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Journal of Food Protection, Vol. 70, No. 2, 2007, Pages 412–418 Copyright ᮊ, International Association for Food Protection

Antibiotic Susceptibility Profiles of Lactobacillus reuteri and Lactobacillus fermentum

MARIA EGERVA¨ RN,1,2* MORTEN DANIELSEN,3 STEFAN ROOS,2 HANS LINDMARK,1 AND SVEN LINDGREN1

1National Food Administration, P.O. Box 622, SE-751 26 Uppsala, Sweden; 2Department of Microbiology, Swedish University of Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Sweden; and 3Chr. Hansen A/S, Bøge Alle´ 10–12, DK-2970 Hørsholm, Denmark

MS 06-201: Received 6 April 2006/Accepted 26 August 2006 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/412/1678833/0362-028x-70_2_412.pdf by guest on 25 September 2021 ABSTRACT

Lactobacillus reuteri and Lactobacillus fermentum, which are commonly used as food processing aids and probiotics, can potentially act as reservoirs of antibiotic resistance genes. Acquired resistance genes may be transferred via the food chain or in the gastrointestinal tract to pathogenic bacteria. Knowledge of the distributions of antibiotic MICs for a species is needed when using a phenotypic method to assess the presence of acquired resistance genes. In the present study, 56 L. reuteri and 56 L. fermentum strains that differed by source and spatial and temporal origin were assessed for antibiotic susceptibility using an Etest kit and a broth microdilution protocol. L. fermentum strains displayed a uniform distribution of MICs for all six antibiotics tested. L. reuteri strains had a bimodal distribution of MICs or a distribution with MICs above the test range for 7 of the 14 antibiotics tested. Genetic relatedness was observed among L. reuteri strains with high MICs for both ampicillin and tetracycline and among strains with high MICs for both erythromycin and clindamycin. Results obtained with the Etest and the broth microdilution method corresponded well with each other. Thus, further research may make it possible to define microbiological breakpoints for distinguishing between strains with and without acquired resistance genes.

Antibiotic-resistant microorganisms are an increasing milk products (20). Both species are used as probiotics (5, medical problem primarily attributed of the overuse of an- 26, 28). tibiotics. The concern is that the use of antibiotics in the Antibiotic susceptibility of L. reuteri and L. fermentum food chain, mainly in food-producing animals, has contrib- has previously been reported for only a few strains in stud- uted to the development and spread of resistant bacteria in ies using various testing procedures (6, 25, 38, 42, 45). the environment (39). Lactic acid bacteria (LAB) have a Thus, there is limited information on the ranges of suscep- long history of safe use as food-processing aids, and as tibility in these species. The main objective of the present probiotics LAB are associated with health benefits (35). study was to determine antibiotic susceptibility profiles of Antibiotic resistance in LAB has garnered increased atten- L. reuteri and L. fermentum of different origins in terms of tion during recent years (11–13, 17, 38, 46). Because of MIC distributions obtained from results of the Etest and a their broad environmental distribution, LAB may function broth microdilution assay. An additional objective was to as reservoirs of antibiotic resistance genes that can be trans- investigate the genetic relatedness of strains with atypical ferred via the food chain or within the gastrointestinal tract MICs by using repetitive extragenic palindromic PCR (rep- to other bacteria, including human pathogens (9, 40). Cur- PCR) genomic fingerprinting. rent knowledge of intrinsic antibiotic resistance of several MATERIALS AND METHODS species of LAB in food, feed, and probiotics is limited, which complicates the recognition of strains harboring ac- Bacterial strains. To achieve high strain diversity, only one quired resistance genes. By comparing a collection of strain from each sample was included in the study unless the ge- strains belonging to the same species, the ranges of anti- notypes of the strains in the samples were known to be different. biotic susceptibility can be established, facilitating the de- The 56 L. reuteri strains included in this study were obtained from termination of rational microbiological breakpoints (44). BioGaia AB (Stockholm, Sweden; 40 strains), the Department of Microbiology at the Swedish University of Agricultural Sciences Lactobacillus reuteri and Lactobacillus fermentum are (9 strains), the BCCM/LMG Bacteria Collection (Ghent Univer- (20) obligate heterofermentative lactobacilli belonging to sity, Ghent, Belgium; 4 strains), and the Chr. Hansen Culture Col- the L. reuteri phylogenetic group (36); these LAB occur lection (Hørsholm, Denmark; 3 strains). The strains were origi- naturally in the gastrointestinal tract of humans and other nally isolated from humans (24 strains), rodents (7 strains), pigs warm-blooded animals (19). L. reuteri and L. fermentum (7 strains), birds (6 strains), cows (4 strains), horses (2 strains), are associated with lactic acid fermentation of sourdough cats (2 strains), dogs (2 strains), and monkeys (1 strain) or were (43), and L. fermentum is commonly present in fermented undefined (1 strain) (Fig. 1). The human strains were isolated from intestines or feces (10 strains), breast milk (8 strains), the vagina (4 strains), and saliva (2 strains). The 56 L. fermentum strains * Author for correspondence. Tel: ϩ46 18 17 53 15; Fax: ϩ46 18 17 14 included were obtained from the Chr. Hansen Culture Collection 94; E-mail: [email protected]. and were originally isolated from Egyptian dairy products (49 J. Food Prot., Vol. 70, No. 2 ANTIBIOTIC SUSCEPTIBILITY OF HETEROFERMENTATIVE LACTOBACILLI 413 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/412/1678833/0362-028x-70_2_412.pdf by guest on 25 September 2021

FIGURE 1. Dendrogram based on the (GTG)5-PCR–generated genomic fingerprints of 56 L. reuteri strains from various sources, time periods, and geographical locations. MICs (micrograms per milliliter) of tetracycline (TET), erythromycin (ERM), and ampicillin (AMP) were determined with the Etest. The letters A and B indicate different genotypic groups. strains), Swiss cheese (1 strain), and French cheese (1 strain) or quencing Kit (Applied Biosystems, Foster City, Calif.). The se- were undefined (5 strains). The Egyptian strains isolated between quences obtained were subjected to a BLAST search in the 1997 and 2003 originated from whey (1 strain), raw buffalo, cow, GenBank database (National Center for Biotechnology Informa- or goat milk (9 strains), fermented milk (zabady) (6 strains), Ras tion, Bethesda, Md.). The identity of strains deposited in the Chr. cheese (19 strains), Domiatti cheese (5 strains), Mish cheese (1 Hansen Culture Collection as L. fermentum was confirmed either strain), and Feta cheese (7 strains) or were undefined (1 strain). by sequencing of approximately 500 bp of the 16S rRNA gene The L. fermentum type strain (ATCC 14931) and the ATCC 9338 using conserved primers 616V (5Ј-AGRGTTTGATYCKGGCT- strain were first reported in 1919 and 1925, respectively. CAG-3Ј) and 610R (5Ј-ACCGCGGCTGCTGGCAC-3Ј) for 37 strains or by a species-specific PCR assay (37) of 19 strains (data Species identification and subtyping. Bacteria were grown overnight on deMan Rogosa Sharpe (MRS; Oxoid, Basingstoke, not shown). UK) agar at 37ЊC in an anaerobic atmosphere (AneroGen, Oxoid). Bacterial strains were subtyped using genomic fingerprinting Chromosomal DNA was extracted with the DNeasy Tissue Kit methods. Chromosomal DNA was extracted as described above. (Qiagen, Valencia, Calif.) according to the manufacturer’s instruc- For the L. reuteri strains, rep-PCR was performed using the prim- tions. Species identification of L. reuteri strains was confirmed by er (GTG)5 according to a program described by Versalovic et al. 16S rDNA sequence analysis (data not shown). A part of the 16S (41). The PCR products were separated by electrophoresis at 2.5 rDNA gene (1,411 bp) was amplified by PCR (30 cycles of 94ЊC V/cm in a 1% agarose gel in 1ϫ Tris-acetate-EDTA buffer. The for 30 s, 54ЊC for 30 s, and 72ЊC for 80 s) using the primers gels were stained with ethidium bromide, and digitized images 16S.S (5Ј-AGAGTTTGATCCTGGCTC-3Ј) and 16S.R (5Ј- were captured under UV transillumination. The obtained rep-PCR CGGGAACGTATTCACCG-3Ј). The resulting PCR products fingerprints were analyzed with GelCompar II V3.5 software (Ap- were purified with the QIAquick PCR Purification Kit (Qiagen). plied Maths, Sint-Martens-Latem, Belgium). Band position toler- The purified fragments were partially sequenced (approximately ance and optimization were both set to 1%. A dendrogram was 500 bp) according to standard methods using the 16S.S primer, constructed using the Pearson correlation and the unweighted pair BigDye Terminator v3.1 sequence analyzer, and the Cycle Se- group method with arithmetic means. L. fermentum strains were 414 EGERVA¨ RN ET AL. J. Food Prot., Vol. 70, No. 2

TABLE 1. Distribution of MICs of six antibiotics for L. reuteri TABLE 2. Comparison of broth microdilution and Etest MICs of as determined by Etest and broth microdilution six antibiotics for the L. reuteri strains (only strains with on-scale MICs are included) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/412/1678833/0362-028x-70_2_412.pdf by guest on 25 September 2021

a Number of strains tested. b Data presented as log2 dilution steps; broth microdilution MICs are used as the reference values. c Shaded fields represent the agreement between the two methods, a ϩ Shaded fields indicate the range of MICs tested for each anti- defined as the MICs falling within 1 log2 dilution step (0, 1, biotic. MICs above the range are given as the concentration clos- and Ϫ1). est to the range. MICs below the range are given as the lowest concentration. b Number of strains tested. strips. For microdilution, the inoculated saline suspension was fur- c MDIL, broth microdilution assay. ther diluted 1:1,000 in LSM broth to obtain a final concentration of 3 ϫ 105 CFU/ml (verified by viable counts), and 100 ␮l was transferred to each well of the panel. The Etest agar plates and subtyped by the rep-PCR method using the box2AR primer as the microdilution panels were incubated anaerobically at 37ЊC for previously described (27). 48 h. The MIC was determined according to the manufacturers’ Antibiotic susceptibility testing. Antibiotic susceptibility recommendations. The end-point was defined as the lowest anti- testing was performed with Etest (AB Biodisk, Solna, Sweden) biotic concentration for which there was no visible bacterial and/or a broth microdilution assay. The susceptibility of a strain growth, i.e., the first microdilution well without pellet or where to a certain antibiotic was determined as the MIC (micrograms the edge of the elliptic inhibition zone intersected the Etest strip. per milliliter). The Etest was performed according to the manu- Etest MICs that fell between the log2 dilution steps were rounded facturer’s instructions. The antibiotic concentration gradient of the to the higher concentration. ␮ Etest strips was 0.016 to 256 g/ml for ampicillin, gentamicin, RESULTS kanamycin, netilmycin, streptomycin, amikacin, tetracycline, erythromycin, clindamycin, chloramphenicol, linezolid, and van- MIC determination. The susceptibility of L. reuteri comycin and 0.002 to 32 ␮g/ml for dalfopristin-quinupristin and to ampicillin, tetracycline, erythromycin, clindamycin, trimethoprim. The broth microdilution assay was performed with streptomycin, and gentamicin was assessed with the Etest ACE-ART VetMIC panels (National Veterinary Institute, Uppsala, and the broth microdilution assay and recorded as MIC dis- Sweden). The 96-well microtiter plates contained six antibiotics tributions (Table 1). A total of 258 antibiotic susceptibility dried in serial log2 dilution steps to determine MICs in the fol- tests for six antibiotics gave results as on-scale MICs with ␮ lowing ranges: 0.5 to 128 g/ml for oxytetracycline, 0.12 to 8 both methods during assessment of the L. reuteri strains ␮g/ml for clindamycin, 2 to 256 ␮g/ml for streptomycin, 0.12 to (Table 2). Comparison of Etest and broth microdilution re- 16 ␮g/ml for erythromycin, 0.5 to 32 ␮g/ml for gentamicin, and 0.12 to 8 ␮g/ml for ampicillin. sults revealed that the same MIC was obtained for 101 Ϯ Because no standard method exists for antibiotic susceptibil- (39%) of these tests. A difference of 1 log2 dilution step ity testing of lactobacilli, the following growth conditions were was obtained for 120 (47%) of the tests, whereas the re- Ϯ used. The strains were cultured on LAB susceptibility test medium maining 37 tests (14%) differed by 2 log2 dilution steps. (LSM) (24) agar at 37ЊC in an anaerobic atmosphere. LSM was The MIC agreement was less pronounced for ampicillin and recently developed for broth microdilution susceptibility testing clindamycin. Higher MICs were generally obtained by the of LAB and ensures adequate growth of the test organisms (24). microdilution method for all antibiotics except erythromy- LSM has no observed antagonistic effect on antibiotics such as cin (Table 2). trimethoprim and aminoglycosides; this effect sometimes occurs For ampicillin, a bimodal distribution was obtained by with commonly used growth media such as MRS. Colonies from Etest: one group (40 strains) had MICs up to 2 ␮g/ml and overnight cultures (20 to 24 h) were suspended in saline solution the other (16 strains) had MICs between 8 and 32 ␮g/ml (0.9% sodium chloride, wt/vol) to a density of McFarland standard 1 (Apogent, Remel, Lenexa, Kans.), which is equivalent to ap- (Table 1). MICs determined by broth microdilution were proximately 3 ϫ 108 CFU/ml (verified by viable counts). For the outside the test range for strains displaying MICs above 8 Etest, the inoculum was swabbed evenly with a cotton swab in ␮g/ml. A bimodal distribution was obtained for tetracycline three directions on 4-mm-thick LSM agar. The agar surface was by both methods, although the distributions were not clearly dried for approximately 15 min before applying the antibiotic separated by broth microdilution. The same 29 strains dis- J. Food Prot., Vol. 70, No. 2 ANTIBIOTIC SUSCEPTIBILITY OF HETEROFERMENTATIVE LACTOBACILLI 415

TABLE 3. Distribution of MICs of eight additional antibiotics TABLE 4. Distribution of MICs of six antibiotics for L. fermen- for L. reuteri as determined by Etest tum as determined by Etest

a Shaded fields indicate the range of MICs tested for each anti- biotic. MICs above the range are given as the concentration clos- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/412/1678833/0362-028x-70_2_412.pdf by guest on 25 September 2021 est to the range. MICs below the range are given as the lowest a Shaded fields indicate the range of MICs tested for each anti- concentration. biotic. MICs above the range are given as the concentration clos- b Number of strains tested. est to the range. MICs below the range are given as the lowest concentration. b Number of strains tested. Subtyping. The genetic relatedness of the L. reuteri c Dalfopristin-quinupristin. strains was investigated by rep-PCR genomic fingerprinting using the primer (GTG)5. The similarity index between the band patterns obtained for the type strain (DSM 20016T) played MICs in the upper range or above the test range was 87% for the four runs of rep-PCR (data not shown). with both methods. For erythromycin and clindamycin, the The DNA fragments obtained ranged in size from approx- majority of strains were clustered with MICs up to 2 ␮g/ imately 0.4 to 5 kb (Fig. 1). Two groups of strains with ml, whereas six strains had MICs above 256 ␮g/ml for both highly similar band patterns were obtained: one (group A) antibiotics (Etest). Two strains exhibited Etest MICs above contained 12 strains (including the type strain) and the other 256 ␮g/ml for erythromycin, clindamycin, and tetracycline (group B) contained 14 strains. The remaining strains dis- (Fig. 1). Independent of method, all strains clustered within played a wide range of band patterns, apart from five pairs of strains with highly similar band patterns. A unique rep- 3 or 4 log2 dilution steps for both streptomycin and gen- tamicin. PCR fingerprint was obtained for strain 5010, with a chlor- ␮ The susceptibility of 49 L. reuteri strains to an addi- amphenicol MIC of 128 g/ml. The majority of the strains tional eight antibiotics was determined by Etest (Table 3). in groups A and B were isolated from humans, including For dalfopristin-quinupristin, the majority of strains had 12 of the 15 strains isolated in Finland in 1997. All strains ␮ MICs between 0.25 and 1 ␮g/ml, whereas three of the in group B had Etest MICs above 256 g/ml for tetracy- ␮ strains (1048, 1068, and 8557:1) had MICs between 8 and cline and from 8 to 32 g/ml for ampicillin. The other two 16 ␮g/ml. These three strains had MICs above the test strains with higher MICs for these antibiotics were evenly scattered throughout the dendrogram. The pig was the dom- range for erythromycin and clindamycin. For chloramphen- inant host for the strains with Etest MICs above 256 ␮g/ icol, MICs ranged from 2 to 4 ␮g/ml, except for one strain ml for erythromycin and clindamycin. All these strains (5010) that had an MIC of 128 ␮g/ml. A uniform distri- were clustered together in the dendrogram (Fig. 1). bution was obtained for amikacin, netilmycin, and linezo- The 56 L. fermentum strains were subtyped by the rep- lid, with MICs of 4 to 64 ␮g/ml, 0.25 to 4 ␮g/ml, and 1 PCR method using the box2AR primer. Despite the diverse to 4 ␮g/ml, respectively. For kanamycin, all strains had origin of the strains, the species appeared homogenous. All MICs in the upper test range, and seven strains had MICs strains had many bands in common, but most strains could above the maximum test concentration. For vancomycin be distinguished by one or several unique bands (data not and trimethoprim, all strains had MICs above the test range. shown). Three L. reuteri strains were included in the rep- The susceptibility of L. fermentum to ampicillin, tet- PCR assay, and in contrast to the L. fermentum strains these racycline, erythromycin, clindamycin, streptomycin, and strains had unique rep-PCR fingerprints (data not shown). gentamicin was assessed by Etest and recorded as MIC dis- tributions (Table 4). For ampicillin, tetracycline, and clin- DISCUSSION damycin, the L. fermentum strains were clustered with Because of the limited information concerning the MICs up to 0.25 ␮g/ml, 4 ␮g/ml, and 0.12 ␮g/ml, respec- comparability of different methods for assessing antibiotic tively, which were below or in the lower range of corre- susceptibility of heterofermentative lactobacilli, the L. reu- sponding MICs for L. reuteri. A wider range was obtained teri strains were tested for their responses to six antibiotics for erythromycin, with MICs up to 4 ␮g/ml. All L. fer- with both the Etest and a broth microdilution assay. The mentum strains were tightly clustered for both streptomycin results of the two methods were in close agreement, with and gentamicin, although with 1 log2 dilution step higher 86% of the MICs within the accuracy limit of MIC standard Ϯ MICs than those for L. reuteri. tests, i.e., 1 log2 dilution step (8). However, the correla- 416 EGERVA¨ RN ET AL. J. Food Prot., Vol. 70, No. 2 tion differed depending on the antibiotic tested, with 58% were made to obtain a wide distribution in terms of source, agreement for ampicillin and up to 98% for the aminogly- time period, geographical location, and clonal diversity, cosides. which is necessary for a risk assessment of antibiotic resis- The susceptibility profiles of L. reuteri to 14 antibiotics tance in a bacterial species (15). The (GTG)5-PCR genomic revealed that the distribution of MICs was uniform for fingerprinting method has previously been successfully ap- streptomycin, gentamicin, amikacin, netilmycin, and line- plied for subtyping strains of lactobacilli (16). Except for zolid and bimodal for ampicillin (by Etest), tetracycline, the two groups of L. reuteri strains that had highly similar erythromycin, clindamycin, dalfopristin-quinupristin, and band patterns (groups A and B), most strains did not form chloramphenicol. For tetracycline and kanamycin, all clusters, indicating genetic heterogeneity and a lack of a strains had MICs in the upper test range or above the max- bias in compilation of the strains. The genetic diversity of imum concentration tested. The high MICs for vancomycin L. reuteri has previously been demonstrated by rep-PCR are in agreement with those from previous studies of L. fingerprinting in strains from murine intestines (32). reuteri (6, 25, 38) and may be due to peptidoglycan dipep- Comparison of MICs and (GTG) -PCR genomic fin- 5 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/412/1678833/0362-028x-70_2_412.pdf by guest on 25 September 2021 tides terminating with D-lactate instead of D-alanine, the gerprinting data revealed that the six strains with high MICs target precursor for vancomycin activity (4, 25). The low for erythromycin and clindamycin were tightly clustered, susceptibility to trimethoprim reported in the present study although they did not form a separate group, and that al- and by others (6, 25) is probably due to thymidine in the most all L. reuteri strains with high MICs for both ampi- growth medium, which antagonizes antibiotic activity (10). cillin and tetracycline were linked to the genotype of group The results for streptomycin and kanamycin are in ac- B. Further research would reveal whether the clustered cordance with previous results for L. reuteri (6, 38, 42) and strains harbor the same resistance determinant. Correlations other Lactobacillus species (11, 23, 46). The high MICs of between genotype and high MICs have been reported in streptomycin, kanamycin, and amikacin for all strains may several studies (1, 30, 31). The remaining strains with high be due to an intrinsic trait, increasing the MICs for ami- MICs for tetracycline that were scattered throughout the noglycosides. A higher susceptibility to gentamicin than to dendrogram appear to be distantly related. streptomycin and kanamycin also has been reported for oth- The susceptibility profiles revealed that L. fermentum, er Lactobacillus species (11). Ampicillin MICs of 8 to 32 in contrast to L. reuteri, had a uniform distribution of MICs ␮g/ml observed for a number of strains is in agreement for the six antibiotics tested. The high MICs of strepto- with previous results obtained by Klein et al. (25) but in mycin for all strains is in accordance with previous findings contrast with results obtained in other Lactobacillus studies, for other Lactobacillus species (11, 23). Even though no in which MICs up to 0.25 ␮g/ml (23) and 2 ␮g/ml (11) atypical MICs were obtained in the present study, the pres- have been reported for this antibiotic. The reason why one ence of plasmids encoding resistance to tetracycline and L. reuteri strain was approximately 30 times less sensitive erythromycin has been reported for L. fermentum (14, 18, to chloramphenicol than were the other strains was not de- 21). termined. However, a resistance determinant (cat-TC) to Phenotypic tests of L. reuteri and L. fermentum were this antibiotic has previously been identified in an L. reuteri applied to establish ranges of susceptibility to a series of strain (29). antibiotics. For strains generating nonuniform distributions The six strains with clearly higher MICs for erythro- of MICs, genetic characterization is needed to further in- mycin than the majority of the strains also deviated for vestigate differences in antibiotic susceptibility. By assess- clindamycin, indicating cross-resistance. Three of these ing the presence of transferable resistance genes in these strains (1048, 1068, and 8557:1) have previously been strains, microbiological breakpoints could subsequently be shown to harbor a plasmid-encoded gene (erm) for resis- defined for individual heterofermentative Lactobacillus spe- tance to these antibiotics (2, 3). The high MICs are due to cies. Current work is focusing on unveiling the underlying methylation of the 50S ribosomal subunit, which causes mechanisms generating atypical antibiotic MICs in L. reu- macrolide, lincosamide, and streptogramin B (MLSB) resis- teri. tance because the binding sites for these drugs overlap (33). Increased MICs to dalfopristin-quinupristin (a mixture of ACKNOWLEDGMENTS streptogramin A and B) might therefore be due to the pres- This work was performed as part of the EU project ACE-ART (CT- ence of an erm gene. 2003–506214). Financial support was provided by the European Com- The wide range of high MICs obtained for tetracycline mission, Sixth Framework Program. BioGaia AB and project partners who provided strains for the study are acknowledged. may indicate that the L. reuteri strains harbor different genes conferring diverse levels of susceptibility, as re- REFERENCES viewed by Chopra and Roberts (7). The high MICs ob- 1. Ahmed, K., G. Martinez, S. Wilson, R. Yoshida, R. Dhar, E. Mo- served for tetracycline are in accordance with previous kaddas, S. Kohno, V. O. Rotimi, and T. Nagatake. 2000. The prev- studies of the species (38, 42). A plasmid-encoded tetra- alence and clonal diversity of penicillin-resistant Streptococcus cycline-resistance gene, tet(W), has recently been identified pneumoniae in Kuwait. Epidemiol. Infect. 125:573–581. in L. reuteri strain ATCC 55730 (22). Several determinants 2. Ahrne´, S. E. Personal communication. 3. Axelsson, L. T., S. E. Ahrne´, M. C. Andersson, and S. R. 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