Journal of Clinical Microbiology, November 1999, p. 3497-3503, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development of a PCR Assay for Rapid Detection
of Enterococci
Danbing
Ke,1,2
François J.
Picard,1
Francis
Martineau,1,2
Christian
Ménard,1
Paul H.
Roy,1,3
Marc
Ouellette,1,2 and
Michel G.
Bergeron1,2,*
Centre de Recherche en Infectiologie de
l'Université Laval, Sainte-Foy, Québec, Canada G1V
4G2,1 and Division de Microbiologie,
Faculté de Medicine,2 and
Département de Biochimie, Faculté des Sciences et
de Génie,3 Université Laval,
Sainte-Foy, Québec, Canada G1K 7P4
Received 22 March 1999/Returned for modification 4 June
1999/Accepted 23 July 1999
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ABSTRACT |
Enterococci are becoming major nosocomial pathogens, and increasing
resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus
level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the
tuf gene from several bacterial species (available in
public databases) and designed degenerate PCR primers derived from
conserved regions. These primers were used to amplify a target region
of 803 bp from four enterococcal species (Enterococcus
avium, E. faecalis, E. faecium, and
E. gallinarum). Subsequently, the complete nucleotide
sequences of these amplicons were determined. The analysis of a
multiple alignment of these sequences revealed regions conserved among
enterococci but distinct from those of other bacteria. PCR primers
complementary to these regions allowed amplification of genomic DNAs
from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority
of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria
species. Furthermore, this assay efficiently amplified all 159 clinical
isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence
comparison of the amplicons for four enterococcal species demonstrated
that there were some sequence variations which may be used to generate
species-specific internal probes. In conclusion, this rapid PCR-based
assay is capable of detecting all clinically important enterococci and
has potential for use in clinical microbiology laboratories.
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INTRODUCTION |
Enterococci are members of the
normal flora of the gastrointestinal tract in humans and animals
(24). The incidence of enterococcal infections has increased
in recent years because of widespread multiresistant enterococcal
strains and increasing numbers of immunosuppressed patients and
catheter-related infections. In fact, enterococci are now the second
most common nosocomial pathogens in the United States (4,
31). There are two major pathogenic species in humans,
Enterococcus faecalis and E. faecium, with occasional infections being caused by E. durans, E. gallinarum, E. casseliflavus, E. avium,
E. hirae, E. mundtii, and E. raffinosus (24, 38). Devriese et al. (8)
suggested that the extensive agricultural use of glycopeptides has
created an animal reservoir of resistant enterococci which may lead to
more enterococcal species resistant to glycopeptides in animal sources
and complicate the control of such infections. Enterococci resistant to
glycopeptides have been isolated with increasing frequency and have
become a major concern worldwide. Rapid identification of enterococci
is important in reducing the spread of multiresistant enterococci (2, 16).
Identification of enterococci through conventional methods, i.e., by
determining phenotypic characters, is complicated and often requires 24 to 48 h (7, 11, 28). The automated methods currently
used are unable to reliably identify enterococci other than E. faecalis and E. faecium (29, 32, 34).
Furthermore, phenotypic identification of some enterococcal species may
be occasionally difficult or even impossible because these species lack
typical characteristics. More rapid and accurate methods would be
helpful for microbiology laboratories. Several DNA-based methods for
the specific detection of E. faecalis or E. faecium have been reported (5, 10, 27, 30). Other
molecular methods, such as contour-clamped homogeneous electric field
electrophoresis patterns, amplified ribosomal DNA spacer polymorphisms,
and randomly amplified polymorphic DNA analysis, have been used to
identify enterococci at the species level (3, 9, 23, 26,
34). However, it is difficult to adapt these tests for use in
clinical microbiology laboratories because of their complexity. An
Enterococcus sp. assay based on the hybridization of rRNA
genes (Gen-Probe, San Diego, Calif.) is commercially available for
culture confirmation (6). The sensitivity of this assay is
unsatisfactory for direct detection from clinical specimens.
A variety of conserved genes, including rRNA genes (3, 19,
22), the heat shock protein 60 (HSP60 or CPN60) gene (13, 14), the major cold shock protein gene (12), and the
sod gene (39), have been exploited for the
detection of bacteria. The tuf gene, encoding elongation
factor EF-Tu, is involved in peptide chain formation and is an
essential constituent of the bacterial genome (15). These
characteristics make it a target of choice for diagnostic purposes.
PCR-based assays in which the tuf gene serves as the target
sequence have been developed for Mycoplasma fermentans
(1) and M. pneumoniae (20). We report
here the development of a PCR-based assay that targets the
tuf gene, that can detect most enterococcal species with
excellent sensitivity and acceptable specificity, and that has
potential for the development of species-specific internal probes.
(This study was presented in part at the 98th General Meeting of the
American Society for Microbiology 1998, Atlanta, Ga., 17 to 21 May
1998.)
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MATERIALS AND METHODS |
Bacterial strains.
Twenty enterococcal strains obtained from
the American Type Culture Collection (ATCC), Manassas, Va., were used
in this study. These strains represent the following species: E. avium (ATCC 14025), E. casseliflavus (ATCC 25788),
E. cecorum (ATCC 43198), E. dispar (ATCC 51266),
E. durans (ATCC 19432), E. faecalis (ATCC 19433, ATCC 29212, ATCC 33186, ATCC 49533, and ATCC 51299), E. faecium (ATCC 19434 and ATCC 51559), E. flavescens
(ATCC 49996), E. gallinarum (ATCC 49573), E. hirae (ATCC 8043), E. mundtii (ATCC 43186), E. pseudoavium (ATCC 49372), E. raffinosus (ATCC 49427), E. saccharolyticus (ATCC 43076), and E. solitarius (ATCC 49428). An additional 159 clinical isolates of
enterococci obtained from various sources were also used in this study
(Table 1).
The specificity of the PCR-based assay was verified by use of a battery
of ATCC reference strains consisting of 44 gram-negative and 50 gram-positive bacterial species (Table
2). The 159 clinical isolates of
enterococci (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus) from
various origins (Table 1) were also tested to further validate the
Enterococcus-specific PCR-based assay. The reference strains
as well as the clinical isolates were all identified by conventional
methods or with an automated MicroScan Autoscan-4 system equipped with
a Positive BP Combo Panel Type 6 (Dade Diagnostics, Mississauga,
Ontario, Canada). Bacterial strains were grown from frozen stocks kept
at 
80°C in brain heart infusion medium containing 10% glycerol and
were cultured on sheep blood agar or in brain heart infusion broth.
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TABLE 2.
Specificity test performed with the
Enterococcus-specific 40-cycle PCR assay and DNA from a
variety of gram-positive and gram-negative bacterial species
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PCR primers.
The tuf gene sequences available
from public databases were analyzed with GCG programs (version 8.0)
(Genetics Computer Group, Madison, Wis.). Based on multiple sequence
alignments, regions of the tuf gene highly conserved among
eubacteria were chosen, and PCR primers were derived from these regions
with Oligo primer analysis software (version 5.0) (National
Biosciences, Plymouth, Minn.). When required, the primers contained
inosines or degeneracies at one or more variable positions.
Oligonucleotide primers were synthesized with a model 391 DNA
synthesizer (Perkin-Elmer Corp., Applied Biosystems Division,
Mississauga, Ontario, Canada). PCR primers used in this study are
listed in Table 3.
DNA sequencing.
An 803-bp portion of the tuf gene
was sequenced for E. avium, E. faecalis, E. faecium, and E. gallinarum. Amplification was performed
with 1 ng of genomic DNA prepared by use of a G NOME DNA kit (Bio 101, Vista, Calif.). The 20-µl PCR mixtures used to generate PCR products
for sequencing contained 1.0 µM each universal primer (U1 and U2;
Table 3), 200 µM each deoxyribonucleoside triphosphate (Pharmacia
Biotech Inc., Baie d'Urfé, Québec, Canada), 10 mM Tris-HCl
(pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 0.5 U of Taq polymerase (Promega Corp., Madison, Wis.), and
TaqStart antibody (Clontech Laboratories Inc., Palo Alto, Calif.). The
TaqStart antibody, which is a neutralizing monoclonal antibody for
Taq DNA polymerase, was added to all PCR mixtures to enhance
the efficiency of the amplifications (18).
The PCR mixtures were subjected to thermal cycling (3 min at 95°C and
then 35 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min
at 72°C, with a 7-min final extension at 72°C) with a PTC-200 DNA
Engine thermocycler (MJ Research Inc., Watertown, Mass.). The amplified
PCR mixtures were resolved by electrophoresis through 1.5% agarose
gels at 4 V/cm for 90 min; the gels were then stained with ethidium
bromide and visualized under 312-nm UV light. Subsequently, PCR
products having the predicted sizes were recovered from the gels with a
QIAquick gel extraction kit (QIAGEN Inc., Mississauga, Ontario, Canada).
The purified DNA fragments were cloned into pCR2.1 vector (Invitrogen
Corp., Carlsbad, Calif.). Plasmids were isolated from transformed
Escherichia coli with a QIAGEN plasmid mini-kit. The presence of DNA inserts in the recombinant plasmids was confirmed by
digesting purified plasmid DNA with EcoRI (New England
Biolabs, Ltd., Mississauga, Ontario, Canada), which allowed excision of the inserted fragments. Both strands of the DNA inserts for each of the
selected recombinant plasmids were sequenced with a PRISM Ready
Reaction DyeDeoxy Terminator cycle sequencing kit and an Applied
Biosystems 373A sequencer (Perkin-Elmer). In order to exclude the
possibility of sequencing errors attributable to misincorporations by
Taq polymerase, each strand of the insert was sequenced from three different clones.
PCR amplification.
For all bacterial species, amplification
was performed from purified genomic DNA or from a bacterial suspension
whose turbidity was adjusted to that of a 0.5 McFarland standard, which
corresponds to approximately 1.5 × 108 bacteria per
ml. One nanogram of genomic DNA or 1 µl of standardized bacterial
suspension was transferred directly to a 19-µl PCR mixture containing
50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM
MgCl2, 0.2 µM each Enterococcus-specific
primer (Ent1 and Ent2; Table 3), 200 µM each deoxynucleoside
triphosphate (Pharmacia Biotech), 3.3 µg of bovine serum albumin
(BSA) (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) per µl,
0.5 U of Taq polymerase (Promega), and TaqStart antibody
(Clontech). PCR amplification and agarose gel analysis of the amplified
products were performed as previously described (21).
The Superlinker phagemid pSL1180 (Pharmacia Biotech) linearized by
digestion with EcoRI (New England Biolabs) and the primers (IC1 and IC2; Table 3) derived from the multiple cloning sites of this
plasmid were used to provide an internal control for all Enterococcus-specific PCR-based assays. These primers can
amplify a 252-bp product. The internal control was integrated into the PCR-based assays to verify the efficiency of the amplifications and to
ensure that significant PCR inhibition was absent. Four thousand copies
of the linearized plasmid were added to each PCR. The concentrations of
the internal control primers were adjusted to ensure that there was no
detrimental effect on the Enterococcus-specific amplification. We found that concentrations of 0.1 and 0.04 µM were
optimal for 30- and 40-cycle PCRs, respectively.
For determination of the sensitivities of the PCR-based assays, twofold
dilutions of purified genomic DNA were used to determine the minimal
number of genomes which can be detected.
Nucleotide sequence accession numbers.
GenBank accession
numbers for the 751-bp partial sequence of the tuf gene
(excluding the sequences of the two universal amplification primers)
are as follows: AF124220 for E. avium, AF124221 for E. faecalis, AF124222 for E. faecium, AF124223 for
E. gallinarum, AF124224 for Abiotrophia adiacens,
and AF124225 for A. defectiva.
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RESULTS |
Sequencing of a portion of the tuf gene from four
enterococcal species.
The tuf sequences from a number
of selected bacterial species, including E. coli (J01690),
M. genitalium (U39732), Haemophilus influenzae
(U32746), Neisseria gonorrhoeae (L36380), Salmonella typhimurium (X55116), and Micrococcus luteus (M17788),
were aligned and compared. Two highly conserved regions were
identified, and a pair of primers (U1 and U2) amplifying a region of
803 bp was designed (Table 3). Several degeneracies and inosines were incorporated into these two primers because some positions are variable
among eubacteria. These primers allowed the amplification of
tuf sequences from a wide variety of bacteria, including 14 enterococcal species. By using these primers, we were able to amplify
the 803-bp portion of tuf for four enterococcal species: E. avium, E. faecalis, E. faecium, and
E. gallinarum. After purification from agarose gels, the
803-bp PCR product was cloned into a TA cloning vector. Subsequently,
the sequence of the inserted DNA fragment was determined by sequencing
of three randomly selected clones for each enterococcal species to
ensure that no sequencing errors were attributable to misincorporation
by the Taq polymerase. In order to facilitate the selection
of Enterococcus-specific primers, we conducted a multiple
sequence analysis using the sequences mentioned above (available in
public databases) as well as partial tuf sequences from
staphylococci, streptococci, enterococci, Listeria spp., and
Abiotrophia spp. (all obtained from our laboratory; unpublished data). By using this approach, we were able to identify regions conserved in the four enterococcal species but variable in the
other bacterial species. The Enterococcus-specific PCR primers were derived from these regions. A similarity comparison of the
tuf sequences for the enterococcal species E. avium, E. faecalis, E. faecium, and E. gallinarum as well as for two Abiotrophia species is
given in Table 4. The sequence
similarities for the 803-bp fragment in these species ranged from 85 to
91%.
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TABLE 4.
Sequence homology for an 803-bp portion of the
tuf gene and Enterococcus-specific PCR products
for four enterococcal species and two Abiotrophia species
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The selected primers revealed no more than two mismatches within the
tuf sequences of the enterococcal species, except for E. faecium and two Abiotrophia species, in which
three mismatches were present at the 5' end of the 5' primer.
Importantly, more than five mismatches were found in the corresponding
regions of the other bacterial species mentioned above. Since the
mismatches in the other bacteria were clustered at the 3' end of the
primers, a position critical for discriminatory PCR amplification, the amplification of bacterial species other than enterococci could be
efficiently prevented.
Amplifications with the Enterococcus-specific PCR
assay.
The specificity of the assay was assessed by performing
30-cycle and 40-cycle PCR amplifications with the panel of
gram-positive (50 species from 9 genera) and gram-negative (44 species
from 21 genera) bacterial species listed in Table 2. The PCR assay was
able to detect 14 of 15 enterococcal species tested in both 30-cycle
and 40-cycle regimens. E. solitarius was the only
enterococcal species tested that was not amplified by the
Enterococcus-specific assay (Fig.
1). For 30-cycle PCR, all bacterial
species tested other than enterococci were negative, except for two
Abiotrophia species, A. adiacens and A. defectiva. For 40-cycle PCR, four Listeria species,
Listeria innocua, L. ivanovii, L. monocytogenes, and L. seeligeri, were also positive in
addition to the enterococci and Abiotrophia species. The
other species tested remained negative. The internal control was always
efficiently amplified when no target DNA was present, thereby showing
the absence of PCR inhibitors. On the contrary, the internal control
was not amplified when target DNA was present in a sample (Fig. 1).
This result is explained by the fact that the concentrations of the
internal control primers were limited in order to favor the
amplification of the target DNA. Tests of a collection of clinical
isolates comprising E. faecalis (n = 77), E.
faecium (n = 61), E. gallinarum (n = 9), and E. casseliflavus (n = 12) showed a
uniform amplification signal with both the 30-cycle and the 40-cycle
PCR assays and a perfect relationship between the genotype and
classical means of identification.
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FIG. 1.
Example of multiplex PCR amplifications with the
Enterococcus-specific PCR assay and the internal control.
PCR assays (40 cycles) were performed with 1 µl of genomic DNA (1 ng/µl) from various bacteria and 4,000 copies of linearized pSL1180
as an internal control. The internal control was not amplified when
target DNA was present due to competitive inhibition by amplification
of Enterococcus DNA. Lanes: 2, E. avium ATCC
14025; 3, E. casseliflavus ATCC 25788; 4, E. cecorum ATCC 43198; 5, E. durans ATCC 19432; 6, E. dispar ATCC 51266; 7, E. faecalis ATCC 19433;
8, E. faecium ATCC 19434; 9, E. flavescens ATCC
49996; 10, E. gallinarum ATCC 49573; 11, E. hirae
ATCC 8043; 12, E. mundtii ATCC 43186; 13, E. pseudoavium ATCC 49372; 14, E. raffinosus ATCC 49427;
15, E. saccharolyticus ATCC 43076; 16, E. solitarius ATCC 49428; 17, E. coli ATCC 25922; 1 and
18, controls to which no DNA was added; M, 100-bp molecular size
standard ladder.
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The sensitivity of our Enterococcus-specific assay with
30-cycle and 40-cycle PCR protocols was determined by using purified genomic DNA from 14 enterococcal species. For PCR with 30 cycles, we
found a detection limit ranging from 330 to 1,700 copies of genomic
DNA, depending on the enterococcal species tested (Table 5). In order to enhance the sensitivity
of the assay, we increased the number of cycles. For PCR with 40 cycles, the detection limit was lowered to two to eight genome copies
(Table 5).
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DISCUSSION |
Enterococci can be encountered throughout the environment, from
human, animal, and food sources. The emergence and dissemination of
vancomycin-resistant enterococci (VRE) underscore the importance of the
rapid detection of these organisms (16). Conventional identification of some enterococci is difficult because no phenotypic criteria are available to unequivocally separate the genus
Enterococcus from the other genera of gram-positive cocci. A
number of tests, such as Lancefield group D antigen, the ability to
grow in 6.5% NaCl broth, the pyrolidonylarylamidase test, the
leucinearylamidase test, and the bile esculin test, are all valuable
for the identification of enterococci. Unfortunately, none of these
tests alone or in combinations provides a phenotype unique to the
enterococci (7). Furthermore, such tests require 24 to
48 h to provide results after pure cultures are obtained. Even
with automated systems, incubation for up to 24 h is routinely
required, and some additional tests may have to be carried out for
species differentiation (29, 32). Since the vancomycin
resistance genes are transferable among different enterococcal species
or even among different genera (38), the inability to detect
enterococci promptly may cause delays in reporting VRE; this situation
may lead to complex and costly containment efforts to eliminate VRE
colonization and infection.
The development of rapid and sensitive DNA-based assays which are
applicable for the direct detection of enterococci from clinical
specimens may improve the rapidity and the accuracy of the diagnosis of
enterococcal infections. The validity and versatility of PCR in
applications for the detection of nucleic acids from a variety of
infectious agents have been well reviewed by Whelen and Persing
(36). In the present study, we have developed a rapid
PCR-based assay to improve the diagnosis of enterococcal infections.
Initially, PCR primers complementary to highly conserved regions of the
tuf gene among eubacteria were designed and used to amplify
an 803-bp portion of the tuf gene from 14 enterococcal species. By sequencing four enterococcal species, which represented four different enterococcal subgroups based on 16S rRNA gene analysis, we have obtained adequate sequence information to design a pair of
Enterococcus-specific PCR primers. Subsequently, a PCR-based assay was set up and optimized to be simple and rapid. Among 50 species
of gram-positive (representing 9 genera) and 44 species of
gram-negative (representing 21 genera) clinically important bacteria
tested, this PCR-based assay was able to detect all enterococcal species tested, except for E. solitarius, which in fact does
not appear to be a member of the genus Enterococcus based on
a phylogenetic analysis (25, 37). Two Abiotrophia
species were also amplified efficiently under the same amplification
conditions. Moreover, amplification of four Listeria species
was also observed when the number of PCR cycles was increased from 30 to 40. It should be noted that DNA from Listeria species was
amplified about 100 times less efficiently than enterococcal DNA.
However, most clinically important bacteria can be easily
differentiated from enterococci by this assay, indicating its
usefulness for the detection of enterococci. The concomitant use of
species-specific internal probes should allow exclusion of
Abiotrophia and Listeria species by increasing
the specificity of the Enterococcus-specific PCR-based assay.
To elucidate the fact that the developed PCR-based assay failed to
detect E. solitarius, which rarely causes infections in humans, we amplified and sequenced an 890-bp portion of tuf
encompassing the 803-bp region by using another pair of primers
(unpublished data). The sequence similarity between E. solitarius and other enterococci ranged from 79 to 81%, while
that between the other enterococci ranged from 89 to 91%. This finding
supports 16S rRNA data reported by others (25, 37)
suggesting that E. solitarius is not a member of the genus
Enterococcus (93.0 to 94.8% homology) but is
phylogenetically more closely related to the genus
Tetragenococcus (97.8% homology). Sequence data revealed
that there were six mismatches at the Enterococcus-specific
3'-primer binding site which led to a failure in the amplification of
E. solitarius by the developed PCR-based assay.
The finding that A. adiacens and A. defectiva,
formerly referred to as nutritionally variant streptococci
(17), were also positive in our
Enterococcus-specific PCR assay indicates a high level of
similarity of the tuf genes at the primer binding sites in
the genus Abiotrophia and the genus Enterococcus.
Sequencing of the 803-bp region of tuf showed that the
sequence similarities between the enterococcal and
Abiotrophia spp. ranged from 85 to 89%, values which are
quite high. For comparison, the tuf sequences among
enterococci are 89 to 91% similar. Therefore, it is not surprising
that PCR primers derived from the tuf sequence amplify DNA
from Abiotrophia spp. as well. In fact, there are only two mismatches at the 5' end of the Enterococcus-specific
primers compared to the sequences of A. defectiva and
A. adiacens. Consequently, the sensitivity level achieved
for the two Abiotrophia species was similar to that obtained
for the enterococcal species. In addition, several Listeria
species were detectable when 40-cycle PCR was performed, even though
three mismatches located near the 3' end of the upper primer were found
between the four Listeria species and enterococci. However,
it should be noted that Listeria DNA was amplified
approximately 100 times less efficiently than enterococcal DNA. The
four Listeria species, L. monocytogenes, L. innocua, L. ivanovii, and L. seelegeri,
showed sequence similarities to enterococci ranging from 82 to 83%
(data not shown). Based on the level of sequence divergence for the
tuf gene, it should be possible to develop enterococcal
species-specific internal probes which allow discrimination of members
of the genera Abiotrophia and Listeria from those
of the genus Enterococcus.
Others have developed E. faecalis-specific and E. faecium-specific PCR-based assays by targeting various genes. The
target genes include (i) ddl (coding for
D-alanine-D-alanine ligase) for the
species-specific detection of E. faecalis and E. faecium (10, 30) and (ii) PBP5 (coding for
penicillin binding protein) for the species-specific detection of
E. faecalis (27). A PCR-based assay amplifying
different vanC genes (coding for intrinsic vancomycin resistance in E. gallinarum, E. casseliflavus,
and E. flavescens) has also been developed to specifically
detect E. gallinarum, E. casseliflavus, and
E. flavescens (10). A sequence of unknown coding
potential, selected from an E. faecium genomic library, has
been used to develop an E. faecium-specific PCR-based assay (5). Although E. faecalis and E. faecium account for a majority of enterococcal infections, other
enterococci may be associated with infections. However, currently
available systems based on culturing for the identification of
gram-positive cocci are often unable to correctly identify these less
frequently encountered enterococci (29, 32, 34).
Tyrrell et al. (35) reported using an internally transcribed
spacer region PCR to identify enterococcal species based on characteristic amplicon profiles and the different patterns of Sau3A-digested PCR amplicons. Donabedian et al.
(9) demonstrated that contour-clamped homogeneous electric
field electrophoresis patterns and DNA-DNA hybridization with
biotin-labeled genomic DNAs from type strains of enterococci used as
probes may be suitable for species differentiation of some enterococci.
However, neither method is feasible for routine use in the clinical
laboratory because of high complexity and must be used in conjunction
with phenotypic tests to provide reliable results (9, 35).
Recently, Quednau et al. (26) and Monstein et al.
(23) identified enterococci to the species or species group
level by using randomly amplified polymorphic DNA methods. A simple
test for the detection of Enterococcus spp. has been
developed by Gen-Probe for use as a culture confirmation assay; this
test was 100% accurate in identifying enterococcal isolates from
cultures by hybridization to rRNA (6). However, this assay
does not offer potential for enterococcal species identification. Moreover, the sensitivity of the Gen-Probe assay is not sufficient for
direct detection of enterococci from clinical specimens.
Besides identification of bacterial species by species-specific
PCR-based assays, identification may also be performed by coupling the
amplification of a highly conserved gene with hybridization to internal
probes or DNA sequencing (1, 14, 20, 22). Many conserved
genes have been selected as targets for this purpose. Among them, the
16S rRNA gene has been used to detect a wide variety of eubacteria
because the presence of conserved regions and variable regions in this
gene provides the possibility of developing PCR-based assays suitable
for detecting and identifying bacteria at the species level or higher
taxonomic levels (22). Other genes have been exploited for
similar purposes. The sod gene, coding for superoxide
dismutase, has been used as a target to amplify 28 species of
mycobacteria and to differentiate one from another with probes
recognizing species-specific regions (39). A similar approach has been used for the detection of staphylococci by targeting the chaperonin 60 (cpn60) gene (13, 14). These
methods are specific, but their sensitivity remains unclear. The
latter is critical when such tests are used to detect bacteria directly from clinical specimens. Berg et al. (1) have
developed a PCR-based detection system for M. fermentans by targeting the tuf gene; the system has a
high sensitivity. A similar assay has also been used for the
identification of M. pneumoniae (20).
The tuf gene acts in translation to bring aminoacylated tRNA
molecules to the ribosome. This gene represents an ideal candidate target for diagnostic purposes because it is highly conserved at the
nucleotide level and ubiquitous in bacteria. By analyzing a rather
small tuf sequence data set available in public databases, we were able to design PCR primers which could amplify an 803-bp portion of the tuf gene from a variety of bacteria,
including members of the genera Enterococcus,
Streptococcus, and Staphylococcus. We found that
there were more nucleotide sequence variations in the tuf
gene sequences than in the corresponding 16S rRNA gene sequences for
four enterococcal species (i.e., E. faecalis, E. faecium, E. avium, and E. gallinarum). An
analysis of the tuf sequences from gram-positive bacteria
allowed the development of a PCR-based assay amplifying 14 of 15 enterococcal species tested. The PCR-based assay described here
differentiates enterococci from most clinically relevant bacteria,
indicating that the tuf gene is a target of choice for the
molecular detection of enterococci. Furthermore, the amplicon sequence
polymorphism should be sufficient to provide discriminatory internal
probes specific for clinically important enterococcal species. The
sensitivity of the 30-cycle PCR-based assay varies from 330 to 1,700 copies of enterococcal genomes for the 14 enterococcal species
detected. This sensitivity level is sufficient for culture confirmation
assays. It is possible to efficiently increase the sensitivity of the
assay. For example, in sensitivity assays performed with 40-cycle PCR,
the detection limit was reduced to about two to eight copies of
enterococcal genomes for the same 14 enterococcal species. This
sensitivity level should be sufficient for the direct detection of
enterococci in clinical specimens, such as fecal or urine samples. The
use of this PCR-based assay coupled with species-specific internal probes specific for clinically important enterococci and other PCR
assays targeting vancomycin resistance genes should provide a useful
screening test for VRE from rectal swabs. Such an assay is currently
under development in our laboratory.
In conclusion, we have developed a PCR-based diagnostic assay which is
simple to conduct and reliable for the detection of enterococci. This
assay offers an alternative to currently used methods and should allow
the identification of clinically important enterococcal species if
coupled with species-specific probes complementary to internal regions
of the amplicons. This new diagnostic tool may lead to the early
diagnosis of enterococcal infections, which is essential for the
prevention and control of transmission of the infections.
| |
ACKNOWLEDGMENTS |
We thank Louise Coté, director of the microbiology
laboratory of CHUQ, Pavillon CHUL, for free access to the laboratory
and for providing enterococcal and other clinical isolates. We thank Jean-Luc Simard, Martin Gagnon, Marie-Josée Boily, Caroline
Paquet, Nicolas Lansac, Marie-Claude Bergeron, and Gisèle
Chassé for help. We also thank Louise Jetté (Laboratoire de
Santé Publique du Québec), Donald E. Low (Mount Sinai
Hospital), Barbara E. Murray (University of Texas, Houston), Fred C. Tenover (Centers for Disease Control and Prevention), Wang Fu (Huashan
Hospital), and Daniela Centron-Garcia (Universidad de Buenos Aires) for
providing enterococcal strains. We thank Maurice Boissinot for critical comments regarding the manuscript.
Marc Ouellette is an MRC scientist and a recipient of the Burroughs
Wellcome Fund new investigator award in molecular parasitology. This
study was supported by grant PA-15586 from the Medical Research Council
of Canada and by Infectio Diagnostic (I.D.I) Inc., Sainte-Foy, Québec, Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre de
Recherche en Infectiologie, Centre Hospitalier Universitaire de
Québec, Pavillon CHUL, 2705 Boul. Laurier, Sainte-Foy,
Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418)
654-2715. E-mail:
Michel.G.Bergeron{at}crchul.ulaval.ca.
| |
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Journal of Clinical Microbiology, November 1999, p. 3497-3503, Vol. 37, No. 11
0095-1137/99/$04.00+0
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Abstract
Introduction
