Antibiotic Resistance and Molecular Characterization of Probiotic and Clinical Lactobacillus Strains in Relation to Safety Aspects of Probiotics

Abstract

The evaluation of the safety of probiotic strains includes the exclusion of antibiotic resistance of clinical importance. Ninety-two strains from the genus Lactobacillus isolated from probiotics, food, and clinical sources were included in the investigation. Species tested were the L. acidophilus group, L. casei group, L. reuteri/fermentum group, and L. sakei/curvatus group. Cell and colony morphology, fermentation patterns, and growth characteristics as well as soluble whole cell proteins were analyzed. Antibiotic resistance against clinically important agents was determined by broth dilution tests. The vanA and tet genes were confirmed. Resistances occurred mainly against gentamicin, ciprofloxacin, clindamycin, sulfonamides, and, in some cases, glycopeptides. The natural glycopeptide resistance within the L. casei group and L. reuteri appears to be not of clinical relevance, as there was no vanA gene present. Therefore, the transfer of this resistance is very unlikely. Tet-(A), -(B), -(C), -(M), or -(O) gene could not be detected. The protein fingerprinting within the L. casei group proved that L. rhamnosus strains of clinical origin clustered together with probiotic strains. For safety evaluations resistance patterns of a broad range of strains are a useful criterion together with the exclusion of known resistance genes (like the vanA gene) and can be used for decision making on the safety of probiotics, both by authorization bodies and manufacturers.

Introduction

L actobacilli are used in a wide range of applications in food and feed fermentation as well as in the production of probiotics for humans and animals. They are also part of the autochthonous microflora of the gut (Holzapfel et al., 1998; Walter, 2008; Jones et al., 2009; Ventura et al., 2009). Probiotic strains derive mainly only from a low number of species, like the Lactobacillus acidophilus group, the L. casei group, and L. reuteri (Casas and Dobrogosz, 1997; Klein et al., 1998).

Evaluation of the safety of probiotic strains relies mainly on the body of knowledge concerning the ingestion via food or on specific criteria like the exclusion of translocation to extraintestinal organs and to cross the intestinal mucosal barrier (Daniel et al., 2006). The exclusion of antibiotic resistance of clinical importance is another criterion, which has recently been challenged, as antibiotic resistance per se is not a virulence factor in itself (Wassenaar and Klein, 2008). So far, no differences between probiotic strains and strains of clinical origin can scientifically be proved. However, the exclusion of transferable resistances is an important safety criterion (EFSA, 2008a).

It is well known that lactobacilli harbor natural resistances against antibiotics and chemotherapeutics (Tynkkynen et al., 1998). Therefore, they have been considered as potential vectors of resistances via the food chain or the environment from animal production to the consumer. This has been shown to be the case for enterococci in food fermentation (Cocconcelli et al., 2003) and must therefore also be considered for lactobacilli. Further, isolation from clinical infections of different sources has been shown (Aguirre and Collins, 1993; Salminen et al., 2006; Martinez et al., 2008). Some species exhibit antimicrobial activity against other pathogens itself by production of bacteriocins (Dortu et al., 2008; Zdolec, 2009).

The strain selection should take the variety of applications into account. The aim of this study was therefore to determine the antibiotic resistance profile against antibiotic substances used in human clinical therapy of strains representing this variety. Therefore, strains from the species of the L. acidophilus group, the L. casei group, and from L. reuteri of different origin (probiotic product; gastrointestinal tract from swine, mouse, rat, monkey, chicken, turkey, and man; human clinical isolate) were chosen. The results should give an overview of the potential risk associated with the application of probiotic Lactobacillus strains in view of the transfer of antibiotic resistances to bacterial pathogens. Some Lactobacillus species are known to be glycopeptide and/or tetracycline resistant (Kastner et al., 2006; Florez et al., 2008). To assess the impact of an antibiotic resistance on the pathogenic potential of a microorganism, it is essential to know the molecular mechanism of the resistance as it has been shown also for other traits like bacteriocin production or stress adaptation (Klaenhammer et al., 2008; Goh and Klaenhammer, 2009). The Qualitative Presumption of Safety concept (QPS) of the European Food Safety Authority (EFSA) also demands knowledge on the antibiotic resistance, especially transferable resistance, and identified some lactobacilli species to be monitored (EFSA, 2008a). A further aim of this study was to determine if the resistance profiles and molecular protein fingerprinting of strains of probiotic lactobacilli versus clinical isolates reveal differences between both groups.

Materials and Methods

Bacterial strains

The selection of strains was based on type and reference strains from culture collections (n = 17) as well as commercially available probiotic strains or production strains from the Institute's collection (n = 23), isolates from the gastrointestinal tract (n = 41), and clinical strains from both culture collections and own isolates (n = 11). Strains investigated in this study included the type and reference strains of all relevant species of probiotic lactobacilli, that is, L. acidophilus group, L. casei group, and L. reuteri/fermentum group. Lyophilized cultures were kept in the culture collection of the Institute and were subcultured before the investigation on MRS Agar (Biotest) under microaerobic conditions at 37°C. The type strains, important reference strains, and probiotic and production strains from dairy or pharmaceutical products, gastrointestinal isolates, or clinical strains are listed in Table 1a–c.

Table 1a.

Type and Reference Strains Used in This Study

Species	Strain	Origin	Source	Strain function
L. acidophilus	DSM 20079^T	DSM	Human	Type strain
L. acidophilus	ATCC 4355	ATCC	Rat	Reference strain
L. acidophilus	ATCC 832	IFH	Feces, rat	Reference strain
L. gasseri	DSM 20243^T	DSM	Human	Type strain
L. johnsonii	LMG 9436^T	LMG	Blood culture, human	Type strain
L. johnsonii	Omniflora	IFH	Pharmaceutical product	Reference strain
L. casei	ATCC 393^T	ATCC	Cheese	Type strain
L. zeae	DSM 20178^T	DSM	Grain	Type strain
L. rhamnosus	DSM 20021^T	DSM	Unknown	Type strain
L. paracasei paracasei	DSM 5622^T	DSM	Unknown	Type strain
L. paracasei paracasei	F 10a	IFH	Feces, human	Reference strain
L. reuteri	DSM 20016^T	DSM	Intestine, human	Type strain
L. reuteri	F 275^T	IFH	Intestine, human	Type strain
L. fermentum	ATCC 14931^T	ATCC	Beetroot, fermented	Type strain

Table 1b.

Strains Used as Probiotic or Therapeutic Culture, Protection Culture, or from Gastrointestinal Tract

Species	Strain	Origin	Source
L. acidophilus	G 3	Paidoflor	Human therapeutic
L. acidophilus	B 101	Producer B	Human therapeutic
L. acidophilus	B 102	Producer B
L. acidophilus	B 103	Producer B
L. acidophilus	B 104	Producer B
L. acidophilus	A 1	IFH	Human therapeutic
L. acidophilus	Acid 601	Producer A	Human therapeutic
L. acidophilus	Acid 676	Producer A	Human therapeutic
L. acidophilus	Acid 74	Producer A	Human therapeutic
L. gasseri	F 164	IFH	Feces, human
L. gasseri	B 201	Producer B	Feces, human
L. gasseri	B 202	Producer B	Feces, human
L. gasseri	B 203	Producer B	Vagina, human
L. gasseri	B 204	Producer B	Vagina, human
L. gasseri	B 205	Producer B	Feces, human
L. gasseri	B 206-I	Producer B	Intestine, proband I
L. gasseri	B 207-I	Producer B	Intestine, proband I
L. gasseri	B 208-I	Producer B	Intestine, proband I
L. gasseri	B 209-I	Producer B	Intestine, proband I
L. gasseri	B 211-III	Producer B	Intestine, proband III
L. gasseri	B 211-III	Producer B	Intestine, proband III
L. gasseri	B 212-III	Producer B	Intestine, proband III
L. gasseri	B 213-III	Producer B	Intestine, proband III
L. gasseri	B 214-III	Producer B	Intestine, proband III
L. gasseri	B 215-III	Producer B	Intestine, proband III
L. gasseri	B 216-III	Producer B	Intestine, proband III
L. gasseri	B 217-V	Producer B	Intestine, proband V
L. gasseri	B 218-V	Producer B	Intestine, proband V
L. gasseri	B 219-VI	Producer B	Stomach, proband VI
L. gasseri	B 220-VI	Producer B	Intestine, proband VI
L. gasseri	B 221-VI	Producer B	Intestine, proband VI
L. gasseri	B 222-VI	Producer B	Intestine, proband VI
L. johnsonii	B 301	Producer B	Joghurt
L. johnsonii	B 302	Producer B	Feces, human
L. johnsonii	Omniflora	IFH	Human therapeutic
L. casei	YS 88	IFH
L. paracasei	Y	IFH	Sour milk product
L. paracasei	P 563	IFH	Sour milk product
L. paracasei	Rv 1p	IFH	Raw sausage
L. rhamnosus	DSM 7133	DSM	Probiotic, pig
L. rhamnosus	P 211	IFH	Sour milk product
L. rhamnosus	P 216	IFH	Sour milk product
L. rhamnosus	P 202/4	IFH	Sour milk product
L. rhamnosus	P 237	IFH	Milk product
L. rhamnosus	GG	Producer D	Milk product
L. rhamnosus	P 972	Producer F	Pharmaceutical product
L. reuteri	F 275	IFH	Intestine, human
L. reuteri	F 275c	IFH	Intestine, human
L. reuteri	K3/47S	IFH	Feces, calf
L. reuteri	D 287	IFH	Feces, calf
L. reuteri	XVII cae	IFH	Caecum, human
L. reuteri	Symbalance	Producer G	Probiotic, joghurt
L. reuteri	PE 3	IFH	Crop, pigeon
L. reuteri	4000	Producer H	Mouse
L. reuteri	4020	Producer H	Mouse
L. reuteri	2010	Producer H	Rat
L. reuteri	M164	Producer H	Monkey
L. reuteri	MV4-2a	Producer H	Human vagina
L. reuteri	MV10-1a	Producer H	Human vagina
L. reuteri	MV11-1a	Producer H	Human vagina
L. reuteri	SD2112	Producer H	Human breast milk
L. reuteri	T-1	Producer H	Turkey
L. reuteri	11284	Producer H	Chicken
L. reuteri	1063	Producer H	Swine
L. fermentum	ATCC 23271	ATCC	Intestine, human
L. fermentum	23	IFH	Human isolate
L. fermentum	F 138a	IFH	Feces, human

Table 1c.

Strains from Clinical Material

Species	Strain	Origin	Source
L. rhamnosus	CCUG 28641	CCUG	Cardiac valve, human
L. rhamnosus	CCUG 28262	CCUG	Sputum, human
L. rhamnosus	CCUG 29281	CCUG	Cervix, human
L. rhamnosus	CCUG 30616	CCUG	Uterus, human
L. rhamnosus	CCUG 22413	CCUG	Blood culture, human
L. rhamnosus	CCUG 27333	CCUG	Hip aspirate, human
L. rhamnosus	CCUG 18011	CCUG	Urethra, human
L. rhamnosus	U 4596-95	Univ. Würzburg	Catheter urine, human
L. rhamnosus	H 213	Clinic Steglitz	Drainage, human
L. rhamnosus	P 898	Clinic Steglitz	Liver abscess, human
L. rhamnosus	P 5397	Virchow-Clinic	Peritoneal dialysis, human

DSM, Deutsche Sammlung von Mikroorganismen; ATCC, American Type Culture Collection; CCUG, Culture Collection, University of Göteborg, Schweden; LMG, Laboratorium voor Mikrobiologie; IFH, own isolate.

Biochemical, physiological, and enzymatic tests

Biochemical properties, such as fermentation of carbohydrates, gas production from glucose, and growth at defined temperatures, were recorded using macrotube tests described by Klein et al. (1995) for all strains. Twenty-one carbon substrates were tested by acidification reactions in a semisolid medium (Klein et al., 1995) after 6 days. Growth at a defined temperature was read after 3 days at 15°C ± 0.1°C, after 2 days at 20°C ± 0.1°C, and after 1 day at 45°C or 50°C ± 0.1°C, respectively. Carbohydrates and their derivates tested were L(+)-arabinose (Merck 1492), D(+)-glucose (Merck 8342), lactose (Merck 7657), D(+)-sucrose (Merck 7651), D(+)-maltose (Merck 5910), D(+)-trehalose (Merck 8353), D(+)-melibiose (Merck 12240), D(+)-cellobiose (Merck 2352), D(+)-raffinose (melitose) (Merck 7549), D(−)-mannitol (Merck 5982), D(+)-salicin [2-o-(-D-glucopyranosido)-benzylalcohol] (Merck 7665), L(+)-rhamnose (Merck 4736), D(+)-xylose (Merck 8692), D(+)-mannose (Merck 5984), D(+)-melezitose (Serva 28550), myo-inositol (Merck 4728), D(−)-sorbitol (Merck 7758), D(+)-inulin (Merck 4733), dextrin (Merck 3006), D(+)-galactose (Merck 4062), D(−)-fructose (Merck 5323), D(−)-ribose (Merck 7605), L-arginine (Merck 1542), amygdalin (Fluka 10050), and Na-gluconate (Merck 822058).

Susceptibility testing

Susceptibility testing was done against the following substances used in human and veterinary clinical therapy—β-Lactams: Penicillin, Ampicillin, Ampicillin/Sulbactam,^* Oxacillin, Cephalothin, and Cefazolin^*; Macrolides: Erythromycin and Clarithromycin^*; Lincosamides: Clindamycin; Aminoglycosides: Gentamicin, Chloramphenicol, and Rifampin; Nitrofuranes: Nitrofurantoin; Quinolones: Ciprofloxacin, Lomefloxacin,^* Ofloxacin,^* Norfloxacin^*; Trimethoprim/Sulfamethoxazol, and Tetracycline; Glycopeptide: Vancomycin and Teicoplanin.^* Testing in ready-to-use microtiter-plates with lyophilized antibiotics was performed according to the manufacturer's instructions (MD Plate Gram Positive, Radiometer, and Sensititre MIC plates MG GP, MCS-Diagnostics, Swalmen/NL, for probiotic L. reuteri strains) and were based on CLSI standards (CLSI, 2008) and other studies (Mayrhofer et al., 2008; Kushiro et al., 2009). The minimum inhibitory concentrations (MICs) were determined after an incubation period of 16–20 h at 37°C ± 0.5°C. For the interpretation of MIC results the MIC ranges proposed by Hummel et al. (2007) based on recommendations of the European Scientific Panel on additives and products or substances used in animal feed (FEEDAP) (EFSA, 2005, 2008b) were used. Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922 were used as reference strains.

Polymerase chain reaction

DNA was isolated from the Lactobacillus strains and the Enterococcus and Staphylococcus control strains as described by Ausubel et al. (1990). Briefly, the bacteria were lysed and the proteins removed by proteinase K treatment. Cell wall debris, polysaccharides, and proteins were removed by precipitation with 10% cetyltrimethylammonium bromide (CTAB)/0.7 M NaCl. Finally, DNA was extracted by isopropanol precipitation. The DNA was resuspended in TE buffer. The DNA concentration was measured by using a DNA Fluorometer (Model TKO 100, Hoefer Scientific Instruments). The same procedure was performed with Aqua bidest (<0.1 μS/cm) for contamination control. The oligonucleotides that were used for polymerase chain reaction (PCR) amplification were synthesized by MWG Biotech GmbH.

vanA gene detection

All vancomycin resistant enterococci (VRE) strains were grown for 24 h at 37°C ± 0.5°C in buffered peptone water (BPW; 10.0 g peptone from casein [Biotest], 5.0g NaCl [Merck], 9.2g Na₂HPO₄x12 H₂O [Merck], 1.5 g KH₂PO₄ [Merck], and 1000.0 mL Aqua demin.). The isolation of bacterial DNA, amplification of a vanA gene fragment by PCR, and agarose gel electrophoresis were done according to Ausubel et al. (1990) and Klare et al. (1995). After lysing the cells with 0.5% SDS, proteins were removed by digestion with proteinase K (Boehringer). CTAB solution (Sigma) was used for removal of cell wall debris, polysaccharides, and the remaining proteins. For extraction of the bacterial DNA for amplification of the vanA gene, DNA was precipitated with isopropanol and transferred to a fresh tube containing 70% ethanol. The DNA concentration was measured with a DNA-Fluorometer TKO 100 (Hoefer). The following oligonucleotides were used as primers for the amplification of the 377 bp fragment of the vanA gene—vanA 1: 5′-TCT GCA ATA GAG ATA GCC GC-3′ (vanA sequence: position 443–462 [13]); vanA 2: 5′-GG AGT AGC TAT CCC AGC ATT-3′ (vanA sequence: position 819–800) (primers from TIB MOL BIOL). The PCR mixture consisted of 10 μL Mg⁺⁺ free buffer, 3 mM MgCl₂, 200 μM dNTP, 0.4 μL primer vanA 1, 0.44 μL primer vanA 2 (50 pmol/100 μL mixture each), 1.25 μL Taq-polymerase (2.5 U), 50 μL mineral oil, 20 ng template DNA, and Aqua bidest. ad 100 μL. The solutions were part of a polymerase kit of Biometra (PrimeZyme™, 100–652). The thermal cycler (TRIO Thermoblock; Biometra) was programmed for 30 cycles: cycle 1 (94°C, 3 min; 55°C, 1 min; 72°C, 1 min), cycles 2–29 (94°C, 1 min; 55°C, 30 sec; 72°C, 30 sec), and cycle 30 (94°C, 1 min; 55°C, 1 min; 72°C, 4 min). Gel electrophoresis was performed for 90 min in an agarose gel (1.4%, Low EEO; Appligene) with 100 V. Enterococcus faecium 64/3, a vanA-negative strain, and the vanA-positive E. faecium 70/90 were used as reference strains.

Tetracycline resistance

Three L. reuteri strains used as probiotics (ATCC 55149, ATCC 53608, and ATCC 55148), and a variety of field strains (human and animal origin) were investigated by PCR analysis to determine if their resistance is related to the presence of the tet-resistance genes tet-(A), -(B), -(C), and -(M/O). Positive controls were as follows: tet-(A); -(M/O); -(M): E. faecalis DSM 2570; tet-(M): E. faecium 70/90; tet-(B): TOPO vector (Fa. Invitrogen, NL) with Amplikon TetB (1170 bp); tet-(C): vector pBR322 (Fa. Sigma, prod. no.: D 9893). Negative control for all tet genes was S. aureus DSM 2569 (Table 2). The primer sequences (Table 3) for tet-(A) genes were constructed according to Hansen et al. (1996), and for tet-(C) primer according to Hansen et al. (1996) and Frech and Schwarz (2000). Primers for tet-(B) genes were used according to Roberts et al. (1996). Primer sequences for tet-(M) were obtained from Trees et al. (2000). Additional primers were designed according to Roberts et al. (1993) for the simultaneous detection of tet-(M) and tet-(O). DNA was isolated from the Lactobacillus strains and the Enterococcus and Staphylococcus control strains as described by Ausubel et al. (1990). Briefly, the bacteria were lysed and the proteins removed by proteinase K treatment. Cell wall debris, polysaccharides, and proteins were removed by precipitation with 10% CTAB/0.7M NaCl. Finally, DNA was extracted by isopropanol precipitation. DNA was resuspended in TE buffer. DNA concentration was measured by using DNA Fluorometer (Model TKO 100; Hoefer Scientific Instruments). The same procedure was performed with Aqua bidest. for contamination control. The oligonucleotides that were used for PCR amplification were synthesized by MWG Biotech GmbH. Ten nanograms of DNA was amplified using the AmpliTaq Gold polymerase (Perkin Elmer) for tet-(M) and tet-(O). For tet-(A), -(B), and -(C), Dynazyme Polymerase-Kit (native enzyme) (Biometra) was used. The reactions were performed in a total volume of 25 μl. The reaction mix was overlaid with mineral oil and the temperature cycles were controlled by a Trio-Thermoblock (Biometra). DNA from controls as listed in Table 2 was used for positive control and from S. aureus DSM 2569 as a negative control, and Aqua bidest. for contamination control. The PCR fragments were separated by horizontal agarose gel electrophoresis. After ethidium bromide staining they were observed under UV light.

Table 2.

Controls (Template DNA) Used for tet Gene Polymerase Chain Reaction-Probing

tet gene	Positive control ^a	Negative control
A	E. faecalis DSM 2570	S. aureus DSM 2569
M/O	E. faecalis DSM 2570	S. aureus DSM 2569
M	E. faecalis DSM 2570; E. faecium 70/90	S. aureus DSM 2569
B	TOPO vector (Fa. Invitrogen, NL) with Amplikon TetB (1170 bp)	S. aureus DSM 2569
C	Vector pBR322 (Fa. Sigma, prod.no.: D 9893)	S. aureus DSM 2569

DSM stands for Deutsche Sammlung von Mikroorganismen und Zellkulturen; the Enterococcus strains were taken from the collection of the Institute of Meat Hygiene and Technology, Free University of Berlin, Germany.

Table 3.

Polymerase Chain Reaction Primers for tet Genes

Primer	Sequence	Gene	Polymerase chain reaction fragment (bp)
TET M A	5′- GGC GTA CAA GCA CAA CTC G	tet-(M)	750
TET M B	5′- TCT CTG TTC AGG TTT ACT CG
TET M 3	5′- ATA GA(CT) ACG CCA GG(AC) CAT AT	tet-(M/O)	528
TET M 4	5′- GGA GCC CAG AAA GGA TT(CT) GG
TET B 1	5′- CAG TGC TGT TGT TGT CAT TAA	tet-(B)	∼500
TET B 2	5′- GCT TGG AAT ACT GAG TGT AA
TET A 1	5′- GTA ATT CTG AGC ACT GT	tet-(A)	∼670
TET A 2	5′- CCT GGA CAA CAT TGC TT
TET C 1	5′- AAC AAT GCG CTC ATC GT	tet-(C)	∼1100
TET C 2	5′- GGA GGC AGA CAA GGT AT

Numerical analysis of the total soluble cytoplasmatic protein patterns with Colloidal Coomassie blue

Culture of strains, protein purification, and determination of protein concentration were done as described by Klein et al. (1995) with the following modifications (Klein et al., 1996): cells were harvested by rinsing two agar plates with 5 mL of 0.9% NaCl each. The protein samples were diluted to a standard concentration of 1.0 μg/μL. Electrophoresis was done with Tris-Glycine-gradient gels (8%–16%, 15 traces, Novex) in a Novex Xcell apparatus, three gels per strain, with markers (Mark12, Novex) in position 1, 4, 8, 12, and 15. Proteins were stained with Colloidal Coomassie blue (Novex) and the two-dimensional gels were dried with a special drying solution (Anarapid; Anamed) between two sheets of cellophane. The stained protein patterns were scanned with 1200 dpi (Highscreen Flatbed color IIs, Laserscanner) and digitized. Normalization of densitometric traces with background substraction (rolling disk), and conversion were done with GelCompar version 3.1 (Applied Maths). Clustering was done by the unweighted average pair group method with “none alignment.”

Results

The phenotypic tests confirmed the species identification as shown in Table 1a–c. Only one strain showed an untypical fermentation pattern, L. rhamnosus GG (Valio) (Table 1b). This strain did not ferment lactose but was otherwise confirmed as L. rhamnosus.

Resistance pattern for the L. acidophilus group

Six different resistance types could be identified within the L. acidophilus group (Tables 4 and 5a). All strains were susceptible to oxacillin, cephalothin, erythromycin (one exception: intermediate, strain B 104), imipenem, chloramphenicol, and tetracycline. All strains were resistant to gentamicin and ciprofloxacin (resistance type 1). Concerning other antimicrobials the resistance pattern was different between strains, especially concerning clindamycin (Tables 4 and 5a), which is a therapeutic of importance in the therapy of bacterial vaginosis (Oduyebo et al., 2009).

Table 4.

Resistance Types Within the Lactobacillus acidophilus Group

Resistance type	Resistances
1	GE CIP
2	GE/GE(I) CIP SXT
3	GE CIP CL/CL(I)
4	GE CIP CL/CL(I) SXT
5	GE CIP CL SXT RIF E (I)
6	GE CIP CL SXT VA

GE, genatmicin; CIP, ciprofloxacin; SXT, sulfamethoxazol/trimethoprim; CL, clindamycin; RIF, rifampin; E, erythromycin; VA, vancomycin; I, intermediate [according to EFSA (2005, 2008b) and CLSI (2008)].

Table 5a.

MIC Values of Strains Belonging to the Lactobacillus acidophilus Group Regarding Human Theraupeutic Antibiotics ^a

Species	L. acidophilus
Strains	DSM 20079^T	ATCC 4355	ATCC 832	A 1	G 3	Acid 74	Ac 601	Ac 676	B 101	B 102	B 103	B 104
Strain origin	R	R	R	G	G	G	G	G	G	G	G	G
Beta-Lactams
Penicillin	0.06	<0.03	0.12	0.06	0.06	0.06	0.12	0.06	0.06	0.12	0.06	<0.03
Ampicillin	0.25	<0.12	0.5	0.25	0.25	0.25	0.5	0.25	0.25	0.5	<0.12	<0.12
Oxacillin	0.5	<0.25	<0.25	<0.25	1	<0.25	1	<0.25	<0.25	1	0.5	0.5
Cephalothin	4	<0.5	1	1	1	1	2	1	1	2	<0.5	1
Macrolides
Erythromycin	0.25	0.5	0.25	0.5	0.25	0.25	0.5	0.25	<0.12	0.5	0.25	1
Lincosamid
Clindamycin	8	8	8	2	0.12	2	2	<0.12	<0.12	4	8	16
Aminoglycoside
Gentamicin	>32	>32	>32	>32	16	32	>32	>32	8	>32	32	>32
Carbapenem
Imipenem	1	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25
Chloramphenicol	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4
Rifampin	<0.5	1	1	<0.5	<0.5	1	<0.5	1	<0.5	<0.5	<0.5	4
Ciprofloxacin	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4
Sulfamethoxazol/Trimethoprim	4/76	<0.25/4.75	>4/76	>4/76	0.5/9.5	>4/76	4/76	>4/76	0.5/9.5	>4/76	>4/76	>4/76
Tetracycline	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2
Glycopeptide
Vancomycin	32	<0.5	<0.5	<0.5	<0.5	1	<0.5	<0.5	<0.5	<0.5	1	1

Species	L. gasseri
Strains	DSM 20243^T	F 164	B 201	B 202	B 203	B 204	B 205	B 206-I	B 207-I	B 208-I	B 209-I	B 210-III	B 211-III	B 212-III	B 213-III
Strain origin	R	G	G	G	G	G	G	G	G	G	G	G	G	G	G
Beta-Lactams
Penicillin	0.06	0.12	<0.03	<0.03	0.06	0.06	0.06	0.06	0.06	0.06	0.06	<0.03	<0.03	0.06	0.06
Ampicillin	0.5	0.5	0.25	0.5	<0.12	0.25	0.5	0.25	<0.12	0.25	0.25	0.5	0.5	0.5	0.5
Oxacillin	1	<0.25	0.5	1	0.5	1	0.5	<0.25	0.5	0.5	0.5	1	1	1	1
Cephalothin	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	1	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	1
Macrolides
Erythromycin	0.5	0.5	0.25	0.25	<0.12	<0.12	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.5	0.5
Lincosamid
Clindamycin	16	8	16	16	<0.12	<0.12	<0.12	2	2	2	2	8	16	4	4
Aminoglycoside
Gentamicin	>32	>32	32	>32	32	>32	32	>32	32	>32	>32	>32	>32	>32	>32
Carbapenem
Imipenem	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25
Chloramphenicol	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4
Rifampin	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5
Ciprofloxacin	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4
Sulfamethoxazol/Trimethoprim	>4/76	2/39	>4/76	>4/76	>4/76	>4/76	>4/76	4/76	4/76	>4/76	>4/76	>4/76	>4/76	4/76	4/76
Tetracycline	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2
Glycopeptide
Vancomycin	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

Species	L. gasseri									L. johnsonii
Strains	B 214-III	B 215-III	B 216-III	B 217-V	B 218-V	B 219-VI	B 220-VI	B 221-VI	B 222-VI	LMG 9436^T	Omni-flora	B 301	B 302
Strain origin	G	G	G	G	G	G	G	G	G	R	P	G	G
Beta-Lactams
Penicillin	<0.03	<0.03	0.06	0.06	<0.03	0.06	0.06	0.06	<0.03	<0.03	0.12	0.12	0.12
Ampicillin	0.5	0.5	0.5	0.5	0.25	<0.12	<0.12	<0.12	<0.12	0.5	1	0.5	0.5
Oxacillin	0.5	1	1	0.5	0.5	0.5	1	<0.25	1	0.5	1	1	1
Cephalothin	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	1	<0.5	<0.5	<0.5
Macrolides
Erythromycin	0.25	0.25	0.5	0.25	0.25	0.25	0.25	0.5	0.25	0.5	0.25		0.25
Lincosamid
Clindamycin	16	16	16	16	2	8	16	16	16	1	0.25		0.25
Aminoglycoside
Gentamicin	>32	>32	>32	>32	32	>32	>32	>32	>32	>32	>32	>32	>32
Carbapenem
Imipenem	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25
Chloramphenicol	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4	<4
Rifampin	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5
Ciprofloxacin	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4
Sulfamethoxazol/Trimethoprim	1/19	>4/76	>4/76	>4/76	4/76	>4/76	2/38	0.5/9.5	2/38	1/19	2/38	4/76	4/76
Tetracycline	<2	<2	<2	<2	<2	<2	<2	4	<2	<2	<2	<2	<2
Glycopeptide
Vancomycin	1	1	1	1	1	1	1	1	1	1	1	1	1

Tested with MD-Gram-positive microtiter plate; > and <, resp. maximum or minimum value of the test plate; MIC values in μg/mL.

R, type or reference strain; P, probiotic strain; G, strain from the gastrointestinal tract.

Resistance pattern for the L. casei group

Strains of the L. casei group showed similar patterns throughout the species (Table 5b). All strains were susceptible to the beta-Lactams penicillin and cephalothin (in part intermediate), to macrolides (erythromycin), to lincosamides (clindamycin), to the carbapenem imipenem, to rifampin, and to tetracycline. Full resistance could be shown for glycopeptides (vancomycin and teicoplanin). For other substances the resistance pattern differed slightly. As a typical resistance pattern the following combination of resistances can be considered: glycopeptides and sulfonamides. Strains were in most cases resistant to less than three antimicrobial classes with the exception of the probiotic strain P 563 and the clinical strain P 898. This probiotic strain exhibited resistances to four classes (with five antimicrobials altogether) and the clinical strain to five classes.

Table 5b.

MIC Values of Strains Belonging to the Lactobacillus casei Group Regarding Human Therapeutic Antibiotics ^a

Species	L. casei		L. zeae	L. rhamnosus
Strains	ATCC 393	YS 88	DSM 20178	DSM 20021	P 211	P216	DSM 7133	GG	P 237	P 972	P 202/4	H 213	P 898	P 5397
Strain origin	R	R	R	R	P	P	P	P	P	P	P	C	C	C
Beta-Lactams
Penicillin	0.06	1	0.5	0.5	0.25	0.5	0.25	0.5	0.5	1	0.25	0.25	0.5	0.5
Ampicillin	0.12	1	1	1	1	1	1	1	1	2	0.5	0.5	1	1
Oxacillin	0.25	4	0.25	1	<0.25	<0.25	1	4	4	0.25	0.5	<0.25	4	0.25
Cephalothin	2	8	8	16	8	8	8	16	8	8	4	8	8	8
Macrolides
Erythromycin	0.12	0.12	0.12	<0.12	0.25	<0.12	<0.12	0.12	0.12	0.12	0.12	<0.12	0.12	0.12
Lincosamid
Clindamycin	0.12	0.12	0.12	0.25	0.25	0.25	<0.12	0.25	0.25	0.12	0.12	<0.12	0.12	0.12
Aminoglycoside
Gentamicin	2	0.25	1	32	8	16	16	1	1	1	0.25	8	4	1
Carbapenem
Imipenem	0.25	0.25	0.25	2	0.5	0.5	1	0.5	0.5	0.25	0.25	<0.5	0.25	0.5
Chloramphenicol	4	4	8	<4	8	8	<4	8	8	32	4	<4	>32	4
Rifampin	0.5	0.5	0.5	<0.5	<0.5	<0.5	<0.5	0.5	0.5	0.5	0.5	<0.5	0.5	0.5
Ciprofloxacin	>4	0.5	1	1	1	2	1	0.5	0.5	2	4	<0.5	4	1
Sulfamethoxazol/Trimethoprim	>4/76	0.25/4.75	0.25/4.75	>4/76	>4/76	>4/76	>4/76	0.25/4.75	0.25/4.75	0.25/4.75	2/38	>4/76	4/76	4/76
Tetracycline	2	2	2	2	<2	<2	<2	2	2	2	2	<2	2	2
Glycopeptide
Vancomycin	>64	>64	>64	>256^b	>256^b	>256^b	>256^b	>64	>64	>64	>64	>256^b	>64	>64
Teicoplanin	nd	nd	nd	>256^b	>256^b	>256^b	>256^b	nd	nd	nd	nd	>256^b	nd	nd

Species	L. rhamnosus								L. paracasei
Strains	U 4596-95	CCUG 28641	CCUG 29281	CCUG 28262	CCUG 18011	CCUG 27333	CCUG 30616	CCUG 22413	DSM 5622	Y	P 563	F 10a	Rv 1p
Strain origin	C	C	C	C	C	C	C	C	R	P	P	G	Sausage
Beta-Lactams
Penicillin	1	0.5	0.5	1	0.5	0.25	0.5	1	0.25	0.5	0.5	0.25	0.06
Ampicillin	2	2	1	2	1	0.5	1	2	1	1	4	0.5	0.12
Oxacillin	16	0.5	0.25	0.25	0.25	1	4	4	4	2	4	2	0.25
Cephalothin	8	8	8	8	8	8	16	8	8	16	16	4	1
Macrolides
Erythromycin	0.12	<0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12
Lincosamid
Clindamycin	0.12	<0.12	0.12	0.12	0.25	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12
Aminoglycoside
Gentamicin	1	16	1	0.5	1	1	0.5	1	1	0.5	4	0.5	0.5
Carbapenem
Imipenem	0.5	1	0.25	0.5	0.5	0.5	0.5	0.5	0.25	0.5	1	0.25	0.25
Chloramphenicol	8	8	4	8	8	4	8	8	16	8	32	8	4
Rifampin	0.5	<0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	1	0.5	>4
Ciprofloxacin	1	1	1	1	2	0.5	1	1	1	0.5	4	2	>4
Sulfamethoxazol/Trimethoprim	4/76	>4/76	>4/76	4/76	2/38	4/76	2/38	>4/76	0.25/4.75	0.25/4.75	1/19	0.25/4.75	>4/76
Tetracycline	2	<2	2	2	2	2	2	2	2	4	4	2	>16
Glycopeptide
Vancomycin	>64	>256^b	>64	>64	>64	>64	>64	>64	>64	>64	>64	>64	0.5
Teicoplanin	nd	>256^b	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd

Tested with MD-Gram-positive microtiter plate and E-Test. resp.; > and <: resp. maximum or minimum value of the test plate; MIC values in μg/mL;

Tested with E-test.

R, type or reference strain; P, probiotic strain; G, strain from the gastrointestinal tract; C, clinical strain; nd, not done.

Resistance pattern for the L. reuteri/L. fermentum group

Within the L. reuteri/L. fermentum group the resistance patterns were quite uniform (Table 5c). Strains were susceptible to macrolides (erythromycin) and lincosamides (clindamycin) with the exception of strain PE 3 from pigeon crop. The same applied for aminoglycosides (gentamicin), imipenem, chloramphenicol (exception strain F 138a from feces, human), and rifampin (exception strain K3/47S). All strains were resistant to ciprofloxacin and glycopeptides (vancomycin). Especially all probiotic strains from producer H exhibited a resistance against the glycopeptide vancomycin and tetracycline (with one exception [strain 4020II, susceptible]), trimethoprim/sulfamethoxazol, against quinolones (ciprofloxacin, lomefloxacin, ofloxacin, and norfloxacin), and against the aminoglycoside gentamicin. They were susceptible to ampicillin/sulbactam, chloramphenicol, and rifampin. Most of those probiotic strains were susceptible to penicillin, ampicillin, cephalothin, and cefazolin. Especially strains MV4-2a, MV10-1a, MV11-1a, and SD 2112 were resistant against these substances. Most of the strains were resistant against oxacillin, differing reactions could be observed for erythromycin (susceptible and intermediate) and for nitrofurantoin (susceptible, intermediate, and one strain resistant [MV11-1a]). Overall, strains were resistant to no more than 3–4 classes of antimicrobials. Exceptions were the probiotic strains from producer H, one further probiotic strain (resistant to five classes with eight substances), the type strain of L. fermentum and L. fermentum strain F 138a.

Table 5c.

MIC Values of Strains Belonging to the Lactobacillus reuteri/Lactobacillus fermentum Group Regarding Human Therapeutic Antibiotics ^a

Species	L. reuteri								L. fermentum
Strains	DSM 20016	K 3/47S	D 287	F 275	F 275c	XVII cae	PE 3	Sym balance	ATCC 14931	DSM 20052	ATCC 23271	F 138a	23
Strain origin	R	R	R	G	G	G	G	P	R	R	R	G	G
Beta-Lactams
Penicillin	1	>4	0.5	0.5	2	1	0.5	>4	0.25	0.25	0.5	0.03	1
Ampicillin	0.5	2	0.5	1	1	0.5	0.12	>16	0.12	0.12	0.25	0.12	0.5
Oxacillin	0.25	32	0.25	2	32	8	0.25	>32	0.25	4	0.25	1	0.25
Cephalothin	4	32	8	16	8	8	8	64	4	4	4	64	8
Macrolides
Erythromycin	0.12	0.12	0.12	0.12	0.12	0.12	>16	0.12	0.12	0.12	0.12	0.12	0.12
Lincosamid
Clindamycin	0.12	0.12	0.12	0.12	0.12	0.12	>16	0.12	0.12	0.12	0.12	0.12	0.5
Aminoglycoside
Gentamicin	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Carbapenem
Imipenem	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Chloramphenicol	8	8	8	16	8	4	4	16	4	16	8	32	4
Rifampin	0.5	4	0.5	0.5	0.5	0.5	0.5	0.5	0.5	2	0.5	1	2
Ciprofloxacin	>4	>4	>4	>4	>4	4	>4	>4	>4	>4	>4	>4	>4
Sulfamethoxazol/Trimethoprim	>4/76	0.5/9.5	>4/76	>4/76	>4/76	1/19	0.25/4.75	>4/76	4/76	>4/76	>4/76	>4/76	0.25/4.75
Tetracycline	16	>16	>16	>16	>16	16	8	>16	>16	>16	8	>16	>16
Glycopeptide
Vancomycin	>64	>64	>64	>64	>64	>64	>64	>64	>64	>64	>64	>64	>64

	Bacterial strains of L. reuteri (all probiotic)
Antibiotic/chemotherapeutic	4000	4020I	4020II	2010	M164	MV4-2a	MV10-1a	MV11-1a	SD 2112	T-1	1063	11284
β-Lactams Penicillin	2	1	0.12	2	0.5	>8	>8	>8	>8	0.75	0.75	1
Ampicillin	1	0.75	0.5	1.5	0.5	>8	>8	>8	>8	0.75	0.75	1.5
Ampicillin/Sulbactam	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4	<8/4
Oxacillin	>2	0.75	>2	>2	2	>2	>2	>2	>2	>2	>2	1.5
Cephalothin	8	2	2	6	4	>16	>16	>16	>16	6	8	4
Cefazolin	8	3	<2	4	4	>16	>16	>16	>16	3	6	4
Macrolides Erythromycin	2	0.5	0.5	0.25	0.5	1	0.75	1	0.75	2.5	3	0.75
Clarithromycin	0.5	0.25	<0.12	<0.12	0.12	0.25	0.25	0.25	0.12	0.75	0.5	0.12
Lincosamides/Clindamycin	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25	<0.25
Aminoglycosides/Gentamicin	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500	>8/<500
Chloramphenicol	8	<4	4	<4	<4	4	8	4	<4	4	4	<4
Rifampin	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5	<0.5
Nitrofuranes/Nitrofurantoin	<32	<32	<32	<32	<32	64	64	>64	64	<32	<32	<32
Quinolones Ciprofloxacin	>2	>2	>2	>2	>2	>2	>2	>2	>2	>2	>2	>2
Lomefloxacin	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4
Ofloxacin	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4	>4
Norfloxacin	>8	>8	>8	>8	>8	>8	>8	>8	>8	>8	>8	>8
Trimethoprim/Sulfamethoxazol	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38	>2/38
Tetracycline	>8	>8	3	>8	>8	>8	>8	>8	>8	>8	>8	>8
Glycopeptide/Vancomycin	>16	>16	>16	>16	>16	>16	>16	>16	>16	>16	>16	>16

Tested with MD-Gram-positive microtiter plate, MG-plate and E-Test, resp.; > and <, resp. maximum or minimum value of the test plate; MIC values in μg/mL.

R, type or reference strain; P, probiotic strain; G, strain from the gastrointestinal tract.

vanA gene detection

The existence of the vanA gene was tested for certain strains of the species L. rhamnosus (DSM 20021^T, DSM 7133 [probiotic], P 237 [probiotic], and H 213 [clinical]) and for several probiotic L. reuteri strains. All these strains were vancomycin resistant phenotypically. None of these strains proved to harbor the vanA gene. The positive control strains E. faecium 70/90 and E. faecalis 1528 showed the typical 377 bp fragment, and the negative control strain E. faecium 64/3 did not.

tet gene detection

The PCR analysis of the three Lactobacillus strains, ATCC 55149, ATCC 53608, and ATCC 55148, showed that none of the strains possessed tet-(A), tet-(B), tet-(C), tet-(M), or tet-(O) genes. With the controls PCR fragments were obtained for the respective tet genes. No specific tet-PCR fragments were obtained with the negative control strain (S. aureus DSM 2569). Unspecific PCR products for tet genes could be observed for some L. reuteri field strains. DNA isolation and PCR analysis with all primer pairs was performed twice with identical results. No relationship between tet-(A), tet-(B), tet-(C), tet-(M), or tet-(O) genes and tetracycline resistance in these probiotic strains could be observed.

Protein fingerprinting

Protein fingerprinting showed clear species-specific clusters for the main taxonomic units investigated, that is, the L. casei group, L. reuteri group, and, for comparison reasons, L. plantarum group, all part of the closely related group of “beta-” and “streptobacteria” within the genus Lactobacillus (Fig. 1). The cluster of the species L. rhamnosus was highly homogenous with r = 93%. Another cluster consisted of the species L. paracasei, including some strains of the species L. casei sensu stricto. The type strains of L. casei and L. zeae were unrelated to any other cluster. L. plantarum exhibited a similarity of r = 71.5% to the L. casei group, both part of the classical streptobacteria. L. reuteri was clearly separated from the other species and strain PE 3 from pigeon crop was unrelated to others. No distinction between clinical strains, probiotic strains, or strains isolated from the gastrointestinal tract could be made. On the contrary, some probiotic strains showed high similarity to clinical strains (Fig. 1).

FIG. 1.

Dendrogram, mainly of probiotic and clinical strains, from the Lactobacillus casei group and the L. reuteri/fermentum group by protein fingerprinting of total soluble cytoplasmatic protein (Colloidal-Coomassie-blue staining). P, probiotic; R, reference strain; C, clinical origin; GIT, gastrointestinal tract.

Discussion

The phenotypic identification was done for each strain, as many strains are misidentified in products or after clinical isolation (Klein et al., 1998). In this study all species identifications could be confirmed.

Only few resistances could be shown for the L. acidophilus group. For the type and reference strains, this was expected, as they are partly isolated several decades ago and are hence maintained in culture collections or as laboratory cultures. On the contrary, the type strain of L. acidophilus DSM 20079^T (ATCC 4356^T) exhibited the most resistances, including vancomycin resistance. Similar results were obtained by Florez et al. (2008). However, freshly isolated strains from the intestinal tract did not show an extended spectrum of resistance. With the exception of the type strain all tested strains remained vancomycin susceptible, which is in accordance with other studies (Klare et al., 2007). Regarding the probiotic strains of the L. acidophilus group, this is an important observation because of isolations from clinical material (Aguirre and Collins, 1993) and therapeutic options in medical therapy. No species-specific pattern could be identified; however, L. gasseri comprised isolates from the vaginal flora (B 203 and B 204) with the lowest number of resistances within this group (resistance type 2, Table 4). Another strain from feces, B 205, was in the same resistance group. In general, strains from feces exhibited more resistances similar to strains from the intestinal tract (resistance group 4 and 4a), with the exception again of strain F 164 (resistance group 3), which was kept in laboratory culture for more than 40 years. Strains from the same origin had usually the same resistance pattern like strains B 206-I to B 209-I and B 210-III to B 216-III (exception B 214-III), which were isolated from the same person (I or III, resp.).

The L. casei group showed full resistance to glycopeptides as noted in other studies too (D'Aimmo et al., 2007). The exclusion of transferable vanA genes allows the usage of strains of this group as probiotics (Klein et al., 2000). The strains used as probiotics or potential probiotic strain, strain P 563 (L. paracasei) and P 202/4 (L. rhamnosus) (sour milk), exhibited similarly to the clinical strain P 898 (L. rhamnosus) (liver abscess) more resistances, including ciprofloxacin. This has also been shown for nalidixic acid in L. casei strains (D'Aimmo et al., 2007). Strain P 563 was additionally resistant against beta-lactam-antibiotics (ampicillin and oxacillin), which may cause difficulties in the safety evaluation of the strain as no defined method for resistance determination in lactobacilli is described (Hummel et al., 2007).

All tested L. reuteri and L. fermentum strains were resistant to the glycopeptide vancomycin and to tetracycline, which has also been shown in other studies (Klare et al., 2007; Egervarn et al., 2009). Again a probiotic strain (L. reuteri) exhibited a higher number of resistances, including beta-lactam antibiotics, which was found uncommon in other investigations (Klare et al., 2007). The type strain was tested with cultures from two different culture collections (DSM 20016^T and F 275^T) and these strains showed slightly different MIC pattern (Table 5c). However, these strains were for ∼15 years cultivated separately in different collections. Together with the error margin of the MIC determination, the differences in the MIC results can be explained.

Resistance genes were expected to be absent in case of vanA genes (Klein et al., 2000; Klare et al., 2007), where the structure of the cell wall leads to intrinsic resistance to glycopeptides (Delcour et al., 1999). Tetracycline resistance genes have been detected in several cases. In Italian fermented dry sausages, tet-(M) was detected in 60% of the tetracycline-resistant lactobacilli, but not among the species of L. rhamnosus (Zonenschain et al., 2009). This is consistent with this study, where tet-(M) was present in the strains of L. reuteri as well. Further, none of the probiotic strains tested possessed tet-(A), tet-(B), or tet-(C) genes as it was expected for Gram-positives, but also tet-(O) genes were absent. In the Italian study (Zonenschain et al., 2009) tet-(L) was not detected and tet-(S) was found only in one L. plantarum strain; however, the tet-(W) gene was more prevalent, but again mainly in L. plantarum and not in L. reuteri. On the contrary tet-(W) genes were found frequently in another study (Egervarn et al., 2009) in L. reuteri strains from diverse origin. In probiotic Lactobacillus strains (Klare et al., 2007) no tet-(W) was found, indicating a great diversity within strains. It also depends on the MIC, with MIC for tetracycline >16 μg/mL more likely to possess a tet-(W) gene than lower MICs (Klare et al., 2007). In another study, strain L. reuteri SD 2112, also tested in this study, harbored the tet-(W) gene; however, the MIC was not determined and a disk diffusion method was applied (Kastner et al., 2006). Starter and probiotic strains, including some Lactobacillus with MICs up to 16 μg/mL species, did not reveal any tet genes (Hummel et al., 2007), confirming the above-mentioned results (Klare et al., 2007). Therefore, the resistance mechanisms of the probiotic L. reuteri strains tested in this study with MICs >16 μg/mL for tetracycline are possibly harboring the tet-(W) gene. However, tet genes like K and L must be monitored as well.

Protein fingerprinting (Fig. 1) proved that clinical strains of L. rhamnosus clustered together with probiotic strains and reference strains. The analysis of the L. reuteri/fermentum group showed type strain DSM 20016^T in close relationship to the probiotic strain Symbalance, which has been shown in a similar way also for type and production strains of the L. rhamnosus group (Klein et al., 1995) and the L. acidophilus group (Klein et al., 1998). A clear differentiation of probiotic strains from clinical ones is therefore not possible, and safety evaluation according to the QPS rules (EFSA, 2008a) is further complicated by missing standards of MIC evaluation and the interpretation of multiple resistances within probiotic strains.

Nevertheless, the QPS approach together with the recommendations of the FEEDAP panel (EFSA, 2005, 2008b) of EFSA will give a framework for better decision making in safety assessments, especially when more data on resistance pattern among probiotic species is available. The current study could identify some commercially applied strains where an extended spectrum of resistance was present, compared to other strains of the same species. These findings can be used for a thorough strain-specific safety evaluation by the producer.

The data on resistance pattern of a broad range of species studied in this investigation can also be used for decision making on the safety of probiotics in food and feed, for example, following the QPS approach, by authorization bodies.

Footnotes

Disclosure Statement

No competing financial interests exist.

*

Only for probiotic L. reuteri strains.

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