Evaluation of Chromocult® enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers
C.M. Franco, Laboratorio de Higiene Inspección y Control de Alimentos, Facultad de Veterinaria pabellón 4 p.b., Campus Universitario, 27002-Lugo, Spain (e-mail: email@example.com).
Aims: To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult® enterococci agar) recently developed by Merck.
Methods and Results: The study was carried out comparing Chromocult® enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed.
Conclusions: A good sensitivity of 98% was obtained for Chromocult® agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P < 0·01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult® enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult® enterococci agar.
Significance and Impact of the Study: The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.
Enterococci are found in a variety of environments. They frequently occur in large numbers in dairy and food products (Giraffa and Sisto 1997) and they are very resistant to adverse environmental conditions and heat treatment. Therefore, they are good indicators of the degree of faecal contamination of foods (Stiles and Holzapfel 1997). From the metabolic point of view, Enterococcus spp. produce lactic acid as the main product of carbohydrate fermentation. Thus, they may be considered to be lactic bacteria (Doming et al. 2003). Some strains are enterotoxigenic, especially those belonging to the species Enterococcus faecium and Enterococcus faecalis (Batish et al. 1984), and several authors have included this genus as a food-borne agent of disease (Grag and Mital 1991). Among all the enterococcal species, E. faecalis and E. faecium are the most important in human infection (Facklam et al. 2002).
The ability to detect the presence of a specific and exclusive enzyme using suitable substrates, in particular fluorogenic and chromogenic enzyme substrates, has led to the development of many methods for the presumptive identification of micro-organisms in their primary isolation media. The incorporation of such substrates into a selective medium may dispense with the need for subculture and further biochemical tests to establish the identity of certain micro-organisms.
The recent introduction of many of these media has led to improved accuracy and faster detection of target micro-organisms (Manafi 1996). In this sense, a new selective and differential medium (Chromocult® enterococci agar; Merck, Darmstadt, Germany) has been designed by Merck for the isolation and enumeration of Enterococcus spp., using a chromogenic mix in a selective agar. Enterococci cleave chromogenic substrates in this medium. Owing to the β-d-glucosidase activity present in enterococci, the chromogenic mix is cleaved and the red colour of the colonies indicates the presence of enterococci. Other β-d-glucosidase-producing organisms are suppressed by the sodium azide content of the media (Manafi 2000), sodium azide being an inhibitor of enzyme systems (catalase, cytochrome c oxidase) in electron transport (Hartman et al. 1966). Although many studies designed to test different chromogenic media have been carried out in recent years (Manafi 2000; Finney et al. 2003; Cárdenes et al. 2004), to our knowledge little work has been done concerning the evaluation and comparison of Chromocult® enterococci agar in highly contaminated samples. The purpose of the present work was to test the use of Chromocult® enterococci agar medium as compared with MRS medium, a nonspecific lactic bacteria medium in current use (De Man et al. 1960; Doming et al. 2003), using faecal samples in order to evaluate its use as an appropriate alternative medium in the processing of highly contaminated samples.
Material and methods
Chromocult® enterococci agar was prepared by suspending 33·0 g of medium in 1 l of distilled water and heating in a boiling water bath. The contents were stirred to aid dissolution for about 45 min, after which the medium was allowed to cool to 45–50°C and was then poured into plates, following the manufacturer's instructions. Chromocult® enterococci agar is composed of (g l−1): peptones 10·0; sodium chloride 5·0; sodium azide 0·2; dipotassium hydrogenphosphate 3·4; potassium dihydrogenphosphate 1·6; ox bile 0·5; Tween®-80 1·0; chomogenic mixture 0·25; Agar-agar 11·0.
The MRS agar was prepared by suspending 66·2 g in 1 l of distilled water and autoclaving for 15 min at 118°C. The medium was allowed to cool to 45–50°C, and used before the surface of the plates had dried, following the manufacturer's instructions. MRS agar is composed of (g l−1): peptone from casein 10·0; meat extract 8·0; yeast extract 4·0; d(+)-glucose 20·0; dipotassium hydrogen phosphate 2·0; Tween®-80 1·0; diammonium hydrogen citrate 2·0; sodium acetate 5·0; magnesium sulfate 0·2; manganese sulfate 0·04; Agar-agar 14·0. Agar medium types were prepared by the same research assistant throughout the study.
Sample preparation and processing
A total of 216 faecal samples were obtained from 18 different batches of six broilers aged 6–10 weeks, using the same feed for all birds. Faecal samples were obtained by swabbing the cloacae of the birds with sterile swabs. A minimum of 1 g of faeces was obtained from each bird and placed aseptically in a sterile tube. Samples were carried to the laboratory in an ice chest in less than half an hour after collection for immediate processing. Samples from three animals were placed in a sterile masticator bag with an appropriate volume (1 : 10) in sterile buffered peptone water (Merck) and subsequently homogenized with a masticator (Aes, Combourg, France) for a maximum of 1 min. After homogenization, 0·1 ml of appropriate dilutions of the homogenates from the masticator bags were surface-plated in triplicate onto Chromocult® enterococci agar and MRS agar plates. Chromocult® plates were incubated at 37 ± 1°C for 48 ± 2 h and MRS agar plates were incubated at 35 ± 1°C for 72 ± 2 h, as specified by the manufacturer. The sampling and processing procedures of poultry faeces described in this paper were always carried out by the same laboratory personnel.
Enterococcus enumeration and identification
Red colonies in Chromocult® enterococci agar with a diameter of 0·5–2 mm were considered to belong to Enterococcus spp. The occasional non-enterococcal strains developing on the medium were colourless, blue, violet, turquoise or green. Circular white colonies with varying diameters were considered to be Enterococcus spp. in MRS agar. Enterococcus faecalis ATCC 29212 was used as control of colony morphology in both media. Only plates containing 20 to 250 colonies were counted. The numbers of Enterococcus spp. were converted to log10 values and expressed as log10 CFU g−1 faeces, after calculating the enterococci population obtained from triplicate assays for each culture media. After incubation, presumptive enterococci colonies were harvested, transferred onto Columbia agar with 5% sheep blood (Biomerieux, Marcy L'Etoile, France), and incubated inverted at 37 ± 1°C for 48 ± 2 h to obtain pure cultures.
One hundred pure cultures of presumptive enterococcus colonies from each medium were characterized by colony and cell morphology, their haemolytic character, Gram stain, and oxidase and catalase activity. Following this preliminary identification, pure cultures were subcultured in Columbia agar with 5% sheep blood, harvested, and used to inoculate galleries of API 20 Strep (Biomerieux) (Appleman et al. 2004) for identification purposes. Readings were taken after 4 and 24 h, as specified by the manufacturer. In the identifications with a reliability of <95%, API 50 CH (Biomerieux) was also used to complete bacterial identification. Readings were taken after 24 and 48 h, as specified by the manufacturer. Data for the API galleries were collected using the Apilab Plus software® (Biomerieux).
A paired Student's t-test was used to compare the overall mean values in both media. The chi-square test was used to compare the different percentages of appearance of the enterococci species in each medium. Differences were considered significant when probabilities were lower than 0·01. All statistical analyses were carried out using Statview version 5.0.1 (SAS Institute, Cary, NC, USA).
Enterococcus spp. colonies appearing on Chromocult® enterococci agar were small (1–1·5 mm diameter), their diameters being slightly larger than those specified by the manufacturer and bright red in colour. These colonies could be readily distinguished from other apparent contaminants. No contaminating colonies with colours related to red were detected, and only in two or three cases were some small pink colonies observed.
One hundred red colonies were randomly selected as described above from Chromocult® agar, as well as another 100 white colonies with varying diameters for the case of MRS agar. The results (Table 1) revealed that 98% (98/100) of the isolates from Chromocult® were Enterococcus spp., and 2% (2/100) were false-positive red colonies on Chromocult® enterococci agar and were always identified as belonging to the genus Leuconostoc. Of all the colonies isolated from MRS agar, 49% (49/100) were Enterococcus spp. There were significant differences between the isolation rates found for each species of Enterococcus in both media (P < 0·01).
Table 1. Distribution and species identities of Enterococcus spp. isolates in Chromocult® agar and MRS agar
|Enterococcus faecalis||65 (66·33)||21 (42·86)|
|Enterococcus faecium||3 (3·06)||18 (36·74)|
|Enterococcus durans/hirae||26 (26·53)||2 (4·08)|
|Enterococcus gallinarum||2 (2·04)||0 (0)|
|Enterococcus casseliflavus||1 (1·02)||4 (8·16)|
|Enterococcus avium||1 (1·02)||4 (8·16)|
From the above results, if it is considered that 98% of the red colonies obtained on Chromocult® enterococci agar were indeed enterococci as well as 49% of the colonies counted in MRS agar, these percentages can be applied to the counts obtained with the 216 bird faecal samples, the results concerning the enterococci populations being shown in Table 2. Thus, the true enterococci recovery values obtained using this correction were significantly greater in MRS agar than the values obtained in Chromocult® enterococci agar (P < 0·01).
Table 2. Estimation of Enterococcus populations in bird faeces obtained with Chromocult® enterococci agar and MRS agar from the specificity determined for each of the two culture media
|Max value (log CFU g−1)||8·0||8·8|
|Min value (log CFU g−1)||4·87||5·17|
Target bacterial colonies obtained from Chromocult® enterococci agar are more easily distinguished than those obtained from other chromogenic media used, mainly those used for the isolation of Salmonella, which shows a higher number of contaminating colonies with colours similar to those produced by the bacteria for which the medium was designed (Dush and Altwegg 1995). This is probably due to the presence in Chromocult® enterococci agar of sodium azide, a powerful inhibitory agent for Gram-negative bacteria as well as for catalase-positive organisms, while in the case of Salmonella, most current chromogenic media do not have such potent selective agents in their formulation.
The results of this study indicate that the specificity of Chromocult® enterococi agar (98%) is good and similar to the best chromogenic media (Manafi 2000). However, the isolation rate for E. faecium was significantly higher for MRS agar than that obtained with Chromocult® enterococci agar. This is important because Enterococcus spp. are the third most common cause of hospital-acquired bacteraemia (Garnier et al. 2000) and E. faecium is the species that has the broadest spectrum of natural and acquired antibiotic resistance (Klare et al. 2003). Therefore, this must be taken into consideration when using this medium for clinical purposes or when performing specific research on E. faecium. Nevertheless, E. faecalis and Enterococcus durans/hirae isolation rates were significantly (P < 0·01) higher in Chromocult® enterococci agar than in MRS.
According to the literature consulted, although the species distribution of the genus Enterococcus in faecal samples from poultry shows important differences, depending on the country where the study was performed or on the authors of the individual work, the species distribution found for MRS in the present work can be said to be more related to the data reported in the literature (Devriese et al. 1991; Butaye et al. 1999; Kühn et al. 2003) than in the case of Chromocult® enterococci agar. Careful scrutiny of these data from the literature reveals that E. faecium isolation rates among enterococci must probably be higher than the 3·06% found for Chromocult® enterococci agar in the present study. Thus, the latter authors reported E. faecium isolation rates in chicken faeces of 37·6, 42 and 15% respectively.
Total enterococci counts obtained from MRS were more in accordance with the reports of other authors such as Netherwood et al. (1999) than those obtained from Chromocult® enterococci agar. To date, there are no data available concerning the use of Chromocult® enterococci agar to process highly contaminated samples, although Manafi and Windhager (1997) have reported that the use of this medium could be a good alternative for the enumeration of enterococci in water samples. According to the present findings, although Chromocult® enterococci agar is an interesting alternative for routine purposes owing to the fact that 98% of the red colonies were finally identified as enterococci, it is necessary to consider that perhaps the whole of the Enterococcus spp. population might not be estimated in very contaminated samples. The lower Enterococcus counts obtained in Chromocult® enterococci agar in comparison with the counts obtained with MRS could derive from the more reduced development of E. faecium observed in Chromocult® enterococci agar.
Special appreciation is extended to Encarnación González and Begoña Sierra for technical assistance.