J
OURNAL OF
C
LINICAL
M
ICROBIOLOGY
, May 1996, p. 1296–1298
Vol. 34, No. 5
0095-1137/96/$04.00
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Copyright
᭧ 1996, American Society for Microbiology
Antimicrobial Susceptibilities of Lactococcus lactis and
Lactococcus garvieae and a Proposed Method
To Discriminate between Them
J. A. ELLIOTT*
AND
R. R. FACKLAM
Streptococcus Reference Laboratory, Childhood and Vaccine Preventable Diseases Branch,
Division of Bacterial and Mycotic Diseases, Center for Infections Diseases,
Centers for Disease Control and Prevention, Atlanta, Georgia
Received 30 October 1995/Returned for modification 22 December 1995/Accepted 16 February 1996
The MICs of antimicrobial agents contained in the SCEPTOR Streptococcus MIC panels (Becton Dickinson
Microbiology Systems) were determined for Lactococcus lactis, L. garvieae, and unknown Lactococcus species.
Several isolates had reduced susceptibilities to many of the antimicrobial agents contained in the panel. For
L. garvieae, the MICs of penicillin and, possibly, cephalothin were higher than for L. lactis, and unlike L. lactis,
L. garvieae was resistant to clindamycin, indicating that knowledge of the Lactococcus species causing an
infection might influence the choice of antimicrobial therapy. Susceptibility to clindamycin can also be used to
differentiate between L. lactis and L. garvieae.
Lactococcus spp. have only recently been reported to cause
infections in humans (5, 15, 23). The clinical information in
these reports indicates that these bacteria cause a variety of
different types of infections similar to the types caused by
enterococci. In fact, before lactococci were recognized as hu-
man pathogens, we often identified them as variants of estab-
lished Enterococcus spp., including Enterococcus faecium, E.
faecalis, E. durans, and E. hirae. There are seven recognized
Lactococcus species and subspecies that can be identified by
physiologic reactions (4, 7–10, 12, 21). The majority of human
source isolates that we have received have been either Lacto-
coccus lactis subsp. lactis or L. garvieae (5).
Distinguishing between these two species solely on the basis
of their physiologic characteristics is very difficult. We have
used molecular tests such as whole-cell protein analysis (5, 7)
and DNA or RNA analysis (1–3, 13, 14, 17–20, 22) to help in
the identification of recent isolates. The value of molecular
techniques for the differentiation between L. lactis and L.
garvieae is demonstrated by our reevaluation of isolates that we
had previously identified by substrate utilization only. Molec-
ular results indicated that some of the isolates we had classified
as L. lactis were actually L. garvieae and that a few that were
identified as L. lactis were L. garvieae (5, 9).
The importance of confirming the identification of L. lactis
and L. garvieae by doing expensive and labor-intensive molec-
ular tests in a clinical microbiology laboratory is unknown. No
information is available about possible differences in antimi-
crobial susceptibility between these species. Such differences
might have an impact on disease therapy and would justify
additional effort in species identification. In this study, we
tested the antimicrobial susceptibilities of L. lactis and L. gar-
vieae to determine if there are differences in antimicrobial
susceptibility between these species.
Six isolates of L. lactis and 13 of L. garvieae, all from humans,
were recovered from the culture collection at the Centers for
Disease Control and Prevention. This is a small group of bac-
teria because of the rarity of infections caused by these bacte-
ria, but their identity is without question, which makes this
group more significant. Species identity was confirmed by mo-
lecular techniques that included whole-cell protein analysis
and DNA and RNA analysis. The type strains for L. lactis
(ATCC 19435) and L. garvieae (ATCC 43921) were also con-
firmed by molecular techniques and included in our tests. Nine
isolates that are presumed to be lactococci but could not be
assigned to a species were also tested.
Antimicrobial susceptibility testing was done by a broth di-
lution procedure. Bacteria were grown on Tryticase soy
agar–5% sheep blood plates (Becton Dickinson Microbiology
Systems, Cockeysville, Md.) for 18 h and suspended in Muel-
ler-Hinton broth to an optical density equivalent to a 0.5 Mc-
Farland turbidity standard. Ten microliters of this suspension
was inoculated into 10 ml of cation-adjusted Mueller-Hinton
broth supplemented with 5% lysed horse blood (16). SCEP-
TOR Streptococcus MIC panels (Becton Dickinson Microbi-
ology Systems) were inoculated by using the automated BD
SCEPTOR preparation station. MIC panels were incubated
for 18 h at 37
ЊC in a CO
2
incubator. Growth was read visually,
and the MIC was defined as the lowest concentration of a drug
that inhibited growth. E. faecalis (ATCC 29212 and ATCC
51299), Staphylococcus aureus (ATCC 29213), and Escherichia
coli (ATCC 35218) were included as controls (16).
The MICs are listed in Table 1. All MICs for the control
bacteria were within the ranges specified by the National Com-
mittee for Clinical Laboratory Standards (16). In addition to
being resistant to clindamycin, L. garvieae appeared to be
somewhat less susceptible to penicillin and cephalothin than
did L. lactis. For some members of both species, the MICs of
several other antimicrobial agents in the panel, including
amoxicillin-clavulanic acid, ampicillin, and norfloxacin, were
higher than those for members of the same species. One of the
unknown Lactococcus spp. had susceptibility results similar to
those of L. garvieae, and eight had results similar to those of L.
lactis.
L. lactis and
L. garvieae differed in susceptibility to clinda-
mycin. L. garvieae was always more resistant to clindamycin
* Corresponding author. Mailing address: Childhood and Vaccine
Preventable Diseases Branch, Centers for Disease Control and Pre-
vention, Atlanta, GA 30333. Phone: (404) 639-2417. Fax: (404) 639-
3123.
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(MIC,
Ն2 g/ml) than was L. lactis, and L. lactis was always
susceptible (MIC,
Յ0.12 g/ml). We used this information to
determine if the two species could be differentiated by a Kirby-
Bauer-like test using clindamycin disks. The test was similar to
the vancomycin disk procedure that is used to identify Leu-
conostoc and Pediococcus spp. (6).
Several colonies of bacteria were spread on a Tryticase soy
agar–5% sheep blood plate, and a clindamycin disk (2
g) was
added to the plate. The diameter of the growth inhibition zone
was measured after 24 h of incubation at 37
ЊC in a CO
2
incu-
bator.
All of the bacteria that we confirmed as L. lactis had zones
of inhibition around the clindamycin disk of
Ն20 mm (average,
24 mm). All of the L. garvieae isolates had no zones of inhibi-
tion. The unknown lactococci that were susceptible to clinda-
mycin (eight isolates) by MIC determination had zones of
inhibition that were similar to that of L. lactis. The other
unknown lactococci had zones of inhibition similar to that of L.
garvieae. We have incorporated the clindamycin disk test in our
identification procedures to differentiate between L. lactis and
L. garvieae.
Our results indicate that there are differences in antimicro-
bial susceptibility between L. lactis and L. garvieae. These dif-
ferences may have an impact on the choice of antimicrobial
therapy, especially if clindamycin is being considered. For both
species, and other human isolates of unidentified lactococci,
the MICs of several of the antimicrobial agents included in the
SCEPTOR Streptococcus MIC panel are higher, but these
organisms are as susceptible to these antimicrobial agents as
are the enterococci (16, 24), the group of bacteria most often
confused with lactococci. Resistance to vancomycin was not
observed in this group of lactococci. Although previous inves-
tigators have reported that vancomycin resistance does occur
in lactococci (11), subsequent reevaluation of the identity of
these bacteria at the Centers for Disease Control and Preven-
tion revealed that they were not Lactococcus species but Pe-
diococcus species. Vancomycin resistance, therefore, has yet to
be found in bacteria that can be confirmed as Lactococcus
species.
The antimicrobial susceptibility results support the impor-
tance of differentiating between L. lactis and L. garvieae. Dif-
ferentiation between these species can be simplified by incor-
porating sensitivity to clindamycin into an identification
scheme. This test must be limited to differentiation between L.
lactis and
L. garvieae, since nearly all of the unknown
Lacto-
coccus isolates had clindamycin susceptibilities that were sim-
ilar to that of L. lactis and one unknown isolate had a suscep-
tibility similar to that of L. garvieae. However, if the physiologic
reactions indicate that the isolate is either L. lactis or L. gar-
vieae, a clindamycin disk test result or the MIC of clindamycin
can be used to differentiate between these two species.
We thank M. D. Collins, Agricultural and Food Research Council,
Reading Laboratory (Shinfield, Reading, United Kingdom), for the
RNA and DNA results.
REFERENCES
1. BeimfohR, C., A. Krause, R. Amann, W. Ludwig, and K. H. Schleifer. 1993.
In situ identification of lactococci, enterococci and streptococci. Syst. Appl.
Microbiol. 16:450–456.
2. Betzl, D., W. Ludwig, and K. H. Schleifer. 1990. Identification of lactococci
and enterococci by colony hybridization with 23S rRNA-targeted oligonu-
cleotide probes. Appl. Environ. Microbiol. 56:2927–2929.
3. Cancilla, M. R., I. B. Powell, A. J. Hillier, and B. E. Davidson. 1992. Rapid
genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed
polymerase chain reaction with
32
P and fluorescent labels. Appl. Environ.
Microbiol.
58:1772–1775.
4. Collins, M. D., J. A. E. Farrow, B. A. Phillips, and O. Kandler. 1983.
Streptococcus garvieae sp. nov. and
Streptococcus plantarum sp. nov. J. Gen.
Microbiol. 129:3427–3431.
5. Elliott, J. A., M. D. Collins, N. E. Pigott, and R. R. Facklam. 1991. Differ-
entiation of Lactococcus lactis and Lactococcus garvieae from humans by
comparison of whole-cell protein patterns. J. Clin. Microbiol. 29:2731–
2734.
6.
Facklam, R. R., and J. A. Washington III. 1991.
Streptococcus and related
catalase-negative gram-positive cocci, p. 238–257. In A. Balows, W. J. Hau-
sler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual
of clinical microbiology, 5th ed. American Society for Microbiology, Wash-
ington, D.C.
7. Garver, K. I., and P. M. Muriana. 1993. Detection, identification and char-
acterization of bacteriocin-producing lactic acid bacteria from retail food
products. Int. J. Food Microbiol. 19:241–258.
8. Garvie, E. I. 1978. Streptococcus raffinolactis (Orla-Jensen and Hansen):
a group N streptococcus found in raw milk. Int. J. Syst. Bacteriol. 28:190–
193.
9. Garvie, E. I., and J. A. E. Farrow. 1982. Streptococcus lactis subsp. cremoris
(Orla-Jensen) comb. nov. and
Streptococcus lactis subsp.
diacetilactis (Ma-
tuszewski et al.) nom. rev., comb. nov. Int. J. Syst. Bacteriol. 32:453–455.
10. Garvie, E. I., J. A. E. Farrow, and B. A. Phillips. 1981. A taxonomic study of
some strains of streptococci that grow at 10
ЊC but not at 45ЊC, including
Streptococcus lactis and Streptococcus cremoris. Zentralbl. Bakteriol. Para-
sitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe C 2:151–165.
11. Green, M., R. M. Wadowsky, and K. Barbadora. 1990. Recovery of vanco-
mycin-resistant gram-positive cocci from children. J. Clin. Microbiol. 28:484–
488.
12. Hashimoto, H., H. Kawakami, K. Tomokane, Z. Yoshii, G. Hahn, and A.
Tolle.
1979. Isolation and characterization of motile group N streptococci. J.
Fac. Appl. Biol. Sci. Hiroshima Univ. 18:207–216.
13. Klijn, N., A. H. Weerkamp, and W. M. de Vos. 1991. Identification of
mesophilic lactic acid bacteria by using polymerase chain reaction-amplified
variable regions of 16S rRNA and specific DNA probes. Appl. Environ.
Microbiol. 57:3390–3393.
14. Ko
¨hler, G., W. Ludwig, and K. H. Schleifer.
1991. Differentiation of lacto-
cocci by rRNA gene restriction analysis. FEMS Microbiol. Lett. 84:307–312.
15. Mannion, P. T., and M. M. Rothburn. 1990. Diagnosis of bacterial endocar-
ditis caused by Streptococcus lactis and assisted by immunoblotting of serum
antibodies. J. Infect. 21:317–326.
16. National Committee for Clinical Microbiology Standards. 1993. Methods
for dilution antimicrobial susceptibility tests for bacteria that grow aerobi-
cally. Approved standard M7-A3. National Committee for Clinical Labora-
tory Standards, Villanova, Pa.
17. Nzouzi, N. L., M. F. Guerin, and D. H. Hayes. 1992. Comparison of elec-
trophoretic distribution patterns of ribosomal RNA gene restriction frag-
ments and of ribosomal subunit proteins of lactococci, streptococci, and
pediococci. Biochimie 74:1007–1017.
18. Ramos, M. S., and S. K. Harlander. 1990. DNA fingerprinting of lactococci
and streptococci used in dairy fermentations. Appl. Microbiol. Biotechnol.
34:
368–374.
19.
Rodrigues, U. M., M. Aguirre, R. R. Facklam, and M. D. Collins. 1991.
Specific and intraspecific molecular typing of lactococci based on polymor-
phism of DNA encoding rRNA. J. Appl. Bacteriol. 71:509–516.
20. Salama, M., W. Sandine, and S. Giovannoni. 1991. Development and appli-
TABLE 1. Antimicrobial susceptibilities of L. lactis and L. garvieae
Antimicrobial agent(s)
MIC range (
g/ml) for strains of:
L. lactis
L. garvieae
Vancomycin
Յ1.0
Յ1.0
Amoxicillin-clavulanic acid
Յ0.12/0.06–1.0/0.48 0.48/0.24–1.0/0.48
Ampicillin
Յ0.12–0.24
0.48
Penicillin
0.12–0.24
1.0
a
Chloramphenicol
2.0–4.0
2.0–4.0
Clindamycin
Յ0.12
Ն8.0
b
Erythromycin
Յ0.12
Յ0.12
Cephalothin
Յ1.0–4.0
4.0–8.0
Cefuroxime
Յ4.0
Յ4.0
Ciprofloxacin
Յ1.0–4.0
Յ1.0–2
Norfloxacin
Յ4.0–16.0
Յ4.0–16.0
Trimethoprim-sulfamethoxazole
Յ1.0/19
Յ1.0/19
Tetracycline
Յ2.0
Յ2.0–Ն8.0
a
One isolate required an MIC of penicillin of 0.24
g/ml.
b
One isolate required an MIC of clindamycin of
Ն2.0 g/ml.
V
OL
. 34, 1996
NOTES
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cation of oligonucleotide probes for identification of
Lactococcus lactis,
subsp. cremoris. Appl. Environ. Microbiol. 57:1313–1318.
21. Schleifer, K. H., J. Kraus, C. Dvorak, R. Kilpper-Ba
¨lz, M. D. Collins, and W.
Fischer.
1985. Transfer of Streptococcus lactis and related streptococci to the
genus Lactococcus gen. nov. Syst. Appl. Microbiol. 6:183–195.
22. Tanskanen, E. I., D. L. Tulloch, A. J. Hillier, and B. E. Davidson. 1990.
Pulsed-field gel electrophoresis of SmaI digests of lactococcal genomic
DNA, a novel method of strain identification. Appl. Environ. Microbiol.
56:
3105–3111.
23. Wood, H. F., K. Jacobs, and M. McCarty. 1985. Streptococcus lactis isolated
from a patient with subacute bacterial endocarditis. Am. J. Med. 18:345–347.
24. Yamane, N., and R. N. Jones. 1991. In vitro activity of 43 antimicrobial
agents tested against ampicillin-resistant enterococci and gram-positive spe-
cies resistant to vancomycin. Diagn. Microbiol. Infect. Dis. 14:337–345.
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