Genus-Specific Identification of Enterococci by PCR Method

Cupáková ·. , M. Pospí‰ i lová, I . Koláãková, R. Karpí‰ková: Genus-Specific Identification of Enterococci by PCR Method. Acta Vet Brno 2005, 74: 633-637. The aim of this study was to verify the application of polymerase chain reaction method focused on the detection of genus-specific tuf-gene for the rapid identification of enterococci. The reaction was as duplex-PCR, when, apart from the detection of genus-specific tuf-gene (amplification product of 112 bp), the internal control for 16S rRNA gene (amplification product of 241 bp) was also inserted. Detection of tuf-gene was positive in Enterococcus spp. isolates from the collections of microorganisms (8 species) and in 283 field isolates from foods and food raw materials, drinking water and various kind of waste water, previously identified by phenotypic methods as enterococci of different species. PCR product of 112 bp was missing in 16 non-enterococci strains and in 14 field isolates thereafter identified as lactococci. The above PCR method can be recommended for the rapid identification of enterococci in the routine use. Enterococcus spp., polymerase chain reaction, tuf-gene The identification of enterococci, using conventional methods and biochemical tests based on their phenotypic characteristics, is complicated and time-consuming (Devriese et al. 1993). Not only in clinical, but also in food microbiology, the methods of molecular biology focused on the genus or species identification of enterococci (Dutka-Malen et al. 1995; Ke et al. 1999), intra-generic typing of enterococci isolated from foods (Suzzi et al 2000; Andrighet to et al. 2001) or faecal samples and rectal swabs of farm and pet animals (Devriese et al. 1996), begin to be applied in the sample examination. Ke et al. (1999) have developed a PCR method for rapid genus-specific identification of enterococci, which is based on the detection of tuf-gene encoding the elongation factor Tu (EF-Tu). EF-Tu is a GTP-binding protein playing a central role in the protein synthesis. In the bacterial genome, there is one up to three tuf-genes present in various copy numbers, when only one is present in the majority of gram-positive bacteria with the low GC content (Ke et al. 2000). During the further study of tuf-gene sequence in 17 species of enterococci, two different sequence variants of tuf-genes (tufA and tufB) were described. The tufA-gene was proved to be present in all tested species, which enables its successful application as the target sequence in drafting the genus-specific PCR method (Ke et al. 2000). The aim of this study was to verify the application of PCR method based on the detection of genus-specific tuf-gene for the rapid identification of enterococci isolated from foods, food raw materials and other environmental samples. Materials and Methods Bacter ia l isolates Total of 297 field isolates of enterococci from foods and food raw materials, drinking water and various kinds of agriculture and sewage waste water were used in the study. The basic isolations were carried out by the plate method streaking 0.1 ml of sample or their dilutions on plates with the Slanetz-Bartley agar (S-B agar, HiMedia ACTA VET. BRNO 2005, 74: 633–637 Address for correspondence: MVDr. ·árka Cupáková, Ph.D. Department of Milk Hygiene and Technology University of Veterinary and Pharmaceutical Sciences Brno Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 5 4156 2713 E-mail: cupakovas@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm

The identification of enterococci, using conventional methods and biochemical tests based on their phenotypic characteristics, is complicated and time-consuming (Devriese et al. 1993).Not only in clinical, but also in food microbiology, the methods of molecular biology focused on the genus or species identification of enterococci (Dutka-Malen et al. 1995;Ke et al. 1999), intra-generic typing of enterococci isolated from foods (Suzzi et al 2000;A ndrighetto et al. 2001) or faecal samples and rectal swabs of farm and pet animals (Devriese et al. 1996), begin to be applied in the sample examination.Ke et al. (1999) have developed a PCR method for rapid genus-specific identification of enterococci, which is based on the detection of tuf-gene encoding the elongation factor Tu (EF-Tu).EF-Tu is a GTP-binding protein playing a central role in the protein synthesis.In the bacterial genome, there is one up to three tuf-genes present in various copy numbers, when only one is present in the majority of gram-positive bacteria with the low GC content (Ke et al. 2000).During the further study of tuf-gene sequence in 17 species of enterococci, two different sequence variants of tuf-genes (tufA and tufB) were described.The tufA-gene was proved to be present in all tested species, which enables its successful application as the target sequence in drafting the genus-specific PCR method (Ke et al. 2000).
The aim of this study was to verify the application of PCR method based on the detection of genus-specific tuf-gene for the rapid identification of enterococci isolated from foods, food raw materials and other environmental samples.

Bacterial isolates
Total of 297 field isolates of enterococci from foods and food raw materials, drinking water and various kinds of agriculture and sewage waste water were used in the study.The basic isolations were carried out by the plate method streaking 0.1 ml of sample or their dilutions on plates with the Slanetz-Bartley agar (S-B agar, HiMedia Laboratories Pvt. Ltd., Bombay, India).Plates were incubated aerobically at 37 °C for 48 hours.The detailed overview of analyzed isolates is in Table 1.The method was verified using the group of 21 different strains (Streptococcus, Staphylococcus, Listeria, Bacillus, Rhodococcus, Escherichia, Proteus, Pseudomonas, Salmonella, and Shigella) acquired from the Czech Collection of Microorganisms (Brno, The Czech Republic) and from the National Institute of Public Health Prague (Czech Republic) (Table 3).
The following tests were used for the identification of isolates: Gram staining, catalase production, morphology of colonies on blood agar (Blood Agar Base No.2, HiMedia Laboratories Pvt. Ltd., Bombay, India; and sheep blood, Bioveta, a.s., Ivanovice na Hané, Czech Republic), growth on S-B agar and Bile Aesculin agar (Oxoid, Basingstoke, Hampshire, England).Selected isolates were identified using the ENCOCCUStest (Pliva-Lachema, a. s., Brno, Isolation of bacterial DNA For each individual isolate, 24-h culture from a blood agar incubated aerobically at 37 °C was used.The DNA isolation was carried out by the boiling the bacterial suspension (density of 1 degree of MacFarland scale) in sterile distilled water at the temperature 100 °C for 20 min with the consecutive spinning at 13000 rpm for 10 min.Supernatant was used as a template.

Polymerase chain reaction
Enterococcus spp.identification was carried out by a modification procedure described by K e et al. (1999).The reaction was designed as duplex-PCR, when, apart from the detection of genus-specific tuf-gene (product size 112 bp), the internal control was also inserted.The sequence of the internal control was derived from the 16S rRNA gene (product size 241 bp) and is universal for any bacterial species (Martineau et al. 1996).The reaction mixture (25 µl) contained: 1 µl of the template, 12.5 µl PPP Master Mix (Top-Bio, s.r.o., Prague, The Czech Republic) with the MgCl 2 to the final concentration of 2.5 mM, 1 µM primers Ent1 and Ent2, 0.2 µM primers InKo1 and InKo2 (Generi Biotech s.r.o., Hradec Králové, Czech Republic).Sequences of applied primers are in Table 2.The strain Enterococcus faecalis CCM 4224 was used as the positive control, the strain Escherichia coli CCM 3954 was used as the negative control.
The PCR amplification passed in the termocycler PTC-200 (MJ Research Watertown, Massachusetts, USA) according to the following program: the initial denaturation 94 °C/2 min, amplification -30 cycles (94 °C/30 s, 55 °C/15 s, 72 °C/30 s), final extension 72 °C/4 min.The products were analyzed by the gel electrophoresis in 2.0% agarose (Serva Electrophoresis GmbH, Heidelberg, Germany) with the consecutive staining with ethidium bromide and visualization on the UV transilluminator.DNA marker 155 -970 bp (Top-Bio, s.r.o., Prague, The Czech Republic) was used as molecular weight standard.

Results and Discussion
Our study is based on the work of Ke et al. (1999) and Martineau et al. (1996).We adjusted the PCR method enabling a rapid genus identification of enterococci based on the detection of the genus-specific tuf-gene and detection of the 16S rRNA gene as internal control.Specifity of the method was verified using the group of 19 gram-positive and 5 gram-negative control collection strains (Table 3 and Plate XIII, Fig. 1).A total of 291 enterococci of various species, isolated from foods, food raw materials, other environmental samples, and acquired from the strain collection (Table 1 and Table 3), were confirmed as enterococci.In none of the non-Enterococcus isolates a product of 112 bp was detected.
Detection of the tuf-gene was negative in group of 14 isolates from carp fillets, namely, 11 isolates identified phenotypically as E. faecium, 1 isolate of E. Group III and 2 isolates of Enterococcus spp.These isolates yielded dubious or negative results of PYRAtest and a poor ability to grow on Bile Aesculin agar.During the initial isolation on S-B agar the isolates grew in small-sized red colonies with a prolonged growing time (48 -72 h).Slower growth in small-sized grey colonies was also observed on blood agar.In the laboratory of the Czech Collection of Microorganisms, these isolates were reclassified as Lactococcus spp.
The plating method is commonly used to establish the number on S-B agar aerobically at 37 °C for the period of 24 -48 h.After incubation red-up-to-brown colonies are being Internal control InKo1 5' -GGA GGA AGG TGG GGA TGA CG -3' InKo2 5' -ATG GTG TGA CGG GCG GTG TG -3' 241 bp Martineau et al., 1996 counted.With subcultivation of characteristic colonies from S-B agar on blood agar, the time necessary to acquire final results, is approximately 30 h, with phenotypic identification it is 48 h.Unambiguous identification by method PCR required only 4 h.
•vec and Sedláãek (1999) state that the morphology of enterococci colonies on S-B agar is significantly variable, even in individual species.Furthermore, the authors state that 17% of strains isolated from S-B agar were not enterococci; these were unspecified grampositive cocci and both gram-positive and gram-negative rods.Devriese et al. (1993), Leclerc et al. (1996) and also Franz et al. (1999) identically point out the close relationship of enterococci, streptococci and lactococci.Patel et al. (1998) describes a close relationship of the species Enterococcus solitarius and bacteria Tetragenococcus spp.For standardization of the enterococci plate count procedure in foods, and to prevent eventual mistakes in reading the results, the application of highly specific confirmation tests is necessary.
The PCR method based on the detection of tuf-gene has been found specific and suitable for the genus confirmation of enterococci isolated from foods and environmental samples.Apart from the initial costs of the equipment, the PCR method itself, owing to the easy DNA isolation by boiling and use of commercial PPP Master Mix, is a low-cost and time saving procedure.The method described in this paper can thus be recommended for the routine identification of bacteria Enterococcus spp.

Table 1 .
Species and origin of isolates tested in the study *A -raw milk, milk products, B -beef, sausage batter, meat products, C -common carp (fillets), D -delicatessen, E -confectionary, F -spices, G -drinking water, H -agricultural and municipal waste water

Table 3 .
Verification of PCR specificity using collection strains of microorganisms Czech Republic), by the production of pyrolidonyl arylamidase (PYRAtest, Pliva-Lachema, a. s., Brno, Czech Republic), by the motility and by the production of pigment.