Evaluation of an alternative chromogenic method for the detection and enumeration of enterococci in waters

1 Departamento de investigaciones de medios de cultivo, Centro Nacional de Biopreparados (BioCen), Carretera a Beltrán, Km 1 1⁄2, Bejucal, Mayabeque, Cuba, CP. 32600. 2 Centro de Investigaciones Pesqueras (CIP), Ave 246 y 5ta avenida, Santa Fe, Playa, Ciudad Habana, Cuba. 3 Laboratorio de Bacteriología Médica, Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas. Instituto Politécnico Nacional. Prolongación de Carpio y Plan de Ayala, S/N. Col. Santo Tomás. Delegación Miguel Hidalgo. CP. 11340. México, DF., México.


INTRODUCTION
The quality of water is routinely monitored to protect human from fecal contamination, using indicator bacteria, such as total coliforms, fecal coliforms, Escherichia coli and enterococci.The bacterial genus Enterococcus is traditionally considered as hygiene indicator due to its ability to persist longer in the environment than other bacteria.Its detection methods are essential elements of safe drinking water regulations worldwide (Frahm and Obst, 2003).Moreover, several studies suggest that bacteria of this genus can be more effective indicators than E. coli and fecal coliforms to assess sanitary water quality (Pepe et al., 2011).
Two procedures are currently used for the detection of such microorganisms in water, the most probable number (MPN) method and the membrane filtration (MF) technique (Volterra et al., 1985).Both include conventional culture media in which the identification of the microorganism is based on its physiological characteristics taking a long time to provide results (Domig et al., 2003).In the last few years, several chromogenic and fluorogenic culture media have been developed as alternative methods for such purposes.These media contain multiple substrates that allow bacteria to form colored colonies depending on their enzyme activity, precluding the need of subsequent cultivation steps and additional biochemical tests to establish the identity of the isolated microorganisms.Furthermore, the chromogenic and fluorogenic culture media also enhance the discrimination of different species in mixed cultures, which provides better sensitivity when compared with conventional culture media (Orenga et al., 2009).
In addition, enterococci are considered important nosocomial pathogens (Hajia et al., 2012).A variety of chromogenic media have been developed for the detection of enterococci in clinical samples (Gander et al., 2013).
The aim of this study was to assess the suitability of a new alternative chromogenic method for the detection and enumeration of enterococci in water samples by the MF technique, in comparison with the ISO 7899:2 method (ISO 7899:2, 2000).

Samples
A total of 135 water samples from different sources were examined with the MF technique from March 2007 to March 2012: 59 industrial water samples (chlorinated), 43 drinking water (chlorinated and coming from an aqueduct), and 33 recreational waters (swimming pools).All samples were collected using sterile bottles, chilled for transportation, and processed within 6 h after collection.

Culture media
The m-CromoCen ENT chromogenic medium (BioCen, Cuba) was used as an alternative method, Slanetz-Bartley (SB) agar (Oxoid, Ltd, Basingstoke, UK) was used as a presumptive reference medium, and bile-esculin azide (BAA) agar (Merck, Darmstadt, Germany) served as confirmatory reference medium.All the media were prepared according to manufacturer's instructions.The composition of the m-CromoCen ENT is shown in Table 1.

Microbiological analysis
A volume of 100 ml of each water sample was filtered through a sterile nitrocellulose membrane (47-mm diameter and 0.45-µm pore size, Sartorius, Gottingen, Germany).The filter was rinsed before and after each filtration with 40 ml of a sterile phosphate buffered solution.The membranes were incubated on plates of m-CromoCen ENT medium during 24 h at 35 ± 2°C.Later, all colonies from light to dark pink color were considered typical.This procedure was compared with ISO 7899-2:2000 for water quality (ISO 7899:2, 2000).
After the corresponding incubation time, enterococcal colonies in each culture medium were enumerated.The results were expressed as colony forming units (cfu) per 100 ml.
Inclusivity and exclusivity tests were performed.For the inclusivity study, 44 strains of target microorganisms were evaluated, including 6 ATCC strains and 38 clinical isolates from hospitals.For the exclusivity assay, 43 strains of non-target microorganisms were tested, mainly Gram-negative bacteria; of them, 26 ATCC strains and clinical isolates.In addition, some representative yeasts strains and Gram-positive bacteria were included in the test.The purity of all clinical isolated microorganisms used in this study was previously confirmed by biochemical tests.

Statistical analysis
The statistical analysis (orthogonal regression), and calculation of the other parameters, such as selectivity (F), efficiency (E), limit of quantification (LOQ) and limit of detection (LOD), were performed according to ISO 16140 (ISO 16140, 2003).Similarly, the ability of the alternative method to measure enterococci in all water matrices was evaluated.Data of enterococci colony counts of both methods were transformed to log10 to obtain a normal distribution of data and facilitate the statistical analysis.Results were analyzed using orthogonal regression to verify the linearity of the relationship between the results obtained with both the m-CromoCen ENT method and the ISO 7899-2:2000 reference.In samples where no growth was observed (absence level), logarithmic transformation was performed by taking the log of value 1; consequently, in the remainder samples, 1 cfu was added to each value of the count obtained and the sums were subjected to logarithmic transformation.The orthogonal regression analysis was undertaken to assess the relative accuracy between both methods.Three regression equations were independently calculated for each matrix and one for the overall matrices.
To determine the relative limit of detection, the water samples were spiked with E. faecalis ATCC 29212 at seven different concentrations and expressed as cfu/100 ml.Six subsamples of each level of contamination were filtered and the growth of enterococci with both methods was reported.The values were calculated by means of a regression analysis, taking into account the number of Petri dishes with growth and the overall number of inoculated plates in each level.To verify the hypothesis that both methods have the same level of detection a 2 × 2 contingency table was created and the exact Fisher test was applied.The analysis was also done according to the Nordval formula (NordVal, 2009).
Determination of LOQ was done using the same procedure used for LOD, but quantifying the number of colonies in each of the six plates of each level.LOQ was calculated for α = 5% and (1-β) = 50% (ISO 16140, 2003).

RESULTS
All strains tested in the inclusivity study exhibited expected typical colony characteristics on both, the alternative and the reference medium with a 100% value.Strains showed pink colonies on m-CromoCen ENT medium; red, maroon or pink colonies on Slanetz-Bartley agar and red or maroon colonies surrounded by a black or brown halo.
In the exclusivity study, most Gram-negative microorganisms and yeasts evaluated on m-CromoCen ENT were inhibited.Some non-target Gram-positive and negative microorganisms grew moderately, showing a different color from that exhibited by enterococci.In the reference method, the characteristic enterococcus colonies or the typical inhibition of non-target microorganisms was observed.
Out of 135 water samples analyzed, 31 showed no microbial growth (absence level) and 102 had enterococci isolated by both methods.Enterococci were isolated by only one method (reference method) in two of all samples.The results of the enumeration of enterococci obtained by the two methods were compared, being enterococci counts, in most of the samples, slightly higher by the alternative method.The range of enterococci counts of all water matrices obtained with alternative and reference methods is shown in Table 2.
Enterococci were isolated by both methods from different sources of natural water, even in samples properly chlorinated.Furthermore, in the artificially spiked samples, several enterococcal species were isolated.In general, most isolates on m-CromoCen ENT corresponded to E. faecalis (7705%), followed by Enterococcus faecium (385%), and Enterococcus solitarius (151%).Other Enterococcus species were isolated in minor proportion (< 1%).
The chromogenic method showed a selectivity of -0.05 and an efficiency of 98%.The percentage of enterococci that grew on m-CromoCen ENT as non-typical colonies did not exceed 10%.The rates of false-positive and falsenegative obtained were 0 and 1.92, respectively.
In all types of water analyzed, the regression analysis for enterococci showed strong correlation between both methods.In all water matrices studied, the intercept was close to zero and the slope value was close to 1, although m-CromoCen ENT gave slightly higher estimates than the reference, demonstrating a high degree of linearity (Figure 1).
The regression slope (b = 1.02) indicates a close relation of the alternative method with regard to the ISO 7899:2 method.The hypothesis verification for the regression showed that the calculated F was lower than the critical F; therefore, there was a linear relation (ISO 16140, 2003).
Concerning LOD, for the five levels studied, critical F was 2.66 and calculated F was 1.36.The relative limit of detection obtained was 0.3 cfu/100 ml.In the exact Fisher test performed to verify the hypothesis, a P = 054 indicates that both methods have the same level of detection.The results of the 2 × 2 contingency table, created to verify the hypothesis that both methods have the same level of detection, are shown in Table 3.
The relative detection limit calculated proved that the confidence intervals of the alternative method are equal or are included in those of the reference method.The detection limit attained was 0.75 cfu/100 ml.The LOQ of the alternative method was 4.08 cfu/100 ml, however the reference method showed a LOQ of 5.48 cfu/100 ml.The chromogenic method studied gave even positive results with as few as 4 cfu/100 ml of enterococci after 24 h.

DISCUSSION
There are many conventional culture methods to detect enterococci in water, but these traditional culture-based methods require a minimum of 24 h incubation period, followed by a confirmation procedure that can take up to several days, which compromises the ability to take the most appropriate and timely action.The need to have rapid detection methods has increased, especially in the water industry and in emergency situations (González et al., 2009).
The alternative method developed to monitor water quality, using enterococci as indicator of fecal pollution, is a chromogenic medium based on the detection of β-Dglucosidase activity present in enterococci (Perry et al., 2007).Several chromogenic and fluorogenic culture media have been described for enterococci detection using the same principle (Manafi, 2000).On m-CromoCen ENT agar, pink colonies allows for easier recognition of enterococci in the different water samples analyzed.
The substrate included in the medium is stable at 115°C for 15 min and thus can be added prior to sterilization, making the preparation of m-CromoCen ENT medium easier and faster.Importantly, with the alternative method only one medium and a rapid test for target bacteria confirmation were necessary.This full procedure requires only 24 h to produce results, in contrast with ISO 7899:2 that takes 48 h and uses two expensive culture media, containing sodium azide, an inhibitor highly harmful to health (Chang and Lamm, 2003;Demircan et al., 2011).Moreover, the chromogenic method can be used for the examination of several types of water samples, such as potable, industrial, and recreational waters, showing a good selectivity and a high efficiency.
The MF technique used in this study allowed the appropriate analysis of these water samples.This procedure has been the method of choice for the detection and enumeration of indicator bacteria in water for many years (Windle-Taylor and Burman, 1964), because it reduces the workload in laboratories when processing a large number of samples.In addition, several statistical studies have demonstrated that this technique is more accurate than the MPN method (APHA, 2012).
Given that enterococci are often associated with a diverse microbiota, selective media are needed for their isolation.Most of these media lack sufficient selectivity, which is necessary to distinguish clearly enterococci from the accompanying microbiota (Domig et al., 2003).In this case, the alternative method showed an appropriate selectivity in relation to the reference one.The value of this parameter was significantly higher than the acceptance value (-1) suggested by the ISO/TR 13843 standard (Messer and Dufour, 1998).The percentage of non-typical colonies, obtained on m-CromoCen ENT, indicates that the chromogenic medium was able to inhibit the accompanying microbiota and foster the growth of enterococci.The rates of false-positive and false-negative samples obtained are well below those reported for other chromogenic method for similar purposes (Messer and Dufour, 1998;Manafi, 2000).Some chromogenic media used for the isolation and preliminary identification of Streptococcus strains in clinical samples reported a high rate of false positives (Tibbs and Creighton, 2013).In addition, the efficiency achieved with the m-CromoCen ENT was slightly higher than that of the reference method (97%).Recent studies with Enterolert, another rapid quantitative detection method of enterococci in the waters, reported a selectivity of -0.3 and 97% of accuracy, therefore it is less selective and less accurate than the m-CromoCen ENT (IDEXX, 2010).
Inclusivity is defined as the ability of a medium to detect the target microorganism from a wide range of strains, being related to with the sensitivity of the analytical method (ISO 16140, 2003).In this study, the inclusivity assay demonstrated a good sensitivity of the chromogenic method to detect enterococci from water samples.In addition, the results of the exclusivity test showed the lack of interference from a relevant range of non-target strains with the alternative method, demonstrating a good specificity of the method.
The most prevalent species isolated from all water sources were E. faecalis followed by E. faecium.Previous studies have reported that E. faecalis and E. faecium are the most frequently enterococci identified in water (Bennani et al., 2012).The regression analysis for enterococci demonstrated a good correlation between both methods.The values of the intercept and the slope obtained for all water matrices studied verified a high degree of linearity.
A reason for higher counts obtained in m-CromoCen ENT was probably based on the ability of this medium to recover injured or stressed target microbial cells due to the absence of sodium azide and the inclusion of highly nutritive peptones and extracts in the formulation.The standard procedure medium includes this inhibitor and it may partially inhibit the growth of the target organisms, particularly if they are sub-lethally injured (Winter et al., 2012).Other chromogenic methods also have shown higher counts in comparison with conventional method (Wohlsen, 2011).
Another possible cause of this difference in recovery may be explained by the high incubation temperature recommended by the reference method that generally reduces the number of enterococci recovered; some strains do not grow or do not grow well at this temperature (Fisher and Phillips, 2009).Another inconvenience was observed when counting on colonies in the BAA agar: the esculin resulting complex spread throughout the medium when this compound was hydrolyzed by enterococci, thus creating difficulties in distinguishing target colonies within a mixed culture and interfering with the counting of enterococci colonies.Levin et al. (1975) reported that diffusion of the precipitate through this medium makes colony counting difficult, if not impossible, mainly when large numbers of colonies are present.
The graphs (Figure 1) illustrate that a greater dispersion of the counts was obtained at lower microbial concentration in all samples.Such findings can be explained by the distribution followed by microorganisms in a sample (Poisson distribution) (Niemelä, 1997).In random distribution, at lower levels, the probability of finding microorganisms in the sample is lower, in other words, the smaller the number of colonies in a sample the greater the uncertainty of measurements (ISO 13843, 2000).
In all cases, the regression lines suggest a strong correlation and an adequate relative accuracy between both methods.A previous work (Noble et al., 2010), where the Enterolert method was compared with the EPA 1600 method for enterococci enumeration in recreational waters, reported a correlation coefficient of r = 0.87 and a slope of 0.92, values similar to those reported in the present study.However, a previously published paper about the Enterolert method for the detection of enterococci in recreational waters reported a lower correlation coefficient (r = 068) (Eckner, 1998).Other rapid methods (PCR) that have been evaluated for the quantification of other bacteria in food reported correlation coefficient comparable to results reported in current experiments (Macé et al., 2013).
The detection limit attained was low but achieved detection of enterococci in most water samples.Another culture method, EPA 1600, claims a detection level of 2.3 cfu/100 ml, a value higher than that obtained in the present study (Maheux et al., 2011).One more study, in which the chromogenic medium m-EI agar was used for the detection and enumeration of enterococci in seawater reported a LOD of 1 cfu/100 ml (Yamahara et al., 2009).Another rapid enterococci detection method in water based on molecular biology has reported values above the detection limit of the present study; however, this technique requires several steps, increasing the overall cost (Maheux et al., 2011).Chromogenic media for detection of other pathogens in spiked potable water samples showed a detection limit (1-20 cfu/100 ml) (Al-Wasify et al., 2013).
The new alternative method improves the LOD with regard to other existing methods for this purpose and allows enumeration of target bacteria even at low enterococci concentrations in the water samples.
The LOQ of the alternative method was also lower than that of the reference method.The capacity to recover enterococci using the m-CromoCen ENT is slightly higher than that of the reference method.Other media used for the enumeration of other bacteria in water samples have yielded a limit quantification of 1 x 10 2 cfu/l, a value higher than that obtained with the medium tested herein (Fitipaldi et al., 2010).
The overall results of the comparative study of the new alternative chromogenic method showed that it is suitable for detecting and counting enterococci in water samples.

Figure 1 .
Figure 1.Scatter plot and orthogonal regression analysis results of log10 cfu per 100 ml of enterococci obtained by alternative versus reference methods for different water matrices: (A) Industrial water samples; (B) potable drinking water samples; (C) recreational water samples; (D) overall water samples.Regression is represented by the continuous line.

Table 2 .
Range of counts for enterococci by the studied level of water samples obtained with alternative and reference methods.

Table 3 .
Contingency table (2 × 2) built to verify the hypothesis that both methods have the same LOD.