Rapid molecular identification of fecal origin-colonies growing on Enterococcus spp.-specific culture methods

In the Province of Québec (Canada), the Programme d'accréditation des laboratoires d'analyse (‘Accreditation program of analytic laboratories’; PALA) is administered by the Centre d'expertise en analyse environnementale du Québec (CEAEQ) which accredits private, municipal and institutional laboratories. The Regulation respecting the quality of drinking water (RRQDW) states that when the water supplied by a distribution system comes in whole or in part from nondisinfected and vulnerable groundwater, the person in charge of the distribution system is bound to control the presence of Escherichia coli, enterococci, and coliphage viruses (Government of Québec 2013). Consequently, a water sample is considered contaminated by fecal pollution if at least one colony of enterococci bacteria is detected. The presence of enterococci in water is also considered by the United States Environmental Protection Agency (USEPA) as an indication of fecal pollution and of the possible presence of enteric pathogens, although some enterococcal species are naturally found in the environment and not necessarily related to fecal pollution (Kjellander 1960; Cabelli et al. 1982; Franz et al. 1999; USEPA 2005).

In fact, detecting Enterococcus spp. is of limited significance for determining the source of contamination in water since the broad spectrum of species cannot be used to distinguish non-fecal (environmental) from fecal contamination (Bonds et al. 2006; Converse et al. 2009). Indeed, there are many possible sources of Enterococcus sp. in water including animal waste (Devriese & De Plesmaecker 1987; Devriese et al. 1991; Sinton et al. 1993; Harwood et al. 2001), soil (Fujioka et al. 1999), invertebrates (Martin & Mundt 1972; Svec et al. 2002), and plants (Müller et al. 2001).

Thus, water quality assessment should more focus on a group of Enterococcus sp. that is associated with sources of fecal pollution rather than relying on the entire Enterococcus genus. Enterococcus faecalis and Enterococcus faecium are the predominant species of the Enterococcus genus found in human feces (Ruoff et al. 1990). All mammals carry these microorganisms in the colon (Noble 1978). Consequently, E. faecalis and E. faecium are potentially good fecal species as they have been consistently identified as predominant enterococcal species in warm-blooded animal feces and sewage, but not from environmental sources (Chenoweth & Schaberg 1990; Ruoff et al. 1990; Manero et al. 2002; Gelsomino et al. 2003). Furthermore, since Escherichia coli is 100 to 1,000 times more concentrated than Enterococcus spp. in feces, the probability of detecting non-faecalis/faecium Enterococcus species in water without any detection of E. coli nor E. faecalis/E. faecium is highly improbable (Slanetz & Bartley 1957; Layton et al. 2010). Thus, a detection method that would allow the specific detection of E. faecalis/E. faecium cells rather than all Enterococcus spp. should be more appropriate to assess water quality by the detection of fecal contamination.

Currently, enterococci in water are detected by different chromogenic culture-based methods comprising USEPA method 1600 on mEI agar (USEPA 2005), Chromocult^® enterococci agar, and method 9230C of the Standard Methods for the Examination of Water and Wastewater manual (membrane filtration on m-Enterococcus agar; American Public Health Association/American Water Works Association/Water Environment Federation (APHA/AWWA/WEF 2012)). However, these methods do not discriminate between enterococci of environmental origin and enterococci of fecal origin.

In this study, we first compared mEI agar, Chromocult^® enterococci agar, and m-Enterococcus agar, in terms of sensitivity, using a panel of different types of strains of enterococci and harvested environmental colonies identified by 16S rRNA sequencing. Secondly, two specific real-time polymerase chain reaction (rtPCR) assays targeting Enterococcus spp. and Enterococcus faecalis/faecium were tested for their ability to confirm, in less than 2 hours, the identity of putative enterococcal colonies grown on chromogenic culture-based agar in order to decrease the high false-positive rate obtained when the entire Enterococcus genus is targeted.

The ability of mEI, Chromocult^® enterococci, and m-Enterococcus agar as well as Enterococcus spp.- and Enterococcus faecalis/faecium-specific rtPCR assays to detect enterococci strains was verified using 41 (for culture-based methods evaluation) and 55 (for rtPCR validation) different strains of Enterococcus spp. (Tables 1 and 2). Species identification was reconfirmed using an automated MicroScan Autoscan-4 system (Siemens Healthcare Diagnostic Inc., Newark, DE, USA) or a Vitek 2 system (bioMérieux SA, Marcy l’Étoile, France). Bacterial strains were grown from frozen stock kept at −80 °C in Brucella medium (Beckton Dickinson, Mississauga, Ontario, Canada), containing 10% glycerol. The strains were cultured on Brain Heart Infusion (BHI) agar. Three passages were performed prior to analysis of each strain with each culture-based method.

Colonies obtained from frozen stocks were suspended in BHI broth and adjusted to a 0.5 McFarland standard (Fisher Scientific Company, Ottawa, Ontario, Canada) before being serially diluted 10-fold in phosphate-buffered saline (PBS; 137 mM NaCl, 6.4 mM Na₂HPO₄, 2.7 mM KCl, 0.88 mM KH₂PO₄, pH 7.4). For each strain, an aliquot of the 10⁻⁵ dilution was spiked in sterile reverse osmosis-purified water (resistivity of 18 MΩ-cm min at 25 °C) to produce suspensions containing approximately 10² colony forming units (CFU) per 100 mL of water. Bacterial counts were verified by filtering three 100 mL volumes of each spiked water sample through Millipore membrane filters (47 mm diameter, 0.45 μm pore size; Millipore Corporation, Billerica, MA, USA) with a standard platform manifold (Millipore Corporation) followed by an incubation on BHI agar for 24 ± 2 h at 35.0 ± 0.5 °C. Tests to confirm the sterility of filter membranes and buffer used for rinsing the filtration apparatus were also performed.

Membrane filtration was performed according to Maheux et al. (2009). Volumes (300 mL) spiked with reference enterococcal bacteria were split into three 100 mL volumes and filtered on Millipore filters with a standard platform manifold. The first filter was incubated on mEI agar (BD, Franklin Lakes, NJ, USA), the second filter on Chromocult^® enterococci agar (Merk KGaA, Darmstadt, Germany) and both were incubated for 24 ± 2 h at 35.0 ± 0.5 °C. The third filter was incubated on m-Enterococcus agar plates (BD Company, Franklin Lakes, NJ, USA) for 48 ± 3 h at 35.0 ± 0.5 °C before determining colony counts and color (Table 1). Each preparation of mEI, Chromocult^® enterococci, and m-Enterococcus was tested for performance using pure cultures of target and non-target microorganisms, as recommended by the USEPA microbiology methods manual. Tests were also performed to confirm the sterility of the filter membranes and buffer used for rinsing the filtration apparatus (APHA/AWWA/WEF 2012).

The sewage water sample used in this study to test environmental colonies was harvested at the discharge of the grit chambers of the west wastewater treatment plant of Québec City, in December 2014. Three 2 μL volumes of sewage water were spiked in 100 mL sterile water and filtered on Millipore filters with a standard platform manifold. The filters were then incubated on mEI agar, Chromocult^® enterococci agar, and m-Enterococcus agar as described in the above section ‘Membrane filtration method’.

Each environmental colony recovered on mEI agar, Chromocult^® enterococci agar, and m-Enterococcus agar was touched with a sterile toothpick and resuspended in 100 μL of sterile reverse osmosis-purified water (resistivity of 18 MΩ-cm min at 25 °C). This suspension was used for rtPCR tests described below.

The identity of the environmental colonies isolated on mEI, Chromocult^® enterococci, and m-Enterococcus plates was confirmed by nucleotide sequencing of 16S rRNA gene using amplification and sequencing primers, SSU27 and SSU534R, an adaptation of Sistek et al. (2012). The SSU534R PCR primer, designed for this study, was developed as follows. First, 16S rRNA gene sequences available from public databases were analyzed with GCG programs (version 8.0; Accelrys, Madison, WI, USA). Based on a multiple sequence alignment and the Oligo primer analysis software (version 5.0; National Biosciences, Plymouth, MN, USA), the SSU534R PCR primer was designed from highly conserved regions of the gene. Primers and probes for Enterococcus spp.- and E. faecalis/faecium-specific rtPCR assays were used as described by Maheux et al. (2011). Sequences of the PCR primers are presented in Table 3. Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA).

For sequencing of the 16S rRNA gene for genotypic identification, 1 μL of each bacterial suspension was transferred directly to 49 μL of PCR mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl2, 0.4 mM of each primer, 200 mM each of deoxyribonucleoside triphosphate (GE Healthcare Bio-Sciences Inc., Baie d'Urfé, Québec, Canada), 3.3 mg per mL of bovine serum albumin (BSA; Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada), 0.06 μg/μL methoxalen (Sigma-Aldrich Canada Ltd), 0.5 enzyme unit (U) of Taq DNA polymerase (Promega, Madison, WI, USA) and TaqStart antibody (Clonetech Laboratories, Mountain View, CA, USA). Decontamination of the PCR mixtures prior to PCR was achieved using the UV crosslinker Spectrolinker™ model XL-1000 (Spectronics Corporation, Westbury, NY, USA; Maheux et al. 2008). For each experiment, 1 μL of sterile water was added to the PCR mixture as a negative control. The PCR mixtures were subjected to thermal cycling (3 min at 95 °C and then 40 cycles of 1 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, with a 5-min final extension step at 72 °C) with a Mastercycler PRO (Eppendorf Canada Ltd, Mississauga, ON, Canada). An agarose gel analysis of the amplified PCR products was performed as previously described (Martineau et al. 1998).

Sequencing of the 16S rRNA gene was performed as described by Picard et al. (2004). Molecular analysis of sequences was conducted using NCBI Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi, Standard Nucleotide BLAST optimized for highly similar sequence) and the Ribosomal database project (http://rdp.cme.msu.edu/, isolates with good quality sequence in Sequence Match search).

For the validation of the Enterococcus spp. and the E. faecalis/faecium rtPCR assays and or confirmation of colony identity, 1 μL of each bacterial suspension was transferred directly to a 24 μL PCR mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM of Enterococcus spp. or E. faecalis/faecium, primers, 0.2 μM of Enterococcus spp. or E. faecalis/faecium probe, 200 μM each deoxyribonucleoside triphosphate (GE Healthcare Bio-Sciences Inc., Baie d'Urfé, Québec, Canada), 3.3 μg per μL of bovine serum albumin (BSA; Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada), 0.025 enzyme unit (U) of Taq DNA polymerase (Promega, Madison, WI, USA), and TaqStart antibody (Clontech Laboratories, Mountain View, CA, USA). For each experiment, 1 μL of sterile water was added to the rtPCR mixture as negative control. The rtPCR mixtures were subjected to thermal cycling (1 min at 95 °C and then 45 cycles of 15 s at 95 °C and 60 s at 60 °C for Enterococcus spp. rtPCR assay; 1 min at 95 °C and then 45 cycles of 15 s at 95 °C, 10 s at 60 °C and 20 s at 72 °C for E. faecalis/faecium rtPCR assay) with a Rotor-Gene Thermocycler (QIAGEN Inc., Mississauga, Ontario, Canada).

The specificity of the mEI, Chromocult^® enterococci, and m-Enterococcus agar methods was demonstrated by testing genomic DNA isolated from 41 enterococcal strains from different species and origin (Table 1). The mEI agar method detected 68.3% (28/41) of the 41 enterococcal strains tested in which only 75% (3/4) of E. faecalis and 66.7% (2/3) E. faecium tested were detected. A positive signal on mEI agar was also detected for Enterococcus caccae, Enterococcus canintestini, Enterococcus canis, Enterococcus casseliflavus, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus mundtii, Enterococcus pallens, Enterococcus phoeniculicoli, Enterococcus quebecensis, Enterococcus sileciacus, Enterococcus sulfureus, Enterococcus termitis, and Enterococcus villorum. These results showed that the recovery of E. faecalis and E. faecium cells could not be optimal with this medium. Furthermore, strictly environmental Enterococcus spp. (E. mundtii and E. sulfureus seem to be strictly associated to plants and soil (Müller et al. 2001)) are also detected by the mEI agar method suggesting that colonies obtained from water samples should be identified to confirm a contamination of fecal origin.

The Chromocult^® enterococci agar method detected 78.0% (32/41) of the 41 enterococcal strains tested in which all (100%) of E. faecalis and E. faecium tested were detected. The only enterococcal species not detected by Chromocult^® enterococci method were E. aquimarinus, E. avium, E. columbae, E. dispar, E. italicus, E. pseudoavium, E. raffinosus, E. ratti, and E. sulfureus. These results showed that the Chromocult^® enterococci method could be a good choice to assess water quality since all E. faecalis and E. faecium tested were detected. However, as for the mEI agar method, strictly environmental Enterococcus spp. are also detected suggesting that colonies obtained from water samples should be identified to confirm a contamination of fecal origin.

Finally, the m-Enterococcus agar method detected only 29.3% (12/41) of the 41 enterococcal strains tested in which all (100%) of E. faecalis but only 33% (1/3) of E. faecium tested were detected. With the exception of E. faecalis and E. faecium, the spectrum of detection of m-Enterococcus agar method was only E. casseliflavus, E. gallinarum, and E. hirae when colonies were subjected to phenotypical confirmation tests (CEAEQ 2014). However, in order to bypass those phenotypic confirmations tests, colonies harvested on m-Enterococcus agar from sewage water described below were only subjected to confirmation using rtPCR assays. These results showed that the recovery of E. faecium cells could not be optimal with this medium.

In traditional water quality assessment, the definition of Enterococcus sp. is phenotypical. Indeed, an Enterococcus is defined as spherical bacterium, in pair or chain, Gram-positive, catalase-negative and facultatively anaerobic. It does not form endospores and some species demonstrate mobility. In addition, an Enterococcus hydrolyzes esculin in the presence of bile and has the ability to grow at 10 °C and 45 °C, pH 9.6 or in the presence of NaCl 6.5% (Mundt 1986; Knudtson & Hartman 1992; APHA/AWWA/WEF 2012). However, since the use of genotypic classification, it has been shown some species of the Enterococcus genus do not express β-glucosidase at 35 °C after 24–48 hours on chromogenic media (Maheux et al. 2008; Sistek et al. 2012). However, that does not mean that they do not express the enzyme at all. But using those culture conditions, the expression of β-glucosidase is not detected.

The ability of Enterococcus spp.- and Enterococcus faecalis/faecium-specific real-time PCR assays to detect enterococcal strains was demonstrated by testing genomic DNA isolated from 55 enterococcal strains including 12 E. faecalis and 5 E. faecium strains (Table 2). The Enterococcus sp.-specific rtPCR primers and probe efficiently amplified DNA from all 55 enterococcal strains tested whereas the multiplexed E. faecalis/faecium rtPCR assay efficiently amplified DNA from 12 of 12 (100%) E. faecalis and 5 of 5 (100%) E. faecium strains tested, respectively. Thus, against all enterococcal strains, the Enterococcus sp. rtPCR assay is 100% sensitive in its ability to detect all enterococcal strains, whereas the multiplex E. faecalis/faecium rtPCR assay is 100% sensitive for the detection of E. faecalis and E. faecium. Maheux et al. (2011) tested 150 closely related non-enterococcal species among the Enterococcus spp.-specific rtPCR assay and showed that there was no specific amplification of the 150 non-enterococcal bacterial species with the exception of Tetragenococcus solitarius. Phylogenetically, T. solitarius is very closely related to enterococci (Ke et al. 1999; Ennahar & Cai 2005) and controversy in its taxonomical classification persists.

Three 2 μL volumes of a 1-L sewage water sample harvested at the discharge of the grit chambers of the west wastewater treatment plant of Québec City were used for testing by mEI agar, Chromocult^® enterococci agar, and m-Enterococcus agar methods to verify their comparative ability to detect Enterococcus spp. (Table 4). All the colonies were harvested and subjected to molecular identification using a 527 base pairs fragment of the 16S rRNA gene. On mEI agar, 23 blue (phenotype +) and 14 pink (phenotype −) colonies were harvested for a total of 37 colonies. On Chromocult^® enterococci and m-Enterococcus agar, 40 pink (phenotype +) and 49 red/pink (phenotype +) colonies were harvested, respectively. All these colonies were harvested and subjected to molecular identification using the 16S rRNA gene. Molecular analysis showed that 1 blue (1/23) colony on mEI agar and 2 pink (2/40) colonies on Chromocult^® enterococci agar were not enterococcal species for a false positive rate of 4.3% and 5.0%, respectively. No false positive results have been found on m-Enterococcus agar. On the contrary, 2 pink (2/12) colonies on mEI agar were enterococcal species for a false negative rate of 16.7%. By using 16S rRNA molecular identification as gold standard, m-Enterococcus agar method has presented the highest specificity (100%), sensitivity (100%), and positive predictive value (100%; Table 4) for the detection of Enterococcus spp. Finally, Chromocult^® enterococci agar and m-Enterococcus agar detected 38 and 39 Enterococcus spp. colonies respectively, whereas mEI agar detected only 24 Enterococcus spp. colonies.

After colonies were identified using 16S rRNA analysis, the population of total coliforms enumerated by mEI agar, Chromocult^® enterococci agar, and m-Enterococcus agar, were classified per species (Table 5). Results showed that the population of enterococcal species detected was more extended for m-Enterococcus agar (7 different species) than mEI and Chromocult^® coliform agar (only 3 and 4 different species, respectively; Table 5). Like the Maheux et al. (2009) study, the results of the present study showed the lack of correlation between test methods based on the same enzymatic principle to recognize a strain as Enterococcus spp. Indeed, our results showed that there is a weak correlation between the three methods tested within the same species. Since all colonies of the present study were isolated from the same water sample and treated in the same way (filtration, incubation, etc.), the difference observed in the population of strains detected by each method cannot just be attributed to environmental factors. The composition of each medium is also involved. As observed in the analytical sensitivity section above, the mEI agar method detected less E. faecalis than the two other methods. Furthermore, m-Enterococcus detected less E. faecium than the two other methods. Consequently, the Chromocult^® enterococci method seems to be the best method among the three tested to recover E. faecalis and E. faecium cells.

It is impossible to exclude that an E. faecalis or an E. faecium detected by a culture-based method was naturally found in the environment. However, some non-faecalis and non-faecium Enterococcus species are not present in feces, but are naturally and highly present in the environment. By detecting these non-fecal Enterococcus species, we cause false-positive results that could be avoided by only detecting the Enterococcus species mostly found in feces rather than relying on the entire Enterococcus genus. Furthermore, since Escherichia coli is 100 to 1,000 times more concentrated than Enterococcus spp. in feces, the probability of detecting non-E. faecalis/E. faecium in water without any detection of E. coli nor E. faecalis/E. faecium is highly improbable (Slanetz & Bartley 1957; Layton et al. 2010). Thus, the sole detection of E. faecalis and E. faecium in water could decrease the false-positive rate obtained with the detection of the entire Enterococcus genus and by this, improve the water quality assessment.

In the present study, colonies harvested on mEI agar, Chromocult^® enterococci agar, and m-Enterococcus agar were identified using two specific-rtPCR assays targeting Enterococcus spp. and Enterococcus faecalis/faecium to confirm, in less than 2 hours, their identity. A positive rtPCR signal obtained with the Enterococcus faecalis/faecium-specific rtPCR assay allows the discrimination between fecal and environmental contamination of water samples.

All colonies harvested, for the present study, on mEI, Chromocult^® enterococci, and m-Enterococcus agar were subjected to Enterococcus spp.- and Enterococcus faecalis/faecium-specific rtPCR assays (Table 6). Both of the rtPCR assays showed a specificity of 100% on the three media tested. Only two (2/19) colonies of E. faecium were not detected by the Enterococcus faecalis/faecium-specific rtPCR assay on mEI agar for a sensitivity of 89.5%. These results showed that the Enterococcus faecalis/faecium-specific rtPCR assay could be used with success to discriminate between fecal and environmental contamination of water samples when testing on colonies isolated on mEI, Chromocult^® enterococci, or m-Enterococcus agar.

The results obtained in the present study were obtained using sewage water samples. Results could differ with other types of water.

The sensitivity evaluation of the three culture-based methods tested using both reference strains and environmental strains identified by 16S rRNA gene sequencing, as well as the validation of the Enterococcus spp.- and Enterococcus faecalis/faecium-specific rtPCR assays showed that the detection and identification of enterococcal colonies on Chromocult^® enterococci agar combined with Enterococcus faecalis/faecium rtPCR assay presents the best combination to decrease the high false-positive rate obtained when the entire Enterococcus genus is targeted.

We wish to thank Dr Steve Charette (IBIS; Institut de Biologie Intégrative et des Systèmes, Université Laval, Québec City (Québec), Canada) for providing laboratory space and equipment as well as Philippe Cantin of the MDDELCC for providing sewage water and plates. This research was supported by AFM Water Consulting.