To the Editor:
Enterococcus
faecalis, which exists commensally in the gut in warm-blooded animals and humans,
is an opportunistic pathogen that causes a variety of community-acquired and health
care–associated infections, such as urinary tract and intraabdominal infections, bacteremia,
and endocarditis (
1
). Only a few studies have assessed the relationships between clinical E. faecalis
strains; strains endemic to the health care setting; and community strains residing
in humans, animals, or animal-origin food (
2
).
Recently we showed that the emergence of high-level gentamicin-resistant (HLGR) E.
faecalis among patients with infective endocarditis (IE) coincided with an increase
in HLGR E. faecalis in the pig population in Denmark (
3
). The majority of isolates belonged to the same clonal group (sequence type [ST]
16), suggesting that pigs constitute a community reservoir of HLGR E. faecalis. We
investigated human and porcine community reservoirs of other E. faecalis clonal types
associated with IE in humans in Denmark.
A total of 20 consecutive gentamicin-susceptible E. faecalis isolates were obtained
from IE patients in North Denmark Region during 1996–2002 (Table A1). Cases of IE
were classified as definite (n = 12) or possible (n = 8) according to the modified
Duke criteria (
4
). A case of community-acquired E. faecalis infection (n = 6) was defined in accordance
with strict criteria applied for methicillin-resistant Staphylococcus
aureus
(
5
); otherwise, cases were deemed to be health care associated (n = 14) (Table A1).
HLGR ST16 isolates recovered from 2 IE patients during the study period have been
characterized (
3
) and were excluded from the present study.
Using multilocus sequence typing (
6
), we identified 14 STs among the 20 IE isolates (Table A1), then compared them with
STs from 2 collections of E. faecalis isolates collected as part of the Danish Integrated
Antimicrobial Resistance Monitoring and Research Program (www.danmap.org): 1) all
14 isolates recovered from community-dwelling humans in North Denmark Region during
2002–2006 with approval from the local ethics committee ([KF] 01-006/02), which were
classified into 10 STs in this study (Table A1); and 2) 19 pig isolates from 2001
that were shown in a previous study to belong to 12 STs (
7
).
Among the 14 STs identified in IE isolates, 4 (ST19, ST21, ST72, and ST306) and 2
(ST40 and ST97) were also found among isolates from community-dwelling humans and
pigs, respectively (Table A1). Isolates belonging to these 6 STs were further characterized
by pulsed-field gel electrophoresis (PFGE) by using SmaI and grouped into PFGE pulsotypes
as described (
3
). STs and PFGE pulsotypes (A–F) were largely concordant (ST97:A, ST72:B, ST19:C,
ST40:D, ST21:E, and ST306:F), except for 2 isolates belonging to ST72 and ST40, for
which PFGE banding patterns (U1 and U2, respectively) were unrelated to the major
PFGE pulsotypes (A–F), and 1 ST306 isolate exhibiting the ST21-like PFGE banding pattern
E (Table A1).
These findings confirm the genetic relatedness of IE isolates with those from community-dwelling
humans (ST72:B, ST19:C, ST21:E, and ST306:F) and pigs (ST97:A and ST40:D). Seven (64%)
of 11 IE isolates belonging to these 6 clonal types originated from IE patients with
health care–associated risk factors (Table A1), which suggests that health care users
are predisposed to colonization and infection with E. faecalis strains residing in
human and porcine community reservoirs.
Previous reports have shown that epidemiologically distinct E. faecalis populations
differ in terms of biofilm formation, virulence gene content, and antimicrobial drug
susceptibility profiles (
2
,
8
). Therefore, we characterized all isolates with respect to these traits. Isolates
were categorized into strong, medium, weak, and nonbiofilm formers by using the method
of Mohamed et al. (
8
). The presence of 12 virulence-associated and pathogenicity island genes (ebpA, gelE,
ef1824, hylA, ef1896, ef2347, ef2505, hylB, ace, cbh, esp, and ef0571) was investigated
by using colony lysates and probes that have been described elsewhere (
9
). The antimicrobial drug susceptibility profiles (ampicillin, chloramphenicol, ciprofloxacin,
erythromycin, gentamicin, kanamycin, linezolid, penicillin, streptomycin, teicoplanin,
tetracycline, and vancomycin) were determined by the Sensititre system (Trek Diagnostic
Systems, East Grinstead, UK) in accordance with Clinical and Laboratory Standards
Institute guidelines (
10
). The isolates were generally homogenous within each clonal type in terms of biofilm
formation, presence of virulence-associated and pathogenicity island genes, and resistance
profiles (Table A1), further supporting that IE isolates are genetically related to
those from community-dwelling humans and pigs, respectively. Notably, most IE isolates
were susceptible to ampicillin (100%), penicillin (100%), vancomycin (100%), high-level
gentamicin (100%), and high-level streptomycin (80%), which are the drugs of choice
in therapeutic regiments for E. faecalis endocarditis.
In conclusion, our results suggest that the normal intestinal microflora of humans
and pigs are community reservoirs of clinical E. faecalis and link 2 porcine-origin
clonal types of gentamicin-susceptible E. faecalis, ST97:A, and ST40:D to IE in humans
in Denmark. This finding strengthens existing evidence that pigs can be a source of
serious infections in humans.