Chromocult Coliform Research Articles

Comparative analysis of classical and molecular microbiology methods for the detection of Escherichia coli and Enterococcus spp. in well water

The microbiological quality of 165 1 litre well water samples collected in the Québec City region was assessed by culture-based methods (mFC agar, Chromocult coliform agar, Colilert(®), MI agar, Chromocult enterococci, Enterolert™, and mEI agar) and by a molecular microbiology strategy, dubbed CRENAME-rtPCR, developed for the detection of Escherichia coli, Enterococcus spp., Enterococcus faecalis/faecium, and Bacillus atrophaeus subsp. globigii. In these drinking water samples, approved culture-based methods detected E. coli at rates varying from 1.8 to 3.6% and Enterococcus spp. at rates varying from 3.0 to 11.5%, while the molecular microbiology approach for E. coli was found to be as efficient, detecting contamination in 3.0% of samples. In contrast, CRENAME-rtPCR detected Enterococcus spp. in 27.9% of samples while the E. faecalis/faecium molecular assay did not uncover a single contaminated sample, thereby revealing a discrepancy in the coverage of waterborne enterococcal species detected by classical and molecular microbiology methods. The validation of the CRENAME-E. coli rtPCR test as a new tool to assess the quality of drinking water will require larger scale studies elaborated to demonstrate its equivalence to approved methods.

The incidence of diarrhoeagenic Escherichia coli in minced beef and boerewors

A survey of diarrhoeagenic Escherichia coli (DEC) was carried out on 21 minced beef and 21 boerewor (a traditional South African fresh sausage) samples purchased from 30% of butcheries in the Bloemfontein District of South Africa. The samples were cultivated on standard plate count agar for aerobic plate counts and chromocult coliform agar for coliform and E. coli counts without enrichment. Diarrhoeagenic E. coli were isolated after enrichment in MacConkey broth followed by cultivation on chromocult coliform agar. The resultant E. coli colonies were selected and serotyped using the slide agglutination test for DEC. Eight (38.10%) minced beef samples were positive for DEC. The 8 positive samples consisted of 5 (23.81%) EPEC and 1 each (4.76%) of EIEC, EAggEC, and VTEC. A total of 100 isolates were serotyped (5 from each sample) and these comprised 6 (6%) EPEC serotypes (3×O18; 1×O125; and 1×O142), and 1 (1%) each of EIEC (1×O128ac), EAggEC (1×O44) and VTEC (1×O26). Boerewors had 6 (28.57%) samples that were positive for DEC. EPEC and VTEC were the virotypes that were isolated at 23.81% (5/21) and 4.76% (1/21) respectively. A total of 105 isolates were serotyped (5 from each sample) and these comprised 7 (6.66%) EPEC (3×O18, 2×O125, 1×O55 and 1×O142); and 1 (0.95%) VTEC (1×O26). EPEC were the most frequent virotype while VTEC were the most virulent virotype recovered from both products. No E. coli O157:H7 were recovered.

Equivalency testing of TTC Tergitol 7 agar (ISO 9308-1:2000) with five culture media for the detection of E. coli in water samples in Greece

In this study ten laboratories in Greece compared the performance of reference method TTC Tergitol 7 Agar (with the additional test of beta-glucuronidase production) with five alternative methods, to detect E. coli in water, in line with European Water Directive recommendations. The samples were prepared by spiking drinking water with sewage effluent following a standard protocol. Chlorinated and non-chlorinated samples were used. The statistical analysis was based on the mean relative difference of confirmed counts and was performed in line with ISO 17994. The results showed that in total, three of the alternative methods (Chromocult Coliform agar, Membrane Lauryl Sulfate agar and Trypton Bilex-glucuronidase medium) were not different from TTC Tergitol 7 agar (TTC Tergitol 7 agar vs Chromocult Coliform agar, 294 samples, mean RD% 5.55; vs MLSA, 302 samples, mean RD% 1; vs TBX, 297 samples, mean RD% -2.78). The other two alternative methods (Membrane Faecal coliform medium and Colilert 18/ Quantitray) gave significantly higher counts than TTC Tergitol 7 agar (TTC Tergitol 7 agar vs MFc, 303 samples, mean RD% 8.81; vs Colilert-18/Quantitray, 76 samples, mean RD% 18.91). In other words, the alternative methods generated performance that was as reliable as, or even better than, the reference method. This study will help laboratories in Greece overcome culture and counting problems deriving from the EU reference method for E. coli counts in water samples.

ISOLASI, KARAKTERISASI DAN PENGELOMPOKAN STRAIN Salmonella typhi ASAL KABUPATEN SUMBA BARAT DAYA NUSA TENGGARA TIMUR BERDASARKAN SIFAT-SIFAT FENOTIP:

Typhoid fever is highly endemic in the South-West Sumba Regency, East Nusa Tenggara. The incidence rate of the diseases is high estimated at 725/100,000. It is an acute systemic infection caused by Salmonella typhi. The clinical symptoms of the disease are extremely diverse, starting from the mild form to severe ones with the most feared complication being perforation within the small intestine. Therefore, it is important to perform isolation, characterization, and grouping of S. typhi strains from the blood culture in order to determined definitely diagnosis and the different phenotypic characteristics in the community. Isolation was done in selective and differential media: BacT/ALERT FA culture media, Selenite Cystine Broth, Chromocult Coliform Agar, MacConkey Agar, and Salmonella Shigella Agar. The typical colony of Salmonella was confirmed on Triple Sugar Iron Agar, Urea agar, and L-Lysine decarboxylation media. Phenotypic characteristics of all isolates were identified using API 20E and API 50CHE diagnostics. Based on biochemical characteristics the result showed that 18 strains obtained from different geographical origins were diverse. Four strains have similarity value 100% while the remained strains have similarity value 86.3–98.4%. All of the strains were categorized in the species of S. typhi.

ISOLASI, KARAKTERISASI DAN PENGELOMPOKAN STRAIN Salmonella typhi ASAL KABUPATEN SUMBA BARAT DAYA NUSA TENGGARA TIMUR BERDASARKAN SIFAT-SIFAT FENOTIP

Typhoid fever is highly endemic in the South-West Sumba Regency, East Nusa Tenggara. The incidence rate of the diseases is high estimated at 725/100,000. It is an acute systemic infection caused by Salmonella typhi. The clinical symptoms of the disease are extremely diverse, starting from the mild form to severe ones with the most feared complication being perforation within the small intestine. Therefore, it is important to perform isolation, characterization, and grouping of S. typhi strains from the blood culture in order to determined definitely diagnosis and the different phenotypic characteristics in the community. Isolation was done in selective and differential media: BacT/ALERT FA culture media, Selenite Cystine Broth, Chromocult Coliform Agar, MacConkey Agar, and Salmonella Shigella Agar. The typical colony of Salmonella was confirmed on Triple Sugar Iron Agar, Urea agar, and L-Lysine decarboxylation media. Phenotypic characteristics of all isolates were identified using API 20E and API 50CHE diagnostics. Based on biochemical characteristics the result showed that 18 strains obtained from different geographical origins were diverse. Four strains have similarity value 100% while the remained strains have similarity value 86.3–98.4%. All of the strains were categorized in the species of S. typhi.

Occurrence and survival of coliform bacteria, Escherichia coli and Salmonella in various manure and compost

pp. 865–874 Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 102 to 106 cfu g1 dry matter. From positive samples, 21 isolates of possible coliform bacteria were purified. Among them, species of coliform bacteria (E. coli, E. vulneria, Pantoea sp. and Buttiauxella agrestis) were identified whereas isolates of Serratia marcescens, not coliform bacteria, were also obtained, suggesting that careful observation was necessary to avoid false positive counting due to the presence of a red colony of S. marcescens that resembled coliform bacteria. Isolates of E. coli were tested for slide aggregation with a set of antiserum against pathogenic E. coli serotypes and negative reaction was obtained for all the isolates tested. Direct detection of E. coli on Chromocult coliform agar and Salmonella on MLCB agar resulted in none and 2 (17%) of 12 samples tested, respectively. The fate of fecal-contamination indicator bacteria as above was followed during compost production on 7 cases at 6 compost facilities and 4 patterns were observed: fecal-contamination indicator bacteria 1) decreased and finally disappeared, 2) decreased once but re-growth was occurred on products, 3) decreased to some extent but remained in products, 4) was not detected throughout production. These results suggest that some fecal-contamination indicator bacteria may survive compost production and appropriate temperature control would be significant for hygiene control of manure and compost.

Detection of Escherichia coli in the sewage influent by fluorescent labeled T4 phage

For a precise estimation of sanitary condition, Escherichia coli detection system using E. coli-specific bacteriophage T4 was constructed. To facilitate E. coli detection, T4e − phage which did not produce the lysozyme was constructed and green fluorescent protein (GFP) was displayed on T4e − small outer capside (SOC) protein. This T4e −/GFP can detect E. coli K12 without cell lysis. In this study, we applied T4e −/GFP to the detection of E. coli in the sewage influent. Chromocult ® coliform (CC) agar plates are generally used for simultaneous detection of total coliforms and E. coli. We investigated the number of coliforms and E. coli in the municipal sewage influent by using CC agar plates for 1 year. There were 10 5–10 6 CFU/ml of total coliforms and 10 4–10 5 CFU/ml of E. coli throughout the year. More than 20 strains of E. coli selected from CC agar were infected by T4e −/GFP. The ratio of clear plaque forming E. coli was different every month and very low (annual average 8%). Most of the E. coli showed turbid plaque or no plaque. E. coli formed the turbid plaque showed no-fluorescence, fluorescence only on cell surface due to phage adsorption, or heterogeneous fluorescence among the cells, while E. coli formed clear plaque could be detected because of T4e −/GFP amplification in the cell.

Evaluation of Chromogenic Enzyme Substrate Mediums, Chromocult Coliform Agar(CCA) and XM-G, by Detection of Freeze-, Heat-, High-Pressure-Injured Coliforms, and Coliforms in Food Samples

The two chromogenic enzyme substrate mediums of chromocult coliform agar (CCA) and XM-G were applied to detect freeze-, heat-, and high-pressure-injured coliforms (Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Klebsiella ozaenae, and Klebsiella pneumoniae) as well as coliforms in various food items. Their detection abilities were then compared with the following three conventional media: tryptic soy agar (TSA), violet red bile agar (VRBA), and VRBA/TSA. The enumerated results of injured coliforms showed that the ability to detect injured cells was in the following descending order: non-selective medium TSA>VRBA/TSA>XMG≥CCA≫VRBA. The recovery rate of injured coliforms, when compared with selective agars, was higher in CCA and XM-G than in VRBA. Investigation of the total coliform counts from 100 food samples showed that the enumeration results of the three selected media (CCA, XM-G, and VRBA) were quite similar. The correlation coefficients were 0.89 for CCA vs. VRBA, 0.91 for XM-G vs. VRBA, and 0.91 for CCA vs. XM-G; indicating that CCA and XM-G are recommendable and can substitute for the conventional selective medium VRBA. In addition to the advantage of simultaneous detection of coliforms and Escherichia coli by CCA and XM-G, their superiority in detecting injured coliforms reveals that these two methods were highly effective and suitable to monitor total coliforms and E. coli including injured cells in food samples.

Growth of Seeded Escherichia coli in Rewetted Cattle Waste Compost of Different Stages

Compost is used mainly as an organic fertilizer, but it is also used as bedding material for cattle. Dairy cattle have been identified as a main reservoir of pathogenic Escherichia coli O157:H7. Further, E. coli is regarded as an environmental pathogen that causes bovine clinical mastitis. Hence, its growth in compost spread or compost bedding should be avoided. Physical and chemical conditions, available nutrients and microflora in compost change greatly during the composting process. Since pathogen growth in compost seems to be related to these changes, we assessed the possibility of E. coli growth in compost samples collected at 0, 7, 13, 22, 41, 190 and 360 d. Cattle waste composts with and without added tofu residue were collected from static piles and immediately air- dried. Compost samples were inoculated with a pure culture of E. coli, the moisture content was adjusted to 50%, and the samples were incubated for 5 d at 30°C. The numbers of E. coli in compost before and after incubation were determined by direct plating on Chromocult coliform agar. Almost all compost samples supported E. coli growth. Samples collected during or immediately after the thermophilic phase (day 7) showed the highest growth. Growth in samples more than 13 d old were not significantly different from those of aged compost samples. The addition of tofu residue gave a higher growth than its absence in younger samples collected prior to 13 d. To minimize the risk of environmental mastitis, the use of compost in the initial stage of the process is better avoided. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 278-282)

Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects

The purpose of this study was to examine the use of Chromocult agar medium for isolation and enumeration of Enterobacteriaceae from human faecal samples, to compare it to MacConkey agar and to evaluate its usefulness as a possible alternative selective medium in human faecal studies. The medium was shown to be effective in identifying Escherichia coli and coliforms in faeces without the need for extensive accompanying biochemical tests for confirmation of identity. A positive correlation ( r=0.86) was found between the recovery of Enterobacteriaceae on the two media, and no significant difference ( P>0.05) between overall mean bacterial counts for the whole study group or at different intervals of faecal collection were observed. Chromocult agar is an effective replacement for MacConkey agar in human faecal studies and has the advantage of differentiating E. coli from other coliforms.

Rapid enzymatic detection of Escherichia coli contamination in polluted river water.

The relationship between the rate of beta-D-glucuronidase hydrolysis (GLUase-HR) and the E. coli concentration in rivers differing in the extent of faecal pollution was investigated. It was hypothesized that the rate of GLUase-HR is a better surrogate parameter for E. coli concentrations than estimated numbers of faecal coliforms (FC). The GLUase-HR of the water sample filter residues was determined as the rate of cleavage of 4-methylumbelliferyl-beta-D-glucuronide. FC and E. coli concentrations were enumerated using mFC and Chromocult Coliform agar, respectively. Regression analysis revealed that a 90% variation of the variable log GLUase-HR was directly related to the variable log E. coli concentrations. The observed relationship between the log of the FC count and the log of the GLUase activity could be explained by the hydrolysis activity of the E. coli population, as E. coli is a part of the FC group. The data suggest that the log of the GLUase-HR can be used as a surrogate parameter for the log of the E. coli concentrations. GLUase-HR determination may provide a rapid alternative technique to estimate E. coli concentrations in freshwaters.

Efficacy of Chromocult Coliform Agar for Coliform and Escherichia coli Detection in Foods

Chromocult coliform agar (CCA) was compared with Petrifilm Escherichia coli count plate (PEC) for identifying coliforms and E. coli in a variety of meat products. Products examined included 45 raw beef samples, 12 sausage emulsion samples, 11 samples of meat-based ready-to-eat appetizers, and 8 pork trimming samples. Coliforms from CCA and PEC were confirmed by gassing in brilliant green lactose broth plus a positive reaction on purple broth agar plus lactose after incubation at 35°C for 48 h. Lauryl sulfate tryptose plus methylumbelliferyl-β-glucuronide and tryptophan broth were used to confirm E. coli from CCA and PEC with 48-h incubations at 35 and 42.5°C, respectively. API 20E test strips were inoculated for final confirmation. The overall respective confirmation percentages (CFU/g) for the PEC and the CCA methods were 93.1 and 93.7% for coliforms and 99.8 and 98.1% for E. coli, although the CCA method yielded significantly (P < 0.001) higher mean CFU/g values for both coliforms and E. coli. Regression analyses of these data indicated a strong positive linear relationship existed between the two methods over a wide CFU/g range for both coliforms and E. coli. The respective correlation coefficients obtained for coliforms and E. coli of 0.89 and 0.86 indicate that the CCA method provides a reliable optional method for these determinations in meat products.

Determination of Escherichia coli contamination with chromocult coliform agar showed a high level of discrimination efficiency for differing fecal pollution levels in tropical waters of Kampala, Uganda.

Escherichia coli, total coliforms, fecal coliforms, and sulfite-reducing anaerobic spore formers from different polluted sites in a tropical environment were determined in order to test for their indication ability for fecal contamination. Quantification of E. coli contamination with Chromocult coliform agar proved to be efficient and feasible for determining fecal pollutions in the investigated area within 24 h. The other microbial parameters showed a lower ability to differentiate sites and cannot be recommended for monitoring fecal pollution in the studied tropical surface waters.

Evaluation of Selected Membrane Filtration and Most Probable Number Methods for the Enumeration of Faecal Coliforms, Escherichia coli and Enterococci in Environmental Waters

Selected methods recommended in national and international water quality guidelines were compared in tests on environmental waters with different levels of faecal pollution. The following methods yielded no statistically significant differences in counts of faecal coliforms and Escherichia coli in raw sewage, semi-treated effluent, polluted urban run-off and stored potable water: Membrane filtration (MF) using MFc Agar or Chromocult Coliform Agar containing X-glucuronide, or a miniaturised microtitre-plate Most Probable Number (MPN) assay using a liquid growth medium containing chromogenic 4-methyl-umbelliferyl-β-D-glucuronide. Significant differences were, however, found between the Chromocult and the other methods for unpolluted river water. Counts of faecal enterococci in raw sewage, semi-treated effluent and polluted urban run-off, obtained by the following methods did not differ significantly: MF using M-Enterococcus Agar, Bile-Esculin Agar or Enterococcus Selective Agar, or a microtitre-plate MPN method with a liquid growth medium containing chromogenic 4-methyl-umbelliferyl-β-D-glucoside. Significant differences were, however, found between the MPN and the other methods for unpolluted river water and stored potable water. MF using Chromocult Coliform Agar had useful benefits for the simultaneous enumeration of coliforms and E coli. However, in view of cost and practical considerations, MF using MFc Agar or M-Enterococcus Agar proved the methods of choice for respectively enumerating faecal coliforms and E coli, or faecal enterococci, in analyses for general water quality surveillance purposes.

Quantitative determination of E. coli, and fecal coliforms in water using a chromogenic medium

A new medium, Chromocult Coliform® Agar (CC agar) developed by E. Merck AG (Darmstadt, Germany) was compared with the Standard Methods, membrane filtration fecal coliform (mFC) medium for fecal coliform detection and enumeration. In the CC agar, non‐E. coli, fecal coliforms (Klebsiella, Enterobacter, and Citrobacter,), (KEC) were identified by the production of a salmon to red colour from p‐galactosidase (LAC) cleavage of the substrate Salmon‐GAL, while E. coli, colonies were detected by the blue colour, produced by the cleavage of X‐glucuronide by β‐ glucuronidase (GUS). Statistically, there was no significant differences between fecal coliform counts obtained with the two media (CC agar and mFC agar) and two incubation procedures (2h‐37°C plus 22h‐44.5°, and 44.5°C) as determined by variance analysis. In our study K coli, represented, on average 70.5–92.5% of the fecal coliform population. A high incidence of false negative KEC (19.5%) and E. coli, (29.6%) colonies was detected at 44.5°C. Two K coli, GUS negative phenotype upon reinoculation into CC agar were GUS+. A total of 31 KEC LAC colonies were streaked onto CC, agar and incubated at 37°C, 29 KEC strains that failed to produce β‐galactosidase at 44.5°C were able to produce the enzyme at 37°C. In our opinion the physiological condition of the fecal coliform isolates could be responsible for the non‐expression of P‐galactosidase and P‐glucuronidase activities at 44.5°C.