PaPer 282 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: antibiotic resistance; Lactobacillus; gastrointestinal tolerance; plasmid; probiotic - Antibiotic susceptibility of potentiAlly probiotic LactobaciLLus strAins JunhuA hAna, DAhuAn chena, shAnshAn lic, Xingfeng lia, Wen-Wen Zhoud, bolin ZhAngb,*, yingmin JiAa,* aCollege of Biological Science and Engineering, Hebei University of Science and Technology, Shijiazhuang, Heibei, 050018, China bSchool of Biological Science and Biotechnology, Beijing Forestry University, Beijing, 100083, China c Dongcheng District Center For Disease Control And Prevention, Beijing, 100009, China dSchool of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou Zhejiang, 310058, China *Corresponding authors: Yingmin Jia, Tel. +86 311 81668010, jjyymm0311@163.com Bolin Zhang, Tel. +86 10 62338221, zhangbolin888@163.com AbstrAct susceptibility of 29 Lactobacilli to 13 antibiotics was assayed by paper disc diffusion method. Plasmids and gastrointestinal tolerance were detected. the relationship between plasmids and antibiotic resistance was discussed. the results showed that all of the strains were resistant to bacitracin, polymyxin b, kanamycin, and nalidixic acid. Many strains were relatively sensitive to chloramphenicol and tetracycline. six strains contained plasmids and showed good gastrointesti- nal tolerance. β-lactam resistance gene blr was found in the plasmid of L. plantarum cIcc 23180 by Pcr. the study will be helpful to promote the safety evaluation and development of potential- ly probiotic lactic acid bacteria. mailto:zhangbolin888@163.com Ital. J. Food Sci., vol. 27 - 2015 283 1. IntroductIon due to the claimed benefits, Lactobacillus bacteria are widely used in food, feed, medical and health related fields. Many lactic acid bac- teria (LAb), such as Streptococcus thermophi- lus and Lactobacillus delbruekii subsp. bulga- ricus, have been used safely for a long history. they are agreed to be secure and do not have the possibility of pathogenic. currently, new beneficial bacteria are being developed contin- uously and will enter the market. However, the security of these new strains has caused great concern. Evaluation of antibiotic sensitivity is an important part of safety assessment. now, overuse of antibiotics has become a serious social problem. this led to the emer- gence of a large number of antibiotic-resistant strains. once the resistance-related factors are tranferred to other microorganisms, especial- ly pathogens via food carrier, it will cause tre- mendous problems. the evolution of antibiotic- resistant foodborne pathogens has been widely reported (tHrELfALL et al., 2000; WALsH et al., 2008; WHItE et al., 2002). Moreover, the resist- ance and resistance-related genes of Bifidobac- terium, Lactobacillus and Pediococcus strains to different antibiotics were studied systematical- ly (HuMMEL et al., 2007; Huys et al., 2004; MA- rIA et al., 2007). the tetM gene transfer of tet- racycline resistance in Lactobacillus plantar- um among strains was reported by nIAMH et al. (2010). In this study, 29 Lactobacillus strains isolat- ed from the food environment with potentially probiotic effects (JIn et al., 2009; LI et al., 2009; LIu et al., 2011; sun et al., 2009; ZHAo et al., 2013) were used. these strains were assayed for susceptibility to 13 antibiotics by agar disc dif- fusion method. furthermore, some strains with higher resistance were analysed for the presence of plasmids. then, the tolerance of the plasmid- containing strains under simulated gastroin- testinal conditions was investigated. by plas- mid elimination and Pcr, the relationship be- tween the plasmid profiles and resistance pat- terns of the strains was explored. this will pro- vide a reference for the safety evaluation meth- od and also will be helpful to improve the eval- uation system of probiotics. 2. MAtErIALs And MEtHods 2.1 Bacterial strains and cultivation 29  Lactobacillus strains  used in the study were listed in table  1. Lactobacillus strains were cultured in Mrs (de Man, rogosa, and sharpe) medium at 37°c for 18h under anaer- obic condition. Quality control strain recommended by clin- ical and Laboratory standards Institute (cLsI) in the antibiotic  sensitivity test was  E. coli Atcc25922 purchased from the Institute of Mi- crobiology, chinese Academy of sciences. the E. coli Atcc25922 was activated and cultivat- ed in Lb medium (yeast extract 5 g/L, tryptone 10 g/L, nacl 10 g/L) at 37°c. 2.2 Testing of antibiotic susceptibility 13 kinds of antibiotics paper discs were pur- chased from the national Institute for the con- trol of Pharmaceutical and biological Products (table 2), each piece with a diameter of 6.5 mm. the quality  was fully complied with  the  WHo criteria. Antibiotic susceptibility was semi-quantita- tively determined with K-b method by antibiot- ic paper disc diffusion referring to the cLsI as described by cHArtErIs et al. (1998a). briefly, 1.0 mL Lactobacillus suspension (ap- proximately 1.5×108 cfu/mL) was added to sterile petri dish with diameter of 90 mm, and then mixed with a 15 mL MH (Muller Hinton, MH) agar (beef extract powder 6g/L, casein ac- table 1 - source of the tested strains for antibiotic susceptibility test. Species Source (original number) Lactobacillus plantarum CICCa 23124 (L11), CICC 23131 (B31), CICC 23135 (B37), CICC 22195 (C35), CICC 23166 (ZJ1), CICC 23138 (C8-1), CICC 23180 (CH8) Lactobacillus rhamnosus CICC 23119 (1132), CICC 22175 (LL), ATCCb 7469, CICC 22151 (LK-Mt), CICC 22173 (R11) Lactobacillus salivarius CICC 23182 (CH-10) Lactobacillus acidophilus CICC 22162 (CH-2) Lactobacillus casei CICC 23184 (Y5-2b) Lactobacillus helveticus CICC 22154 (LLB) Lactobacillus pentosus CICC 23116 (SN23), CICC 22161 (Lp-4), CICC 22160 (Lp-5), CICC 22159 (Lp-B), CICC 22156 (Ind-3), CICC 22157 (Lp-A) Lactobacillus paralimentarius CICC 22148 (412), CICC 22149 (413) Lactobacillus delbrueckii CICC 22153 (LB), CICC 22163 (LC) Lactobacillus paracasei CICC 22165 (5M1), CICC 22167 (5M7), CICC 23183 (D-400) aCICC, China center of industrial culture collection. bATCC, American type culture collection. 284 Ital. J. Food Sci., vol. 27 - 2015 ids hydrolysate  17.5 g/L,  soluble  starch,  1.5 g/L, agar 17 g/L, pH 7.3±0.1) until the medi- um solidified. the antibiotic paper discs were pasted closely onto the solidified medium with sterile tweezers after 5min at room temper- ature. three discs were pasted in each dish. the distance was more than 24 mm of each disc center and more than 15 mm from disc edge to the inner edge of dish. next, the dishes were placed at room temperature for 1.5 h and then incubated at 37°c. After 24 h, the inhibi- tion zone diameter was measured around the antibiotic disc with vernier caliper and record- ed. for one tested strain, each antibiotic disc was done 3 times. the inhibition zone diame- ter was averaged standard sensitive strain of E. coli Atcc25922 was used as the control. the operation was the same as the above. the antibiotic susceptibility of the tested strains was evaluated according to the cLsI cri- teria (table 2). 2.3 Plasmid DNA extraction 10 mL of Lactobacillus suspension cultured overnight was centrifugated at 10000 rpm for 5 min. then the precipitation was suspended with 500 μL of lysozyme solution (10 mg/mL). the mixture was placed in a water bath for 45 min at 37°c. then plasmid dnA of Lactobacilli strains was extracted and purified with dnA ex- traction and purification kit of tiangen biotech (beijing) co., Ltd. Plasmid dnA was observed by agarose gel electrophoresis. Antibiotic susceptibility and plasmid stabil- ity were tested after cultivated 30 generations at 37°c in Mrs medium according to the above methods. table 2 - the content of antibiotic paper discs and criteri- on for judgement. Antibiotics Content/disc inhibition zone diameter (mm) Rc I S Vancomycin 30 μg ≤9 10-11 ≥12 penicillin G 10 U ≤14 15-17 ≥18 ampicillin 10 μg ≤21 22-28 ≥29 Bacitracin 0.04 U ≤10 10-12 ≥12 cephalothin 30 μg ≤14 15-17 ≥18 streptomycin 10 μg ≤11 12-14 ≥15 kanamycin 30 μg ≤13 14-17 ≥18 tetracycline 30 μg ≤11 12-14 ≥15 chloramphenicol 30 μg ≤12 13-17 ≥18 gentamicin 10 μg ≤12 13-14 ≥15 nalidixic acid 30 μg ≤13 14-18 ≥19 multi-polymyxin B 300 μg ≤11 12-14 ≥15 rifampicin 5 μg ≤16 17-19 ≥20 cNote: R-Resistant; S-Susceptible; I-Intermediate. 2.4 Gastrointestinal tolerability test  In order to explore the application safety in  human, the gastrointestinal  tolerability of those lactic acid bacteria containing the plas- mids were tested. for acid tolerance test, Lactobacillus cells were harvested by centrifugation at 6000 rpm for 15 min, washed twice with 0.01 mol/L Pbs, pH 7.2 after cultured for 18 h at 37°c in Mrs broth, and then suspended in 20 mL sterile sa- line (0.85%, w/v) adjusted to pH 2.5 with ster- ile hydrochloric acid. for bile tolerance test, the modified method of LEE et al. (1999) was referred to test bile tol- erance. the Lactobacillus cells were centrifuged (6000 rpm, 15 min) after cultivated for 18 h at 37°c in Mrs broth and suspended in 20 mL ster- ile saline (0.85 %, w/v) supplemented with 0.3% (w/v) bile salts (taurocholate, sigma) at pH 6.8. for pepsin and trypsin tolerance test, Lactoba- cillus cells were centrifuged (6000 rpm, 15 min) after cultivated for 18 h at 37°c in Mrs broth, then suspended in 20 mL sterile simulated gas- tric and pancretic juices. fresh simulated gas- tric and pancreatic juices were prepared daily according to charteris et al (1998b). Pepsin (sig- ma) was added into the simulated gastric juice with a final concentration of 5 mg/mL. then the pH was adjusted to 2.5 with sterile hydrochloric acid. trypsin (sigma) was added into the sim- ulated pancreatic juices with a final concentra- tion of 10 mg/mL. then pH was adjusted to 8.0 with 0.1 M naoH. All of the tolerability detection, the initial bac- terial counts were adjusted to about 108 cfu/ mL and were checked by viable count determi- nation on Mrs agar. for the tolerance assay, the bacterial suspensions were incubated and counted at 37°c for 0,1,2,3,4,5,6 h, respectively. All tests were repeated three times to estimate the standard error. 2.5 Detection of antibiotic resistance genes Part of the antibiotic-resistant genes of those lactic acid bacteria containing both plasmids and high tolerance were investigated. the β-lactam resistance-related gene sequence of blr, ECP- 1569, nps-1 and the chloromycetin resistance- related gene sequence of cmlA, cat, cmlA1 in plasmids were found in national center for bi- otechnology Information (ncbI). the primers were designed and synthesized by beijing sun- biotech co., Ltd (table 3). the Pcr programmes were performed with the plasmid template of the tested strains accord- ing to the following procedures: initial heating at 94°c for 4 min was followed by 34 cycles of the following sequence: 94°c for 30 s, 72°c for 1 min, and 72°c for 1 min. final extension took place at 72°c for 7 min. the amplification products were separated Ital. J. Food Sci., vol. 27 - 2015 285 by conventional 1.0% (w/v) agarose gel electro- phoresis  (100V, 4°c) in tAE (tris-acetate-Ed- tA) buffer and visualised by ethidium bromide staining. the target fragment was recovered and sequenced by taKara biotechnology (dali- an, china) co., Ltd. the resistance-related gene of plasmid was determined by comparison with the known fragment. 3. rEsuLts And dIscussIon 3.1 Antibiotic susceptibility Antibiotic susceptibility of the tested strains was evaluated according to the anti-microbi- al  drug sensitivity standard of cLsI criteria. the sensitivity of the tested Lactobacillus to 13 kinds of antibiotics was shown in table 4. the tested 29 strains were generally resisitant to multi-polymyxin b, bacitracin, kanamycin, na- lidixic acid, and were mostly sensitive to chlo- ramphenicol and tetracycline. the same species of Lactobacillus generally had similar resistance patterns. but there was species specificity such as the different antibiotic sensitivity in L. plan- tarum, L. rhamnosus, and L. pentosus. Moreover, the antibiotic-resistant level of different strains is also different. Antibiotic resistance of the foodborne lactic acid bacteria had heen reported in the 1980s. the researchers generally believed that the re- sistance was a result of the long evolution and it was generally endogenous resistance and ob- tained resistance (Zeng et al., 2004). so, the re- sistant lactic acid bacteria of natural or isolat- ed from human intestinal can indirectly reflect the habitat of used antibiotic. It can be seen  from table 5, the  29  strains s h o w e d   d i f f e r e n t p a t t e r n s o f r e s i s t - table 3 - the primers of the resistance genes in the experiment. Gene Sequence of the primer Annealing temperature Fragment size blr--up 5’-CGTCTTATTGAATTAACAGGTTGG -3’ 53°C 125 bp blr--down 5’-CACGAAGCCATGTTGTGTTC -3’ ECP-1569--up 5’-CAATCAACAGAGATGTGGGCTG-3’ 57°C 155 bp ECP-1569--down 5’-GTACCGTAGTACTCTGTTCAGGTGG-3’ nps-1--up 5’-TCATTCTTCTGGCCTGTAGC-3’ 54°C 782 bp nps-1--down 5’-GGCGATACCGCTCAGTTAC-3’ cmlA--up 5’-CAAGGAGATGGTTTCGTGCG-3’ 56°C 551 bp cmlA--down 5’-CATGCCCAAACCTAGAAACGC-3’ cat--up 5’-GGCATTTCAGTCAGTTGCTC-3’ 55°C 530 bp cat--down 5’-TGGAAGCCATCACAAACG-3’ cmlA1--up 5’-GCTGAAGCCAAGCTGAGAC-3’ 56°C 492 bp cmlA1--down 5’-CTACGTTGTGGCGTCAATG-3’ ance  to  13  kinds of antibiotics. to bacitra- cin, polymyxin b, kanamycin and nalidixic acid, the resistance rate of the 29 tested strains was 100%. to β-lactam and aminoglycosides, the resistance  percentage  was 20.7%-37.9% and 86.2%-100%, respectively. All of the 29 strains were mostly sensitive to  chloramphenicol and tetracycline. Among of the tested antibiotics, the nali- dixic acid and polymyxin b can inhibite dnA synthesis and interfer cell membrane forma- tion, respectively. the resistance of lactobacil- lus to these kinds of antibiotics may be due to the thicker cell wall of Gram-positive bacteria. While the tested strains showed different sen- sitivity to the antibiotics, such as streptomy- cin, kanamycin, tetracycline, chloramphenicol, gentamicin with protein synthesis inhibitition effect. Most lactobacillus strains showed re- sistance to those antibiotics against gram-neg- ative bacteria, for example, streptomycin, gen- tamicin, kanamycin. this was consistent with report of Zhang et al (2007). 3.2 Plasmid DNA extraction of antibiotics-resistant lactobacillus strains 16 cIcc strains with relatively strong antibi- otic resistance were screened for plasmid extrac- tion. As can be seen from fig. 1, among these strains, only cIcc 23180, 22161, 22175, 22157, 23124, and 22154 contained plasmids. L. plantarum cIcc 23180 showed 6 plasmid dnA bands and there is one band greater than 23 kb. L. pentosus cIcc 22157 showed two plas- mid dnA bands of 10 kb and 5 kb, respective- ly. L. rhamnosus cIcc 22175 and L. plantarum cIcc 23124 contained respectively 2 and 4 of plasmid dnA bands and both of the two strains contained a 10 kb plasmid. L. helveticus cIcc 286 Ital. J. Food Sci., vol. 27 - 2015 t a b le 4 - t h e se n si ti vi ty r es u lt s o f 2 9 L a ct o b a ci ll u s s tr a in s to 1 3 a n ti b io ti cs . L. pla nta rum L. pe nto su s L. rha mn os us L. sa liv ari us L. ac ido ph ilu s L. ca se i L. he lve tic us L. pa rac as ei L. de lbr ue ck ii L. pa ral im en tar ius CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC AT CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC CI CC 23 16 6 23 13 1 23 18 0 23 13 5 23 12 4 23 13 8 22 19 5 22 16 1 22 15 7 22 16 0 22 15 9 22 15 6 23 11 6 22 17 5 23 11 9 74 69 22 15 1 22 17 3 23 18 2 22 16 2 23 18 4 22 15 4 22 16 5 22 16 7 23 18 3 22 15 3 22 16 3 22 14 8 22 14 9 Va nc om yc in R R R R R R R R R R R R R R R R R R R S R R R R R S S R R pe nic illin G R R S R R R R S R I R S S I R R I I S S S S S S S S S S R ce ph alo thi n R R R R S S S I R I I S S R S I I I I I S S S S S S S I I Ba cit rac in R R R R R R R R R R R R R R R R R R R R R R R R R R R R R am pic illin R I I I I I I I I R I R I R I I R I R I I R I I R I I R R Mu ltip oly my sin B R R R R R R R R R R R R R R R R R R R R R R R R R R R R R str ep tom yc in R R R R R R R R R R R R R R R R I R R R R R R R R R I R R ka na my cin R R R R R R R R R R R R R R R R R R R R R R R R R R R R R tet rac yc lin e R I S R S I I I S S S S I R S S S S S S S S S S S S S S S ch lor am ph en ico l I S R S S S S R S I I I I R S I I I I S I I S S S S S I S ge nta mi cin R R R R R R R R R R R R R R R R I R R R R R R R S S I R R na lid ixic ac id R R R R R R R R R R R R R R R R R R R R R R R R R R R R R rifa mp icin R R R I R R S R I R I S S I S S S S R R S S S S S S S S R table 5 - the percentage of the antibiotic resistance of 29 Lactobacillus strains. Antibiotics Quantity Percentage of resistant strains of resistance (%) vancomycin 26 89.7 penicillin G 11 37.9 cephalothin 6 20.7 bacitracin 29 100 ampicillin 10 34.5 multi-polymyxin B 29 100 streptomycin 27 93.1 kanamycin 29 100 tetracycline 3 10.3 chloramphenicol 3 10.3 gentamicin 25 86.2 nalidixic acid 29 100 rifampicin 10 34.5 fig. 1 - the plasmids in Lactobacillus (1.cIcc 23180, 2.cIcc 22161, 3.cIcc 22175, 4.cIcc 22157, 5.cIcc 23124, 6.cIcc 22154. M. λHindIII marker). 22154 showed only one plasmid dnA band of about 10 kb. Lactic acid bacteria generally contain plas- mids. the plasmid size was usually 1.9 kb-84.8 kb. Most of the plasmid was less than 20 kb (WAnG and LEE, 1997). In the culture process from generation to generation, many plasmids might disappear from the bacterial cell, but most of the plasmids were stable. In the study, the plasmids of the above six strains and the anti- biotic susceptivity showed no changes after cul- tivated 30 generations. 3.3 Gastrointestinal tolerability resistance to gastrointestinal stress is very important for one strain to play the potential probiotic function (GuGLIELMottI et al., 2007). If the strains have a high tolerance to gastroin- testinal stress, it will have the chance to  sur- vive and play the probiotic effects in the gastro- intestinal environment. the tolerance of the selected six strains to low Ital. J. Food Sci., vol. 27 - 2015 287 pH, bile salt, pepsin and trypsin is presented in fig. 2. As shown in fig. 2A, the viable counts of L. pentosus cIcc 22161 strain reduced to be- low 106 cfu/mL after 4 h and 1.32 × 104 cfu/ mL  after  6 h. However, the viable numbers of L. helveticus cIcc 22154,  L. pentosus cIcc 22157,  L. plantarum cIcc 23124,  23180 and L. rhamnosus cIcc 22175 were still more than 106 cfu/mL after 6 h in the gastric acid of pH 2.5. thus, these five strains showed higher tol- erance in acid environment.  for bile tolerance, except the L. pentosus cIcc 22161, the viable counts of the other five strains were still more than 106 cfu/mL after 6 h in the medium containing bile salt (fig. 2b). However, the viable cells of L. pentosus cIcc 22161 had decreased to 2.0×106 cfu/mL within 3 h. And it declined to only 1.8 × 104 after 6 h. for pepsin tolerance, among of six strains, the viable cells of L. pentosus cIcc 22161 and L. helveticus cIcc 22154 decreased significant- ly in 6 h and it is less than 104 cfu/mL and 106 cfu/mL after 6 h exposure to 5 mg/mL pepsin solution (pH 2.5), respectively (fig. 2c). for trypsin tolerance, as can be seen in fig. 2d, the viable counts of the tested six strains still re- mained at 106 cfu/mL or more after 6 h exposure to 10 mg/mL trypsin solution (pH 8.0). 3.4 Detection of Resistance genes According to the above results, except strain L. pentosus cIcc 22161 and L. helveticus cIcc 22154, the tested strains may be able to sur- vive in the simulated gastrointestinal environ- ment. However, if the above strains contain an- tibiotics-resistant plasmids, there is the possi- bility of resistance transfer to other bacteria, es- pecially pathogenic bacteria. It will be a poten- tial hazard to human health and be a serious so- cial problem. so, the plasmid-determined resist- ant gene should be checked firstly before sub- sequent utilization. After 0.02% sds combined with heat treat- ment of the four strains (cIcc 22175, 22157, 23124, 23180), only the plasmids of L. plantar- um cIcc 23180 were removed and the resist- ance to cephalothin and chloromycetin disap- peared simultaneously (unpublished results). so, the primers of β-lactam resistance-relat- fig. 2 - the viable counts of strains ccIcc 22154, 22175, 22161, 22157, 23180 and 23124 in the gastrointestinal environ- ment after 6 hs at 37°c. A: pH 2.5; b: 3 mg/mL bile; c: 5mg/mL pepsin; d: 10 mg/mL trypsin. 288 Ital. J. Food Sci., vol. 27 - 2015 ed genes including blr, ECP-1569 and nps-1 as well as chloromycetin resistance-related genes including cmlA, cat and cmlA1 were designed. the plasmid-determined resistant genes of L. plantarum cIcc 23180 were detected by Pcr. As shown in fig. 3, the plasmid of L. plantar- um cIcc 23180 contained β - lactam resist- ance gene blr, excluding other resistant genes. blr gene encodes beta-lactamase, which can hydrolyze β-lactam ring and then make the β-lactam antibiotic inactivation. this is proba- bly the main reason of the bacteria resistant to β-lactam antibiotics. In the present study, the successful amplification of blr gene in L. plan- tarum cIcc 23180 indicated that its cefalotin fig. 3 - the Pcr result in the genome and plasmid of cIcc 23180. M. Marker; 1. blr; 2. EcP-1569; 3. nps; 4. cmlA; 5. cat; 6. cmlA1; 7.control. uct types. And studies have shown that more genes associated with antibiotic resistance are located in plasmids and transposons (doucEt et al., 1992; MAyyA et al., 2011). but unlike the chromosome dnA, both plasmids and transpo- sons can provide the possibility of transferabili- ty for resistance genes between bacteria. PIEr et al. (2003) proved the high transferability of plas- mid pcf10 that encodes tetracycline resistance from Enterococcus faecalis oG1rf to Enterococ- cus faecalis bf3098c during cheese and sausage fermentation. JoAnnA et al. (2008) reported the transferability of erythromycin resistant plasmid (pAMβ1) from Lactococcus lactis sH4174 to Lac- tococcus lactis bu2-60. A similar study also indi- cated that the transferability of tetracycline re- sistance in E. italicus LMG 22195 from ferment- ed milk (MIrIAM et al., 2010). so, the assessment of antibiotic resistant of potentially probiotic lactic acid bacteria used in food industry, especially the resistance-relat- ed genes and the transferability are very neces- sary. We can also say that,  exploring the pro- biotic property and safety of  lactic acid bacte- ria are equally important. 4. concLusIons the tested 29 strains of potential probiotic lac- tobacillus showed different resistance to antibi- otics.  those  resistant strains containing both plasmids and high tolerance to gastrointesti- nal condition may cause food safety problems. so these strains need to be re-assessed carefully. the study found that the plasmid of L. plantar- um cIcc 23180 exactly carried the cephaloth- in-related gene blr. However, the transferibility of the resistance-related gene remains to be fur- ther studied. this study provides a reference in investigating the relationships between antibi- otic resistance spectrum and the plasmids and evaluating the safety of probiotics. AcKnoWLEdGEMEnts this work was supported by the science and technolo- gy research youth fund Project (2010240) and the natu- ral science foundation of Hebei Province (c2013208161, c2010000863). 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Paper Received January 16, 2014 Accepted August 25, 2014 http://med.wanfangdata.com.cn/Paper/PeriodicalInfo.aspx?periodicalID=N2008EPST0010808 http://med.wanfangdata.com.cn/Paper/PeriodicalInfo.aspx?periodicalID=N2008EPST0010808 http://jac.oxfordjournals.org/search?author1=Ingo+Klare&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Carola+Konstabel&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Guido+Werner&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Geert+Huys&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Vanessa+Vankerckhoven&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Vanessa+Vankerckhoven&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Gunnar+Kahlmeter&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Bianca+Hildebrandt&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Sibylle+M%C3%BCller-Bertling&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Sibylle+M%C3%BCller-Bertling&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Wolfgang+Witte&sortspec=date&submit=Submit http://jac.oxfordjournals.org/search?author1=Herman+Goossens&sortspec=date&submit=Submit http://www.cabdirect.org:80/search.html?q=au%3A%22Lee+Yuan+Kun%22 http://www.cabdirect.org:80/search.html?q=au%3A%22Nomoto%2C+K.%22 http://www.cabdirect.org:80/search.html?q=au%3A%22Salminen%2C+S.%22 http://www.cabdirect.org:80/search.html?q=au%3A%22Gorbach%2C+S.+L.%22 http://www.ncbi.nlm.nih.gov/pubmed?term=Zhao H%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Zhou F%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Qi Y%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Dziugan P%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Bai F%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Walczak P%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Walczak P%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang B%5BAuthor%5D&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed/23747815/