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Antimicrobial Agents and Chemotherapy, December 1999, p. 3039-3041, Vol. 43, No. 12
Area Bioquímica y Biología
Molecular, Universidad de la Rioja, 26004 Logroño,1 and Servicio de
Microbiología, Hospital Ramón y Cajal, 28034 Madrid,2 Spain
Received 14 December 1998/Returned for modification 29 April
1999/Accepted 19 September 1999
Testing of susceptibility to 13 antibiotics was performed with 90 isolates of Lactobacillus, Leuconostoc, and
Pediococcus. MICs at which 90% of the isolates tested were
inhibited by HMR3647, erythromycin, and ciprofloxacin were 0.015, 0.125 and 32 µg/ml, respectively. The penicillin MIC was Lactobacillus,
Leuconostoc, and Pediococcus spp. are commonly
found as natural microflora in the mucous membranes of humans and
animals, in dairy products, and on some plant surfaces (2, 21). Nevertheless, they are increasingly recognized as
opportunistic pathogens involved in human infections (1, 3, 5, 8, 10, 11, 14, 17, 22). Intrinsic resistance to glycopeptides is
well documented in these genera of lactic acid bacteria (LAB) (13,
26, 31); however, there are few reports on susceptibilities of
LAB to other antibiotics. The purpose of this work was to determine the
susceptibility pattern to 13 antibiotics in 90 LAB isolates: 60 Lactobacillus isolates (L. plantarum, 28;
L. paracasei, 17; L. brevis, 8; L. rhamnosus, 3; L. casei, 1; L. fermentum, 1; L. curvatus, 1; L. pentosus, 1); 18 Leuconostoc isolates (L. mesenteroides, 10; L. pseudomesenteroides, 1; L. oenos, 1; Leuconostoc spp., 6); and 12 Pediococcus isolates (P. pentosaceus, 10;
P. acidilactici, 2). They were identified as previously
recommended (6, 15, 15a) and by using the API50 CH system
(Biomérieux). MICs were determined by the National Committee
for Clinical Laboratory Standards agar dilution method
(19) using brain heart infusion agar (BHIA) and an atmosphere of 5% CO2. Results in BHIA were
compared with those obtained in Mueller-Hinton agar supplemented
with 5% sheep blood (MHA-B). Strains grew better in BHIA
than in MHA-B, and therefore, the former agar medium was used for
MIC determinations. Control strains used were Staphylococcus
aureus ATCC 29213 and Enterococcus faecalis ATCC 29212. Tested antibiotics were the following: ketolide HMR3647,
erythromycin A, clarithromycin, roxithromycin, azithromycin,
levofloxacin, and teicoplanin (all supplied by Hoechst Marion Roussel);
spiramycin and penicillin (Sigma); pristinamycin I (Rhône-Poulenc
Rorer); vancomycin (Elli Lilly); and ciprofloxacin (Bayer).
The susceptibility testing results are shown in Table
1 and 2.
The MICs at which 90% of isolates were inhibited (MIC90s) of the macrolides tested for 60 Lactobacillus isolates were
in the range of 0.03 to 0.5 µg/ml (Table 1). Similar data have been obtained by others (23, 26). The MIC90 of
HMR3647 for these isolates was 0.015 µg/ml, 3 dilutions lower than
that of erythromycin. One of the studied Lactobacillus
isolates, L. paracasei J19, was resistant to all macrolides
(MICs in the range of 16 to 256 µg/ml). The MIC of HMR3647 for this
isolate was 0.5 µg/ml, lower than those of macrolides and higher than
those found for susceptible isolates. Erythromycin resistance mediated
by plasmids has been previously reported for Lactobacillus
(7, 9, 20, 28), and different genes encoding methylases,
such as ermGT (28), ermBC
(7), and ermAM (20), have been
reported. Only one Lactobacillus isolate was susceptible to
vancomycin, and five were susceptible to teicoplanin. MICs of
penicillin for 16 of 60 Lactobacillus isolates (26.6%) were
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Activities of Ketolide HMR3647, Macrolides, and Other
Antibiotics against Lactobacillus,
Leuconostoc, and Pediococcus
Isolates
ABSTRACT
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16 µg/ml
against 26.2% of the studied Lactobacillus sp. isolates
and 50% of Lactobacillus plantarum. HMR3647 showed
excellent activity against these genera.
TEXT
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16 µg/ml, but no 
-lactamase activity was detected. MICs of
penicillin or ampicillin for most Lactobacillus isolates
referred to in the literature were 
2 µg/ml (12, 26). Ciprofloxacin had a poor activity against our Lactobacillus
isolates (MIC 4 µg/ml in 60% of them). L. plantarum showed the highest level of resistance to all tested
antibiotics except macrolides (Table 2). The respective
MIC90s of penicillin and ciprofloxacin were 128 and
32 µg/ml for L. plantarum and were 1 and 2 µg/ml for
L. paracasei. The modal MICs of penicillin and
ciprofloxacin for L. brevis were 4 µg/ml and 16 to
32 µg/ml, respectively (Fig. 1).
TABLE 1.
In vitro activities of different antibiotics against
LAB isolates from various generaa
TABLE 2.
In vitro activities of different antibiotics against
Lactobacillus spp.a
View larger version (21K):
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FIG. 1.
Graph of penicillin and ciprofloxacin MICs for 53 Lactobacillus isolates. White bars, L. plantarum;
black bars, L. paracasei; stippled bars, L. brevis.
All studied Leuconostoc isolates were susceptible to
macrolides, with MIC90s in the range of 0.03 to 0.5 µg/ml
(Table 1). The MIC90 of HMR3647 was 0.015 µg/ml, 3 dilutions lower than that of erythromycin. Similar results have
previously been reported (18, 26). Penicillin MICs were
always 2 µg/ml. Ciprofloxacin and levofloxacin were poorly active
against Leuconostoc, showing MICs of 4 µg/ml for 72.2 and 61.1% of the isolates, respectively.
For all but one of the studied Pediococcus isolates
erythromycin MICs were 0.25 µg/ml. The MIC90 of HMR3647
for these isolates was 0.015 µg/ml. Marie-Bigot et al.
(18) have reported similar results. P. acidilactici AR63 was the only macrolide-resistant Pediococcus isolate found in our study (MICs of 64
µg/ml). The MIC of HMR3647 for this isolate was 4 µg/ml. The
mechanism of resistance involved was studied by PCR, and a positive
result was obtained when degenerate erm primers were used
(4). Nevertheless, negative results were obtained when
specific primers for amplification of ermA, ermB,
ermC (25), or ermTR
(16), as well as msrA (30) and
mefA/E (24), were assayed. A
high-molecular-weight plasmid was observed in P. acidilactici AR63, and its putative relationship with macrolide
resistance was studied by filter conjugation using erythromycin-sensitive E. faecalis JH2-2 and E. faecium GE-1 as recipients. Results were negative in all cases.
Erythromycin resistance has previously been reported for
Pediococcus strains (26, 31). One
P. acidilactici strain with macrolide resistance related to a determinant homologous to the ermAM gene located on a
nontransferable 46 kb plasmid has previously been reported
(27).
For all Leuconostoc and Pediococcus isolates
penicillin MICs were 2 µg/ml. MIC ranges of ciprofloxacin for
Pediococcus and Leuconostoc were 32 to 64 and 1 to 32 µg/ml, respectively. All isolates of both genera showed
high-level glycopeptide resistance (MIC 512 µg/ml). This
group of organisms is sometimes mistaken for enterococci, but the
latter genus is usually susceptible to vancomycin.
MICs were determined in MHA-B medium and were compared with those obtained in BHIA for 78 LAB isolates in this study. MICs were similar in both media, although they tended to be 1 dilution higher in BHIA (data not shown).
In summary, with few exceptions, the LAB tested in this work were
susceptible to all macrolides and the ketolide HMR3647. For all tested
Pediococcus and Leuconostoc isolates
penicillin MICs were 2 µg/ml, whereas for 26% of
Lactobacillus isolates MICs were 16 µg/ml. Ciprofloxacin
MICs for all Pediococcus isolates, 60% of
Lactobacillus isolates, and 72% of Leuconostoc
isolates were 4 µg/ml. Ecological balance modifications of the
natural species in the human gut flora by antibiotic action are a
matter of growing concern. This paper shows that differences in
susceptibility to widely used groups of antibiotics occur among
different genera, as well as among species within a given genus.
Therefore, population replacement derived from antibiotic pressure
would not come unexpectedly. Thus, for instance, L. plantarum (resistant to penicillin and ciprofloxacin) might
replace L. paracasei (susceptible to both types of drugs).
The clinical or evolutionary implications of these changes are
currently unknown. More attention should be paid to the selection of
naturally (or acquired) resistant microorganisms that are members of
the normal human microbiota.
(Part of this work was presented at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997 [29].)
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant of Hoescht Marion Rousell and a grant from the Fondo de Investigaciones Sanitarias of Spain (FIS 98/282).
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FOOTNOTES |
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* Corresponding author. Mailing address: Area de Bioquímica y Biología Molecular, Avenida de la Paz 105, Universidad de La Rioja, 26004 Logroño, Spain. Phone: 34-941-299284. Fax: 34-941-299274. E-mail: carmen.torres{at}daa.unirioja.es.
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