JAC Advance Access originally published online on March 16, 2007
Journal of Antimicrobial Chemotherapy 2007 59(5):900-912; doi:10.1093/jac/dkm035
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use
1 Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany 2 Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium 3 Laboratory of Medical Microbiology, University of Antwerp, Antwerp, Belgium 4 Department of Clinical Microbiology, Central Hospital, Växjö, Sweden 5 Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
* Corresponding author. Tel: +49-3943-679-247; Fax: +49-3943-679-207; E-mail: i.klare{at}rki.de
Received 30 October 2006; returned 12 December 2006; revised 17 January 2007; accepted 22 January 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
Objectives: To determine MICs of 16 antimicrobials representing all major classes for 473 taxonomically well-characterized isolates of lactic acid bacteria (LAB) encompassing the genera Lactobacillus, Pediococcus and Lactococcus. To propose tentative epidemiological cut-off (ECOFF) values for recognizing intrinsic and acquired antimicrobial resistances in numerically dominant species.
Methods: On the basis of depositors' information, LAB were grouped in categories of probiotic, nutritional, probiotic or nutritional research, human and animal isolates and tested for their antibiotic susceptibilities by broth microdilution using LAB susceptibility test medium (LSM). Tentative ECOFFs were defined according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing. Isolates showing acquired antimicrobial resistance(s) were selected for PCR-based detection of resistance gene(s) and in vitro conjugative transfer experiments.
Results: Tentative ECOFF values of 13 antibiotics were determined for up to 12 LAB species. Generally, LAB were susceptible to penicillin, ampicillin, ampicillin/sulbactam, quinupristin/dalfopristin, chloramphenicol and linezolid. LAB exhibited broad or partly species-dependent MIC profiles of trimethoprim, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and fusidic acid. Three probiotic Lactobacillus strains were highly resistant to streptomycin. Although erythromycin, clindamycin and oxytetracycline possessed high antimicrobial activities, 17 Lactobacillus isolates were resistant to one or more of these antibiotics. Eight of them, including six probiotic and nutritional cultures, possessed erm(B) and/or tet(W), tet(M) or unidentified members of the tet(M) group. In vitro intra- and interspecies filter-mating experiments failed to show transfer of resistance determinants.
Conclusions: Finding of acquired resistance genes in isolates intended for probiotic or nutritional use highlights the importance of antimicrobial susceptibility testing in documenting the safety of commercial LAB.
Keywords: lactic acid bacteria , antimicrobial susceptibility testing , broth microdilution , MIC ranges , epidemiological cut-off values
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
The genera Lactobacillus, Pediococcus and Lactococcus belong to the lactic acid bacteria (LAB) and are part of the commensal intestinal flora of humans and animals.1,2 Strains of these genera are frequently used on a large-scale as starter cultures in food industries (e.g. in the production of fermented milk products or sausages) or as probiotics.3–6 In probiotic applications, selected LAB strains mainly belonging to the genera Lactobacillus and Bifidobacterium are used as food supplements that may favourably influence the intestinal flora of human and animal hosts, e.g. by competitive exclusion of gastrointestinal pathogens, stimulation of the immune response or antimutagenic and anticancerogenic activities.7–9 On the other hand, lactobacilli, pediococci and lactococci have also been implicated in rare cases of human infections.10–14
Because of their long-time use in various food and feed preparations, LAB have been given the so-called GRAS status (generally recognized as safe).15–19 In practice, this means that such LAB strains are food-grade organisms without imposing a health risk for the consumers or the environment. However, there are several studies that have documented the presence and expression of virulence genes and/or antibiotic resistance genes in food-associated LAB.16,19–23 When located on mobile genetic elements such as plasmids or (conjugative) transposons, antibiotic resistance traits can potentially be transferred to the human or animal commensal flora and to pathogenic bacteria temporarily residing in the hosts. Therefore, it is very important to verify that probiotic and nutritional LAB strains consumed on a daily basis worldwide lack acquired antimicrobial resistance properties prior to considering them safe for human and animal consumption.
Antimicrobial susceptibility testing of LAB can be performed by several methods, including agar disc diffusion and agar overlay disc diffusion, Etest, agar dilution and broth macro- and microdilution.24 In general, dilution methods and the Etest are preferred over diffusion tests providing inhibition zones, as the former techniques allow determination of MICs of antimicrobials that result in a more reliable indication of the intrinsic or acquired nature of a given resistance phenotype. However, since many LAB require special growth conditions in terms of medium acidity and carbohydrate supplementation, conventional media such as Mueller–Hinton and Iso-Sensitest (IST) agar or broth are often not suitable for susceptibility testing of lactobacilli, pediococci and lactococci. Previously, we developed a broth formula referred to as the LAB susceptibility test medium (LSM) for determining MICs of antibacterial agents of all major antibiotic classes for Lactobacillus, Pediococcus and Lactococcus species.24 The aim of the present study is to further validate the use of the LSM formulation for susceptibility testing by broth dilution of an extensive collection of 473 well-characterized isolates encompassing 24 species of the genera Lactobacillus, Pediococcus and Lactococcus against 16 antimicrobials. Isolates were of human, animal or nutritional category and also included several cultures that are currently under investigation as probiotic candidates or that are already on the market in commercial probiotic products. Secondly, from the large data set obtained from this study, tentative species- or group-specific epidemiological cut-off (ECOFF) values of MICs were defined for most of the antimicrobial agents tested, to allow better differentiation between wild-type (WT) isolates (lacking acquired antimicrobial resistance traits) and non-wild-type (NWT) isolates (containing one or more acquired antibiotic resistance traits). Recognition of isolates with acquired antibiotic resistances is very important because of the potential transferability of resistance traits to other bacteria, including pathogenic microbes.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
Bacterial isolates
We tested 473 isolates belonging to the genera Lactobacillus (416 isolates of 21 species) and Pediococcus (49 isolates of 2 species) and to Lactococcus lactis (8 isolates). Species designations and categories of the 473 non-enterococcal LAB isolates included in this study are listed in Table 1. Isolates were obtained in the framework of the EU project ‘Biosafety Evaluation of Probiotic Lactic Acid Bacteria Used for Human Consumption’ (PROSAFE) and were classified into probiotic (i.e. isolates effectively used in probiotic products; n = 129), nutritional (i.e. isolates used in food products as starter cultures without a specific probiotic claim; n = 27), research (i.e. isolates under investigation as probiotic or nutritional candidates; n = 24), human (i.e. isolates from healthy humans and human clinical isolates; n = 288) and animal (i.e. isolates from healthy animals; n = 5) categories based on the descriptive information provided by the respective depositors.25 The full official designations of the PROSAFE (PRSF-) isolates were abbreviated for practical reasons in the present paper and only the abbreviations of the corresponding genus (e.g. L, Lactobacillus) and the running number of the isolates are named here. For instance, the short designation of the Lactobacillus crispatus isolate ‘L-295’ stands for ‘PRSF-L-295’. All isolates were re-identified up to the species level using a polyphasic identification strategy as previously described.26 Isolates were routinely cultured at 37°C on de Man, Rogosa, Sharpe (MRS) agar plates (Oxoid) under aerobic conditions (but a 5% CO2-enriched atmosphere can be favourable for some isolates) from which fresh cultures were prepared for inoculation of the broth microdilution test.24
|
Antimicrobial susceptibility testing
MICs of 16 antimicrobial agents encompassing nearly all important classes were determined by microdilution using the newly developed and standardized LSM broth formulation essentially consisting of a mixture of IST broth (90%) and MRS broth (10%) adjusted to pH 6.7 as previously described.24
The following antimicrobials were tested in the concentration ranges (mg/L) given in parentheses: penicillin G (0.032–64), ampicillin (0.032–64), sulbactam/ampicillin (sulbactam was tested as fixed concentration of 8 mg/L: 0.032–64), gentamicin (1–2048), streptomycin (2–4096), vancomycin (0.125–256), teicoplanin (0.125–256), quinupristin/dalfopristin (tested as 30:70 ratio: 0.032–64), erythromycin (0.016–32), clindamycin (0.032–32), oxytetracycline (0.063–128), chloramphenicol (0.125–256), fusidic acid (0.063–128), linezolid (0.016–32), trimethoprim (0.25–512) and trimethoprim/sulfamethoxazole (tested as 1:19 ratio: 0.25–512). In Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)], concentrations that were outside the test range of the corresponding antimicrobial are marked in grey.
Epidemiological MIC cut-off values for differentiation of WT and NWT isolates
MIC50s, MIC90s and tentative ECOFF values for differentiation between WT and NWT isolates were determined to the antimicrobials named above for the following 12 LAB species represented by 10 or more isolates: Lactobacillus acidophilus, L. crispatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Pediococcus acidilactici and Pediococcus pentosaceus. MIC50 and MIC90 are defined as MICs inhibiting 50% and 90% of the isolates tested, respectively, and ECOFF values were determined from MIC distributions for each species–drug combination as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)27–30 (Table S1). WT and NWT isolates are characterized by their MIC values as follows: WT 
z mg/L, NWT > z mg/L; z is the ECOFF value of the corresponding antibiotic for the species in question. According to EUCAST definitions, a WT isolate of a microbial species is characterized by the absence of acquired and/or mutational resistance mechanisms to the antimicrobial agent in question, whereas a NWT isolate is defined by the presence of such resistance mechanisms. WT and NWT microorganisms may or may not respond clinically to treatment with antimicrobial agents.27
Detection of antibiotic resistance genes in NWT LAB isolates
Isolates displaying MICs that were situated in the NWT subpopulation of the MIC distribution profiles were selected for PCR-based detection of genes conferring resistance to the aminoglycoside streptomycin [aad(E) gene], to erythromycin (erm genes) and oxytetracycline (tet genes), respectively (Table 2). DNA of LAB isolates was isolated by using a DNeasy Tissue Kit (Qiagen) and amplification of the corresponding gene fragments was performed in a DNA Engine Thermal Cycler (PTC-200, MJ Research). The PCR mixtures consisted in each case of 0.25 µL of the two primers, 0.25 µL of DNA-containing DNeasy beads and 24.25 µL of Aqua bidest. The primer sequences, annealing temperatures and amplicon sizes are listed in Table 2. PCR-based detection of the genes aad(E), erm(A), erm(B), erm(C), tet(K), tet(L) and tet(M), and of further members of the tet(M) group, was performed under the following conditions: 95°C for 1 min; 94°C for 30 s, 55°C for 30 s and 72°C for 30 s (30 cycles); and 72°C for 4 min. Detection of the genes tet(O), tetB(P), tet(Q), tet(S), tet(T) and tet(W) was conducted using the PCR conditions previously described.33 Amplification products were detected by electrophoresis in a 1.4% agarose gel (Type II: Medium EEO, Sigma) and subsequent staining in ethidium bromide solution.
|
In vitro experiments on intra- and interspecies transfer of antibiotic resistance genes
Selected LAB isolates of probiotic, nutritional and research categories from the PROSAFE strain collection (marked in Table 5 by footnote e) that represented NWT isolates with acquired antibiotic resistance(s) were used as donors for intra- and interspecies in vitro transfer experiments by conjugation (filter-mating). Intraspecies recipients were generated by a multi-step approach to obtain strains with high-level resistance to rifampicin and fusidic acid, starting from suitable susceptible isolates of the corresponding Lactobacillus species from the PROSAFE collection. The well-documented strains Enterococcus faecium 64/3 and Enterococcus faecalis JH2-2 displaying high-level resistance to rifampicin/fusidic acid were chosen as recipients for interspecies gene transfer experiments. Possible transconjugants were identified in several steps, selecting for the selective and non-selective markers.
In each case, 1 mL of the fresh logarithmic growth phase cultures of the donor and recipient strains were mixed and filtered (sterile 0.45 µm Millipore membrane filter). The filtrate was filtered again by the addition of 2 mL of sterile peptone physiological saline (PPS = 0.85% saline with 0.1% neutralized bacterial peptone; useful for tight contact of the cells36) using the same membrane filter. Subsequently, the bacteria-loaded filter was cultured overnight on an antibiotic-free nutrient agar plate optimized for the recipient's growth. Bacteria were washed from the filter with 2 mL of PPS, and suitable dilutions of this mating mixture were spread on selective agar plates (containing oxytetracycline/rifampicin or erythromycin/rifampicin or streptomycin/rifampicin, respectively). After incubation for up to 72 h, single colonies were streaked on agar plates containing the selective antimicrobial (oxytetracycline, erythromycin or streptomycin) and thereafter on agar plates with the non-selective antibiotic (fusidic acid). The colonies grown on the latter nutrient medium were considered possible transconjugants and were further characterized by MIC determination, PCR-based detection of resistance genes, PFGE and fingerprinting by PCR-based amplification of repetitive bacterial DNA elements using (GTG)5 primers [(GTG)5-PCR].37,38
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
MIC distribution profiles
The complete distribution of MICs of 16 antimicrobial agents tested for 473 LAB isolates has been made available as online supplementary data (Table S1). Additionally, MIC50s, MIC90s, tentative ECOFF values and the presence of intrinsic and/or acquired antibiotic resistance(s) in the corresponding LAB species are indicated in Table S1. Table 3 summarizes the data from Table S1 for the 12 Lactobacillus and Pediococcus species represented by 10 or more isolates. For some of these species, ECOFFs could not be defined because the MIC distributions were poorly delineated (e.g. for trimethoprim and trimethoprim/sulfamethoxazole) or truncated at the low end of dilutions (Table S1).
|
Altogether, of those 12 LAB species that are represented by at least 10 isolates, acquired antibiotic resistances were only found to 4 out of 16 antimicrobials tested (streptomycin, erythromycin, clindamycin and oxytetracycline). Of the 383 Lactobacillus isolates listed in Table 4 belonging to different categories, in each case, 3 isolates (0.8%) were resistant to streptomycin, erythromycin or clindamycin and 12 isolates (3.1%) were resistant to oxytetracycline. In contrast, none of the pediococci and lactococci tested showed acquired antibiotic resistances (Table S1 and Table 4). In the following text, these data are described in more detail.
|
The three penicillins tested in this study exhibited comparable antimicrobial activities against all LAB species examined. In general, most of the LAB isolates were inhibited by a maximum of 2 or 4 mg/L of the corresponding penicillin and no NWTs were identified among the isolates examined. However, species of the L. acidophilus group (L. acidophilus, L. gasseri, L. crispatus, L. johnsonii, L. delbrueckii and Lactobacillus amylovorus) were relatively more susceptible to penicillins in comparison with other LAB species. The L. acidophilus group was also relatively more susceptible to penicillin than to aminopenicillins when compared with pediococci, L. rhamnosus, L. paracasei, L. plantarum and, to a lesser extent, also with L. reuteri. The aminoglycosides gentamicin and streptomycin showed well-defined MIC distributions between
1 and 8 mg/L for gentamicin and between 2 and 128 mg/L for streptomycin. However, most isolates displayed MICs at the low end of the concentration ranges (1 mg/L for gentamicin and 2 mg/L for streptomycin, respectively), whereas three probiotic Lactobacillus isolates (L. rhamnosus L-015 and L-455 and L. paracasei L-005) displayed high-level resistance to streptomycin with MICs of 
2048 mg/L. Species of the L. acidophilus group were relatively more susceptible to streptomycin than other Lactobacillus species. In contrast, pediococci and members of several Lactobacillus species (including L. paracasei and L. plantarum and to some extent also L. rhamnosus and L. fermentum) appeared to be less susceptible to streptomycin. The glycopeptides vancomycin and teicoplanin exhibited heterogeneous profiles in their in vitro activities. Intrinsic high-level resistance to glycopeptides was found in pediococci (MICs: vancomycin > 256 mg/L and teicoplanin 64 mg/L) and in several Lactobacillus species such as L. rhamnosus, L. paracasei, L. plantarum, L. reuteri (MICs: vancomycin 128 mg/L and teicoplanin 32 mg/L) and L. fermentum with a broader spectrum of susceptibilities to teicoplanin (MICs: vancomycin 32– > 256 mg/L and teicoplanin 2– > 256 mg/L). In contrast, the tested lactococci and species of the L. acidophilus group were clearly susceptible to these antibiotics, displaying MICs between 0.25 and 1 mg/L for vancomycin and between 0.125 and 0.25 mg/L for teicoplanin, respectively. Quinupristin/dalfopristin exhibited activities against all LAB tested in this study, with MIC ranges of 0.032–2 mg/L. Erythromycin and clindamycin were also very active against most LAB isolates examined, and clindamycin showed high activities especially against P. acidilactici, P. pentosaceus, L. lactis and several Lactobacillus species for which most isolates generated MICs of 0.125 mg/L (erythromycin) and 0.25 mg/L (clindamycin). However, there were also three isolates that showed MICs of erythromycin and clindamycin outside their corresponding ranges for WT organisms, namely, the probiotic L. rhamnosus strain L-455 and the two research isolates L. crispatus L-295 and L-296 (Table 5). Oxytetracycline was generally active against the majority of LAB isolates examined, especially against L. lactis. In contrast, pediococci showed relatively high MICs of 4–16 mg/L of this antibiotic. However, some isolates of different Lactobacillus species exhibited MICs higher than their corresponding ECOFF values for this antibiotic, i.e. the probiotic isolates L. plantarum L-437, L. reuteri L-285 and L-285-2, L. rhamnosus L-016 and L-341 and L. paracasei L-343, the research isolates L. crispatus L-295 and L-296, the human isolates L. rhamnosus L-078, L. johnsonii L-073 and L-155 and the animal strain L. johnsonii L-153. Additionally, four isolates with MICs of oxytetracycline between 8 and 128 mg/L were observed in three species that were presented by less than 10 isolates and for which no ECOFF could be determined, i.e. the probiotic isolates Lactobacillus brevis L-405 and Lactobacillus buchneri L-366 and the two nutritional isolates Lactobacillus curvatus L-377 and L-344 (Table 5 and Table S1). Chloramphenicol showed efficient in vitro antibacterial activities, nearly all LAB isolates tested were inhibited by 4 mg/L and no NWT isolates were observed. In vitro susceptibilities to fusidic acid were very different among the LAB species tested. Within the genus Lactobacillus, members of L. fermentum and L. reuteri appeared to be susceptible to fusidic acid (MICs 2 mg/L), whereas other species (e.g. L. rhamnosus, L. paracasei, L. plantarum and those of the L. acidophilus group) seem to be intrinsically resistant. Although two isolates of P. acidilactici displayed an MIC of fusidic acid of 16 mg/L, pediococci and L. lactis were mostly inhibited by 2 or 4 mg/L. The oxazolidinone antibiotic linezolid showed a unique pattern of high antibacterial activities against the different LAB species tested in this study. Nearly, all strains were inhibited by 1–2 mg/L, and only three isolates of L. acidophilus possessed an MIC of linezolid of 4 mg/L. Trimethoprim and trimethoprim/sulfamethoxazole showed broad spectra and no Gaussian curves of their MIC profiles, which did not allow determination of ECOFF values for the LAB species tested; the MICs for many LAB species ranged between 0.25 (or 0.5) and > 512 mg/L for both antibiotics (Table S1).
|
Detection of antimicrobial resistance genes
The NWT Lactobacillus isolates that possessed MICs higher than the corresponding species-specific ECOFF values of the tested antibiotics were selected for PCR detection of the corresponding resistance gene(s). Species for which no ECOFF values were determined, but containing isolates displaying high MICs when compared with NWT ranges in other Lactobacillus species, were also tested. Altogether, 19 out of 416 (4.6%) Lactobacillus isolates tested in the present study displayed MICs exceeding the respective ECOFF values. However, only some of these NWT isolates also possessed the corresponding antibiotic resistance gene(s) (Table 5). Three probiotic isolates (L. paracasei L-005 and L. rhamnosus L-015 and L-455) displaying high-level resistance to streptomycin were negative for the aad(E) gene. The latter isolate was also resistant to erythromycin and clindamycin, but did not contain the corresponding resistance genes erm(A), erm(B) or erm(C) (Table 5). Eight, mostly probiotic Lactobacillus isolates that generated MICs of oxytetracycline between 8 and 16 mg/L, did not possess any of the tested tet genes. However, two isolates of L. johnsonii with MICs of this antibiotic in the range of 8–16 mg/L were tet(W)-positive. All Lactobacillus with higher MICs of oxytetracycline (64– > 128 mg/L) contained tet(M), tet(W) or a non-specified member of the tet(M) group. In addition, the research isolates L. crispatus L-295 and L-296 that showed MICs of erythromycin and clindamycin of > 32 mg/L were positive for the erm(B) gene (Table 5).
Probiotic, nutritional or research NWT isolates containing one or two antibiotic resistance genes (Table 5; isolates are marked by footnote e) were selected as donors for experiments. Rifampicin- and fusidic acid-resistant isolates of the corresponding Lactobacillus species and the strains E. faecium 64/3 and E. faecalis JH 2-2 served as recipients. However, none of the intra- and interspecies donor–recipient combinations tested produced transconjugants under the experimental in vitro conditions applied (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
In the present study, MICs of 16 antimicrobial agents of nearly all important classes were determined for a collection of 473 isolates encompassing 24 species of the genera Lactobacillus, Pediococcus and Lactococcus by microdilution test using LSM broth.24 Because most isolates were identified by fingerprinting techniques that also allow determination of the relationship of isolates at the individual strain level, there is reasonable evidence to assume that the human, animal and research isolates were not duplicate isolates of the same strain. Within the probiotic or nutritional categories, however, a number of isolates received from different depositors produced very similar fingerprints (data not shown). In these cases, it cannot be ruled out that duplicate cultures were included in the present study.
Overall, our results are in good agreement with data from other studies for a broad range of LAB species and antibiotics, although different nutrient media, incubation conditions and/or susceptibility testing methods were used.22,23,39–47 In some studies, higher MICs of the aminoglycosides gentamicin and streptomycin were reported for LAB, which is probably due to the fact that susceptibility testing was performed on MRS agar (e.g. by Etest).22,48 In the latter two studies, streptomycin-resistant Lactobacillus spp. were encountered with MICs of streptomycin of > 256 mg/L. The reason for the increased MICs of the aminoglycosides on MRS agar may be due to the medium's low pH (6.2 ± 0.2), because the pH optimum of aminoglycosides is in the alkaline range (pH 7.8).49 In contrast, the pH value of LSM broth is adjusted to pH 6.7, which appears to favour the antibacterial activities of aminoglycosides, resulting in lower MICs. The observed difficulties in determining MICs of trimethoprim and trimethoprim/sulfamethoxazole, in contrast, are obviously linked to the presence of antagonistic components such as p-aminobenzoic acid (against sulfamethoxazole) and/or thymidine (against trimethoprim)50 in LSM broth as the consequence of its 10% portion of MRS broth.24 The synthetic IST broth as the main component (90%) of LSM broth24 is known for its poor content of antagonists and, therefore, the portion of MRS broth in LSM broth obviously caused the non-evaluable MIC profiles of trimethoprim and trimethoprim/sulfamethoxazole. However, we did not find negative effects of LSM broth on the activities of other antibiotics examined and the LAB species tested did not show growth problems.24
On the basis of the MIC data obtained for 16 antimicrobial agents, we were able to define tentative ECOFF values for 13 antibiotics in up to 12 LAB species represented by at least 10 isolates. These tentative ECOFFs may offer an objective basis to update and/or modify specific guidelines, such as those outlined by the Feed Additives and Products (FEEDAP) panel of the European Food Safety Authority (EFSA).51 Among the 432 LAB isolates tested (Table 4), low resistance frequencies were found to oxytetracycline (2.8%) and to streptomycin, erythromycin and clindamycin (in each case 0.7%), resulting in a total frequency of 4.6% NWT isolates (19 out of 416) for the genus Lactobacillus (Tables 1, 4 and 5). However, when looking in more detail at the frequencies of Lactobacillus isolates with acquired antibiotic resistances according to their categories, striking differences were observed (Tables 1, 4 and 5), e.g. 11 isolates among 113 (9.7%) probiotic, 2 among 23 probiotic/nutritional research (8.7%), 2 among 26 nutritional (7.7%), 1 among 5 (‘20%’) animal isolates, but only 3 among 249 human isolates (1.2%) possessed acquired antibiotic resistances. However, duplicate cultures of probiotic or nutritional isolates cannot be ruled out as discussed above. Furthermore, several intrinsic antibiotic resistances (or probable intrinsic resistances) were recorded in different LAB species tested, as indicated in Table S1 and Tables 3 and 4. Although 44% of the human isolates originated from blood cultures and 41% from faecal and vaginal flora of healthy and hospitalized persons, the low frequency of acquired antibiotic resistance found in human Lactobacillus isolates was surprising. But in a retrospective review on pathogenic relevance of over 200 Lactobacillus-associated infections, the isolates were also mostly susceptible to antibiotics such as erythromycin and clindamycin, except the species-dependent intrinsic resistance to vancomycin.14 Additionally, the low frequency of NWT in human isolates may be due to the biased selection of human isolates within the PROSAFE project. The probiotic NWT isolates investigated mainly belong to species most commonly used in probiotic applications, i.e. L. plantarum, L. paracasei and L. rhamnosus. This finding reinforces the antimicrobial susceptibility testing in the safety assessment procedure of strains intended for probiotic or nutritional use, especially in humans.17,52
Several studies have reported the prevalence of tet genes in Lactobacillus isolates. The most widespread of these genes, tet(M), has been detected in members of L. plantarum, L. curvatus, Lactobacillus casei, L. acidophilus, L. gasseri and L. crispatus.23,53,54 The tet(W) gene appears to be less widely distributed in lactobacilli and has so far only been reported in the probiotic strain L. reuteri SD 2112.55 In the present study, tet(M), tet(W) and unidentified members of the tet(M) group were detected in five Lactobacillus species. Of these, the occurrence of tet(W) in strains of L. crispatus and L. johnsonii is a novel finding. Erythromycin resistance displayed by two isolates of L. crispatus was attributed to the presence of the erm(B) gene. This gene has previously been detected in various Lactobacillus species including L. reuteri, L. fermentum, L. casei, L. plantarum, L. acidophilus, L. gasseri, L. rhamnosus and L. johnsonii.54,56–58 The gene aad(E) responsible for high-level streptomycin resistance was not detected in the three Lactobacillus isolates displaying MICs of streptomycin of 2048 mg/L. Possibly, another mechanism may be responsible for the high-level resistance to this antibiotic, such as mutations in genes encoding ribosomal proteins and/or rRNA. Previously, a Lactobacillus isolate from yoghurt was reported with an MIC of streptomycin of 1024 mg/L, but no details were provided on species identity and the presence of resistance genes.45
None of the in vitro transfer experiments using NWT isolates with confirmed presence of erm(B) or different tet genes as donor strains and susceptible isolates of the corresponding Lactobacillus species and Enterococcus laboratory strains (E. faecium 64/3 and E. faecalis JH 2-2), respectively, as recipient strains yielded confirmed transconjugants. However, interspecies transfer experiments from the control strains L. plantarum LMG 21684 [containing erm(B) and tet(M)] and L. plantarum LMG 21687 [containing tet(M)] to E. faecalis JH2-2 produced transconjugants but in low frequencies of 
10–8 per recipient cell (data not shown), compared with higher frequencies of 10–4 – 10–6 previously reported.59 Possibly, higher transfer frequencies could be obtained with the NWT isolates tested under modified in vitro conditions or as demonstrated recently under in vivo conditions.60,61
In conclusion, the finding of acquired resistance genes in isolates intended for probiotic use or in isolates that are already used as probiotics for a longer time raises the question whether such cultures should be deliberately released in the food chain. The precautionary principle would plead against the use of such strains, independent of the fact of whether intra- and interspecies transfer can be proven or not. According to the opinion of the FEEDAP panel on the updating of criteria used in the assessment of bacteria for resistance to antibiotics of human and veterinary importance, strains carrying an acquired resistance to antimicrobial(s) should not be used as feed additives, unless it can be demonstrated that it is a result of chromosomal mutation(s).51 In this respect, however, it is important to realize that acquired antibiotic resistances can be transferred not only by conjugation but also by other mechanisms, such as transformation or transduction, that are even more difficult to study under controlled laboratory conditions.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
None to declare.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
|
Acknowledgements |
|---|
Klaas D'Haene is thanked for excellent technical assistance. We also wish to acknowledge all depositors of strains. This work was supported by a grant from the European Commission ‘Biosafety Evaluation of Probiotic Lactic Acid Bacteria Used for Human Consumption’ (PROSAFE; QLRT-2001-01273). G. H. is a post-doctoral fellow of the Fund for Scientific Research, Flanders, Belgium (F.W.O.—Vlaanderen).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsSupplementary dataReferences |
|---|
1 Vaughan EE, Heilig HG, Ben-Amor K, et al. (2005) Diversity, vitality and activities of intestinal lactic acid bacteria and bifidobacteria assessed by molecular approaches. FEMS Microbiol Rev 29:477–90.[CrossRef][Web of Science][Medline]
2 Zoetendal EG, Vaughan EE, de Vos WM. (2006) A microbial world within us. Mol Microbiol 59:1639–50.[CrossRef][Web of Science][Medline]
3 Holzapfel WH, Haberer P, Snel J, et al. (1998) Overview of gut flora and probiotics. Int J Food Microbiol 41:85–101.[CrossRef][Web of Science][Medline]
4 Klein G, Pack A, Bonaparte C, et al. (1998) Taxonomy and physiology of probiotic lactic acid bacteria. Int J Food Microbiol 26:103–25.
5 Carr FJ, Chill D, Maida N. (2002) The lactic acid bacteria: a literature survey. Crit Rev Microbiol 28:281–370.[CrossRef][Web of Science][Medline]
6 Temmerman R, Masco L, Huys G, et al. (2004) In search of new probiotics: diversity, selection and exploitation of health-promoting microorganisms. In Kurtböke I and Swings J (Eds.). Microbial Genetic Resources and Biodiscovery. A World Federation of Culture Collections (WFCC) Publication(Queensland Complete Printing Services, Queensland) pp. 220–33.
7 Mercenier A, Pavan S, Pot B. (2003) Probiotics as biotherapeutic agents: present knowledge and future prospects. Curr Pharm Des 9:175–91.[CrossRef][Web of Science][Medline]
8
Reid G, Jass J, Sebulsky MT, et al. (2003) Potential uses of probiotics in clinical practice. Clin Microbiol Rev 16:658–72.
9 Servin AL. (2004) Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev 28:405–40.[Web of Science][Medline]
10 Davies AJ, James PA, Hawkey PM. (1986) Lactobacillus endocarditis. J Infect 12:169–74.[CrossRef][Web of Science][Medline]
11
Barton LL, Rider ED, Coen RW. (2001) Bacteremic infection with Pediococcus: vancomycin-resistant opportunist. Pediatrics 107:775–6.
12 Arpi M, Vancanneyt M, Swings J, et al. (2003) Six cases of Lactobacillus bacteraemia: identification of organisms and antibiotic susceptibility and therapy. Scand J Infect Dis 35:404–8.[CrossRef][Web of Science][Medline]
13 Antolin J, Ciguenza R, Saluena I, et al. (2004) Liver abscess caused by Lactococcus lactis cremoris: a new pathogen. Scand J Infect Dis 36:490–1.[CrossRef][Web of Science][Medline]
14 Cannon JP, Lee TA, Bolanos JT, et al. (2005) Pathogenic relevance of Lactobacillus: a retrospective review of over 200 cases. Eur J Clin Microbiol Infect Dis 24:31–40.[CrossRef][Web of Science][Medline]
15 Adams MR and Marteau P. (1995) On the safety of lactic acid bacteria from food. Int J Food Microbiol 27:263–4.[CrossRef][Web of Science][Medline]
16 Salminen S, von Wright A, Morelli L, et al. (1998) Demonstration of safety of probiotics—a review. Int J Food Microbiol 44:93–106.[CrossRef][Web of Science][Medline]
17 Saarela M, Mogensen G, Fondén R, et al. (2000) Probiotic bacteria: safety, functional, and technological properties. J Biotechnol 84:197–215.[CrossRef][Web of Science][Medline]
18 Ishibashi N and Yamasaki S. (2001) Probiotics and safety. Am J Clin Nutr 73:Suppl 2.
19 Borriello SP, Hammes WP, Holzapfel W, et al. (2003) Safety of probiotics that contain lactobacilli or bifidobacteria. Clin Infect Dis 36:775–80.[CrossRef][Web of Science][Medline]
20 Perreten V, Schwarz F, Cresta L, et al. (1997) Antibiotic resistance spread in food. Nature 389:801–2.[Medline]
21 Teuber M, Meile L, Schwarz F. (1999) Acquired antibiotic resistance in lactic acid bacteria from food. Antonie Van Leeuwenhoek 76:115–37.[CrossRef][Web of Science][Medline]
22 Danielsen M and Wind A. (2003) Susceptibility of Lactobacillus spp. to antimicrobial agents. Int J Food Microbiol 82:1–11.[CrossRef][Web of Science][Medline]
23
Gevers D, Danielson M, Huys G, et al. (2003) Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl Environ Microbiol 69:1270–5.
24
Klare I, Konstabel C, Müller-Bertling S, et al. (2005) Evaluation of new broth media for microdilution antibiotic susceptibility testing of lactobacilli, pediococci, lactococci, and bifidobacteria. Appl Environ Microbiol 71:8982–6.
25 Vankerckhoven V, Van Autgaerden T, Huys G, et al. (2004) Establishment of the PROSAFE collection of probiotic and human lactic acid bacteria. Microb Ecol Health Dis 16:131–6.[CrossRef]
26 Huys G, Vancanneyt M, D'Haene K, et al. (2006) Accuracy of the species identity of commercial bacterial cultures intended for probiotic or nutritional use. Res Microbiol 157:803–10.[Medline]
27 European Committee on Antimicrobial Susceptibility Testing (EUCAST). Clinical Breakpoints and Epidemiological Cut-off Values: Definitions of Clinical Breakpoints and Epidemiological Cut-off Values http://www.srga.org/eucastwt/eucastdefinitions.htm 28 February 2007.
28 Kahlmeter G, Brown DF, Goldstein FW, et al. (2003) European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria. J Antimicrob Chemother 52:145–8.[Medline]
29 Turnidge J, Kahlmeter G, Kronvall G. (2006) Statistical characterisation of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values. Clin Microbiol Infect 12:418–25.[CrossRef][Web of Science][Medline]
30 European Committee on Antimicrobial Susceptibility Testing (EUCAST). Antimicrobial Wild Type Distributions of Microorganisms http://217.70.33.99/eucast2/ 28 February 2007.
31
Strommenger B, Kettlitz C, Werner G, et al. (2003) Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 41:4089–94.
32
Werner G, Willems RJL, Hildebrandt B, et al. (2003) Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium. J Clin Microbiol 41:1499–1506.
33
Aminov RI, Garrigues-Jeanjean N, Mackie RI. (2001) Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl Environ Microbiol 67:22–32.
34 Charpentier E, Gerbaud G, Courvalin P. (1993) Characterization of a new class of tetracycline resistance gene tet(S) in Listeria monocytogenes BM4210. Gene 131:27–34.[CrossRef][Web of Science][Medline]
35 Clermont D, Chesneau O, de Cespedes G, et al. (1997) New tetracycline resistance determinants coding for ribosomal protection in streptococci and nucleotide sequence of tet(T) isolated from Streptococcus pyogenes A498. Antimicrob Agents Chemother 41:112–6.[Abstract]
36
Sasaki Y, Taketomo N, Sasaki T. (1988) Factors affecting transfer frequency of pAMß1 from Streptococcus faecalis to Lactobacillus plantarum. J Bacteriol 170:5939–42.
37 Klare I, Konstabel C, Mueller-Bertling S, et al. (2005) Spread of ampicillin/vancomycin-resistant Enterococcus faecium of the epidemic-virulent clonal complex-17 carrying the genes esp and hyl in German hospitals. Eur J Clin Microbiol Infect Dis 24:815–25.[CrossRef][Web of Science][Medline]
38 Gevers D, Huys G, Swings J. (2001) Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett 205:31–6.[CrossRef][Web of Science][Medline]
39
de la Maza L, Ruoff KL, Ferraro MJ. (1989) In vitro activities of daptomycin and other antimicrobial agents against vancomycin-resistant gram-positive bacteria. Antimicrob Agents Chemother 33:1383–4.
40
Swenson JM, Facklam RR, Thornsberry C. (1990) Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species. Antimicrob Agents Chemother 34:543–9.
41 Yamane N and Jones RN. (1991) In vitro activity of 43 antimicrobial agents tested against ampicillin-resistant enterococci and gram-positive species resistant to vancomycin. Diagn Microbiol Infect Dis 14:337–45.[CrossRef][Web of Science][Medline]
42
Tankovic J, Leclercq R, Duval J. (1993) Antimicrobial susceptibility of Pediococcus spp. and genetic basis of macrolide resistance in Pediococcus acidilactici HM3020. Antimicrob Agents Chemother 37:789–92.
43
Felten A, Barreau C, Bizet C, et al. (1999) Lactobacillus species identification, H2O2 production, antibiotic resistance correlation with human clinical status. J Clin Microbiol 37:729–33.
44
Zarazaga M, Saenz Y, Portillo A, et al. (1999) In vitro activities of ketolide HMR3647, macrolides, and other antibiotics against Lactobacillus, Leuconostoc, and Pediococcus isolates. Antimicrob Agents Chemother 43:3039–41.
45 Katla AK, Kruse H, Johnsen G, et al. (2001) Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. Int J Food Microbiol 67:147–52.[CrossRef][Web of Science][Medline]
46 Temmerman R, Pot B, Huys G, et al. (2003) Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol 81:1–10.[CrossRef][Web of Science][Medline]
47 Perez Pulido R, Omar NB, Lucas R, et al. (2005) Resistance to antimicrobial agents in lactobacilli isolated from caper fermentations. Antonie Van Leeuwenhoek 88:277–81.[CrossRef][Web of Science][Medline]
48 Charteris WP, Kelly PM, Morelli L, et al. (2001) Gradient diffusion antibiotic susceptibility testing of potentially probiotic lactobacilli. J Food Prot 64:2007–14.[Web of Science][Medline]
49 Amsterdam D. (2005) Susceptibility testing of antimicrobials in liquid media. In Lorian V (Ed.). Antibiotics in Laboratory Medicine(Williams & Wilkins, Baltimore) pp. 61–143.
50 Turnidge JD and Bell JM. (2005) Antimicrobial susceptibility on solid media. In Lorian V (Ed.). Antibiotics in Laboratory Medicine(Williams & Wilkins, Baltimore) pp. 8–60.
51 European Food Safety Authority (EFSA). (2005) Opinion of the scientific panel on additives and products or substances used in animal feed on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance (question no. EFSA-Q-2004-079). EFSA J 223:1–12 adopted on 25 May 2005.
52
Huys G, D'Haene K, Swings J. (2006) Genetic basis of tetracycline and minocycline resistance in the potentially probiotic Lactobacillus plantarum strain CCUG 43738. Antimicrob Agents Chemother 50:1550–1.
53 Danielsen M. (2002) Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure. Plasmid 48:98–103.[Medline]
54 Cataloluk O. (2004) Gogebakan B. Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey. FEMS Microbiol Lett 236:7–12.[Web of Science][Medline]
55 Kastner S, Perreten V, Bleuler H, et al. (2006) Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food. Syst Appl Microbiol 29:145–55.[CrossRef][Web of Science][Medline]
56 Lin CF and Chung TC. (1999) Cloning of erythromycin resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors. Plasmid 42:31–41.[CrossRef][Web of Science][Medline]
57 Gfeller KY, Roth M, Meile L, et al. (2003) Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1. Plasmid 50:190–201.[CrossRef][Web of Science][Medline]
58 Cauwerts K, Pasmans F, Devriese LA, et al. (2006) Cloacal Lactobacillus isolates from broilers show high prevalence of resistance towards macrolide and lincosamide antibiotics. Avian Pathol 35:160–4.[CrossRef][Web of Science][Medline]
59 Gevers D, Huys G, Swings J. (2003) In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria. FEMS Microbiol Lett 225:125–30.[CrossRef][Web of Science][Medline]
60 Jacobsen L, Wilcks A, Hammer K, et al. (2007) Horizontal transfer of tet(M) and erm(B) resistance plasmids from strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats. FEMS Microbiol Ecol 59:158–66.[CrossRef][Medline]
61
Dahl KH, Mater DDG, Flores MJ, et al. (2007) Transfer of plasmid and chromosomal glycopeptide resistance determinants occurs more readily in the digestive tract of mice than in vitro and exconjugants can persist stably in vivo in the absence of glycopeptide selection. J Antimicrob Chemother 59: pp. 478–86.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Rosander, E. Connolly, and S. Roos Removal of Antibiotic Resistance Gene-Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the Resulting Daughter Strain, L. reuteri DSM 17938 Appl. Envir. Microbiol., October 1, 2008; 74(19): 6032 - 6040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Vankerckhoven, G. Huys, M. Vancanneyt, C. Snauwaert, J. Swings, I. Klare, W. Witte, T. Van Autgaerden, S. Chapelle, C. Lammens, et al. Genotypic Diversity, Antimicrobial Resistance, and Virulence Factors of Human Isolates and Probiotic Cultures Constituting Two Intraspecific Groups of Enterococcus faecium Isolates Appl. Envir. Microbiol., July 15, 2008; 74(14): 4247 - 4255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mayrhofer, K. J. Domig, C. Mair, U. Zitz, G. Huys, and W. Kneifel Comparison of Broth Microdilution, Etest, and Agar Disk Diffusion Methods for Antimicrobial Susceptibility Testing of Lactobacillus acidophilus Group Members Appl. Envir. Microbiol., June 15, 2008; 74(12): 3745 - 3748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Feld, S. Schjorring, K. Hammer, T. R. Licht, M. Danielsen, K. Krogfelt, and A. Wilcks Selective pressure affects transfer and establishment of a Lactobacillus plantarum resistance plasmid in the gastrointestinal environment J. Antimicrob. Chemother., April 1, 2008; 61(4): 845 - 852. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||