplies.

Serotyping of campylobacters by co-agglutination on the basis of heat-stable antigens.

A new and simple method of serotyping campylobacters has been developed which utilises co-agglutination to detect the presence of heat-stable antigens. Campylobacters are heated at 75 degrees C for 30 min to destroy antigenic protein and allowed to react on a glass slide with staphylococci coated with antibody. Of 74 isolates, 67 gave the same result by co-agglutination and the previously described passive haemagglutination method. The co-agglutination technique may be used as a rapid screening test before serotyping by passive haemagglutination.

Aeromonas spp. in foods: a significant cause of food poisoning?

From a total of 563 samples of various foodstuffs purchased from retail outlets in the Reading area 287 were found to contain mesophilic Aeromonas spp. The types of sample which were most frequently contaminated were poultry (79.3%) and offal (84.3%). Of three media compared for their efficiency in recovering Aeromonas spp. after enrichment in alkaline peptone water, Difco Aeromonas was the most efficient. Cytotoxin was produced by approximately 50% of the A. hydrophila and A. sobria strains but by none of A. caviae strains. It is concluded that both raw and cooked foods are potential sources for infecting human beings with Aeromonas spp.

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.

A note on the effect of different storage procedures on the ability of Preston medium to recover campylobacters.

The effect of different storage procedures on the ability of Preston medium to recover campylobacters was investigated. Freshly poured media was shown to recover more campylobacters than media stored under aerobic or anaerobic conditions at room temperature or at 4 degrees C. The growth of Campylobacter laridis was greatly reduced by storage of media and although most strains of C. jejuni and C. coli were not markedly affected, the growth of one strain of C. jejuni was considerably reduced. It is recommended that freshly prepared media be used whenever possible, but if storage is necessary, then plates should be held at 4 degrees C, preferably under anaerobic conditions. These precautions may not be necessary for workers interested solely in C. jejuni or C. coli, but are essential for the optimum isolation of C. laridis.

A note on salmonella excretion in the black headed gull (Larus ribibundus) feeding at sewage treatment works.

The range of salmonella serotypes found in sewage sludge and in the faeces of black headed gulls (Larus ribibundus) feeding on the sludge was investigated. A close association was demonstrated between the serotypes found in both types of sample. Salmonella takoradi (a serotype which is uncommon in Scotland) appeared in the sludge for two short periods during the twelve week study and on both occasions it was later found in the gull faeces. It was shown that gulls become infected after feeding on contaminated sewage sludge but that the infection is probably short-lived.

A comparison of methods for the isolation of salmonellae from sewage sludge.

Methods for the isolation of salmonellae from sewage sludge were compared. Buffered peptone water and lactose broth were compared to determine their efficiencies as preenrichment media and temperature and duration of incubation were also investigated. In addition five enrichment and five plating media were compared together with the effects of multiple plating of enrichment broths. Buffered peptone water incubated at 43 degrees C for 24 h was shown to be the pre-enrichment method of choice with enrichment in the RB 10 form of Rappaport's broth incubated at 43 degrees C for 48 h and plating at 24 and 48 h onto brilliant green agar containing sulphamandelate supplement and Hynes' modification of desoxycholate citrate agar. Of the 100 samples used in the study. 96 were found to be positive for salmonella by at least one of the procedures used. A total of 15 Salmonella serotypes were isolated. Salmonella virchow being the most common.

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium.

The efficiency of Rappaport's broth ( RB10 ) and Rappaport's broth containing novobiocin ( NRB10 ) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used: no significant difference in their ability to recover salmonellae was demonstrated.

Application of the polymerase chain reaction to the identification of Escherichia coli and coliforms in water.

The polymerase chain reaction (PCR) has been applied to the identification of Escherichia coli and coliforms after overnight growth using two sets of primers described previously. The primer set for E. coli, which was derived from the uid A gene, correctly identified all E. coli strains tested. The sequence was also identified in five non-E. coli coliforms. The coliform primer set correctly identified approximately 70% of the coliforms tested. We conclude that PCR can be used for the rapid identification of E. coli using the primers described here but that further work is required accurately to define a new primer set for the coliform group.

A two-year study of the distribution of 'thermophilic' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype.

The incidence of 'thermophilic' campylobacters in foods and environmental samples has been studied over a two-year period. Of 781 environmental samples, 529 (67%) were found to contain campylobacters, and campylobacters were isolated from 835 (39%) of 2116 food samples. Sewage was almost always contaminated with campylobacters (96.6% of samples) and of the food samples both poultry (55.5%) and offal (47.0%) were commonly contaminated. Determination of the heat-stable serotypes of all strains isolated from these sources and of 921 strains isolated from human faeces showed that there was a wide distribution of serotypes in most types of sample. Serotype Pen 2 was the commonest type found in human faeces (18.9%) and it was also commonest in offal (21.3%), beef (40.0%), sewage (17.7%) and was the third commonest type in poultry. A comparison of culture media and conditions for optimal production of both cytotoxic and cytotonic enterotoxins showed that Brucella Broth incubated under microaerobic conditions for 24 h at 42 degrees C was suitable for both toxins. Detection of cytotoxic activity was most sensitive using HeLa cells. The sensitivities of two ELISA systems and a Chinese Hamster Ovary tissue culture assay for detection of cytotonic enterotoxin were comparable. Not all strains isolated from cases of enteritis in human beings produced toxin; 23.1% produced cytotonic enterotoxin and 17.5% produced cytotoxin. There was no correlation between serotype and toxin production. The wide distribution of campylobacters, indistinguishable from those isolated from cases of enteritis in human beings, leads us to conclude that simplistic statements suggesting that one particular type of food is primarily responsible for cases of human disease should not be made.

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.

The effect of the use of different selective media on the ability to recover salmonellae from seagull faeces.

Solid media were compared for their ability to recover salmonellae from seagull faecal material after pre-enrichment in buffered peptone water and enrichment in Rappaport's broth. Of the 847 specimens examined 96 were found to be positive for salmonellae. Use of Brilliant Green agar containing sulphamandelate supplement resulted in the detection of salmonellae from each of the 96 samples found to be positive and was the most efficient medium tested. Brilliant Green agar lacking the supplement was the least effective medium, salmonellae being isolated from only 80 samples using this medium. All of the media tested were shown to support the growth of a wide range of salmonella serotypes, although Salmonella typhi and S. dublin did not form colonies on those media which contained Brilliant Green. Hynes' modification of deoxycholate citrate agar was shown to be considerably less inhibitory to salmonellae after ageing for four days. Ageing of other media had no significant affect on their ability to support the growth of salmonellae.

Campylobacters in wading birds (Charadrii): incidence, biotypes and isolation techniques.

311 birds from four species of the charadrii group were examined for the carriage of Campylobacter spp. Cloacal swabs or "washouts" were taken from birds captured by cannon netting and cultured using enrichment in Preston broth followed by plating onto Preston agar. Incubation of enrichment cultures for 48 h and of solid media for a minimum of 48 h is recommended for culture of avian faecal material. Of the birds examined, 222 were found to be carrying Campylobacter spp., 145 strains of which belonged to the C. jejuni/C. coli group. Of the four species examined, Oystercatcher were significantly more frequently associated with the carriage of C. jejuni biotype 1 than were the other three species (P less than 0.05). The high carriage rate of Campylobacter spp. coupled with the behaviour of this group of birds may have implications for human health.