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, *Corresponding authors: Yingmin Jia, Tel. +86 311 81668010, [email protected] Bolin Zhang, Tel. +86 10 62338221, 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.
- Keywords: antibiotic resistance; Lactobacillus; gastrointestinal tolerance; plasmid; probiotic - 282 Ital. J. Food Sci., vol. 27 - 2015
testinal conditions was investigated. by plas- mid elimination and Pcr, the relationship be- tween the plasmid profiles and resistance pat- due to the claimed benefits, Lactobacillus terns of the strains was explored. this will pro- bacteria are widely used in food, feed, medical vide a reference for the safety evaluation meth- and health related fields. Many lactic acid bac- od and also will be helpful to improve the eval- teria (LAb), such as Streptococcus thermophi- uation system of probiotics.
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 2. MAtErIALs And MEtHods the possibility of pathogenic. currently, new beneficial bacteria are being developed contin- 2.1 Bacterial strains and cultivation uously and will enter the market. However, the security of these new strains has caused great 29  Lactobacillus strains  used in the study concern. Evaluation of antibiotic sensitivity is were listed in table  1. Lactobacillus strains an important part of safety assessment.
were cultured in Mrs (de Man, rogosa, and now, overuse of antibiotics has become a sharpe) medium at 37°c for 18h under anaer- serious social problem. this led to the emer- obic condition.
gence of a large number of antibiotic-resistant Quality control strain recommended by clin- strains. once the resistance-related factors are ical and Laboratory standards Institute (cLsI) tranferred to other microorganisms, especial- in the antibiotic  sensitivity test was  E. coli ly pathogens via food carrier, it will cause tre- Atcc25922 purchased from the Institute of Mi- mendous problems. the evolution of antibiotic- crobiology, chinese Academy of sciences. the resistant foodborne pathogens has been widely E. coli Atcc25922 was activated and cultivat- reported (tHrELfALL et al., 2000; WALsH et al., ed in Lb medium (yeast extract 5 g/L, tryptone 2008; WHItE et al., 2002). Moreover, the resist- 10 g/L, nacl 10 g/L) at 37°c.
ance and resistance-related genes of Bifidobac- terium, Lactobacillus and Pediococcus strains to 2.2 Testing of antibiotic susceptibility different antibiotics were studied systematical- ly (HuMMEL et al., 2007; Huys et al., 2004; MA- 13 kinds of antibiotics paper discs were pur- rIA et al., 2007). the tetM gene transfer of tet- chased from the national Institute for the con- racycline resistance in Lactobacillus plantar- trol of Pharmaceutical and biological Products um among strains was reported by nIAMH et (table 2), each piece with a diameter of 6.5 mm. al. (2010).
the quality  was fully complied with  the  WHo In this study, 29 Lactobacillus strains isolat- ed from the food environment with potentially Antibiotic susceptibility was semi-quantita- probiotic effects (JIn et al., 2009; LI et al., 2009; tively determined with K-b method by antibiot- LIu et al., 2011; sun et al., 2009; ZHAo et al., ic paper disc diffusion referring to the cLsI as 2013) were used. these strains were assayed for described by cHArtErIs et al. (1998a).
susceptibility to 13 antibiotics by agar disc dif- briefly, 1.0 mL Lactobacillus suspension (ap- fusion method. furthermore, some strains with proximately 1.5×108 cfu/mL) was added to higher resistance were analysed for the presence sterile petri dish with diameter of 90 mm, and of plasmids. then, the tolerance of the plasmid- then mixed with a 15 mL MH (Muller Hinton, containing strains under simulated gastroin- MH) agar (beef extract powder 6g/L, casein ac- table 1 - source of the tested strains for antibiotic susceptibility test.
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 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.
Ital. J. Food Sci., vol. 27 - 2015 283
table 2 - the content of antibiotic paper discs and criteri- 2.4 Gastrointestinal tolerability test  on for judgement.
Content/disc inhibition zone diameter (mm)
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 ≤14 15-17 ≥18 were harvested by centrifugation at 6000 rpm μg ≤21 22-28 ≥29 for 15 min, washed twice with 0.01 mol/L Pbs, pH 7.2 after cultured for 18 h at 37°c in Mrs μg ≤14 15-17 ≥18 broth, and then suspended in 20 mL sterile sa- line (0.85%, w/v) adjusted to pH 2.5 with ster- μg ≤13 14-17 ≥18 ile hydrochloric acid. for bile tolerance test, the modified method chloramphenicol 30 μg ≤12 13-17 ≥18 of LEE et al. (1999) was referred to test bile tol- μg ≤12 13-14 ≥15 erance. the Lactobacillus cells were centrifuged (6000 rpm, 15 min) after cultivated for 18 h at multi-polymyxin B 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. cNote: R-Resistant; S-Susceptible; I-Intermediate.
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- ids hydrolysate  17.5 g/L,  soluble  starch,  1.5 tric and pancretic juices. fresh simulated gas- g/L, agar 17 g/L, pH 7.3±0.1) until the medi- tric and pancreatic juices were prepared daily um solidified. the antibiotic paper discs were according to charteris et al (1998b). Pepsin (sig- pasted closely onto the solidified medium with ma) was added into the simulated gastric juice sterile tweezers after 5min at room temper- with a final concentration of 5 mg/mL. then the ature. three discs were pasted in each dish. pH was adjusted to 2.5 with sterile hydrochloric the distance was more than 24 mm of each acid. trypsin (sigma) was added into the sim- disc center and more than 15 mm from disc ulated pancreatic juices with a final concentra- edge to the inner edge of dish. next, the dishes tion of 10 mg/mL. then pH was adjusted to 8.0 were placed at room temperature for 1.5 h and with 0.1 M naoH.
then incubated at 37°c. After 24 h, the inhibi- All of the tolerability detection, the initial bac- tion zone diameter was measured around the terial counts were adjusted to about 108 cfu/ antibiotic disc with vernier caliper and record- mL and were checked by viable count determi- ed. for one tested strain, each antibiotic disc nation on Mrs agar. for the tolerance assay, was done 3 times. the inhibition zone diame- the bacterial suspensions were incubated and counted at 37°c for 0,1,2,3,4,5,6 h, respectively.
standard sensitive strain of E. coli Atcc25922 All tests were repeated three times to estimate was used as the control. the operation was the the standard error.
same as the above.
the antibiotic susceptibility of the tested 2.5 Detection of antibiotic resistance genes strains was evaluated according to the cLsI cri- teria (table 2).
Part of the antibiotic-resistant genes of those lactic acid bacteria containing both plasmids and 2.3 Plasmid DNA extraction high tolerance were investigated. the β-lactam resistance-related gene sequence of blr, ECP- 10 mL of Lactobacillus suspension cultured 1569, nps-1 and the chloromycetin resistance- overnight was centrifugated at 10000 rpm for related gene sequence of cmlA, cat, cmlA1 in 5 min. then the precipitation was suspended plasmids were found in national center for bi- with 500 μL of lysozyme solution (10 mg/mL). otechnology Information (ncbI). the primers the mixture was placed in a water bath for 45 were designed and synthesized by beijing sun- min at 37°c. then plasmid dnA of Lactobacilli biotech co., Ltd (table 3). strains was extracted and purified with dnA ex- the Pcr programmes were performed with the traction and purification kit of tiangen biotech plasmid template of the tested strains accord- (beijing) co., Ltd. Plasmid dnA was observed ing to the following procedures: initial heating by agarose gel electrophoresis.
at 94°c for 4 min was followed by 34 cycles of Antibiotic susceptibility and plasmid stabil- the following sequence: 94°c for 30 s, 72°c for ity were tested after cultivated 30 generations 1 min, and 72°c for 1 min. final extension took at 37°c in Mrs medium according to the above place at 72°c for 7 min.
the amplification products were separated 284 Ital. J. Food Sci., vol. 27 - 2015
table 3 - the primers of the resistance genes in the experiment.
Sequence of the primer
Annealing temperature
5'-CGTCTTATTGAATTAACAGGTTGG -3' blr--down 5'-CACGAAGCCATGTTGTGTTC -3' ECP-1569--up nps-1--up nps-1--down 5'-GGCGATACCGCTCAGTTAC-3' cmlA--down 5'-CATGCCCAAACCTAGAAACGC-3' cat--down 5'-TGGAAGCCATCACAAACG-3' cmlA1--up cmlA1--down 5'-CTACGTTGTGGCGTCAATG-3' by conventional 1.0% (w/v) agarose gel electro- ance  to  13  kinds of antibiotics. to bacitra- phoresis  (100V, 4°c) in tAE (tris-acetate-Ed- cin, polymyxin b, kanamycin and nalidixic acid, tA) buffer and visualised by ethidium bromide the resistance rate of the 29 tested strains was staining. the target fragment was recovered 100%. to β-lactam and aminoglycosides, the and sequenced by taKara biotechnology (dali- resistance  percentage  was 20.7%-37.9% and an, china) co., Ltd. the resistance-related gene 86.2%-100%, respectively. All of the 29 strains of plasmid was determined by comparison with were mostly sensitive to  chloramphenicol and Among of the tested antibiotics, the nali- dixic acid and polymyxin b can inhibite dnA 3. rEsuLts And dIscussIon synthesis and interfer cell membrane forma- tion, respectively. the resistance of lactobacil- 3.1 Antibiotic susceptibility lus to these kinds of antibiotics may be due to the thicker cell wall of Gram-positive bacteria. Antibiotic susceptibility of the tested strains While the tested strains showed different sen- was evaluated according to the anti-microbi- sitivity to the antibiotics, such as streptomy- al  drug sensitivity standard of cLsI criteria. cin, kanamycin, tetracycline, chloramphenicol, the sensitivity of the tested Lactobacillus to 13 gentamicin with protein synthesis inhibitition kinds of antibiotics was shown in table 4. the effect. Most lactobacillus strains showed re- tested 29 strains were generally resisitant to sistance to those antibiotics against gram-neg- ative bacteria, for example, streptomycin, gen- lidixic acid, and were mostly sensitive to chlo- tamicin, kanamycin. this was consistent with ramphenicol and tetracycline. the same species report of Zhang et al (2007).
of Lactobacillus generally had similar resistance patterns. but there was species specificity such 3.2 Plasmid DNA extraction as the different antibiotic sensitivity in L. plan- of antibiotics-resistant lactobacillus strains tarum, L. rhamnosus, and L. pentosus. Moreover, the antibiotic-resistant level of different strains 16 cIcc strains with relatively strong antibi- is also different.
otic resistance were screened for plasmid extrac- Antibiotic resistance of the foodborne lactic tion. As can be seen from fig. 1, among these acid bacteria had heen reported in the 1980s. strains, only cIcc 23180, 22161, 22175, 22157, the researchers generally believed that the re- 23124, and 22154 contained plasmids.
sistance was a result of the long evolution and L. plantarum cIcc 23180 showed 6 plasmid it was generally endogenous resistance and ob- dnA bands and there is one band greater than tained resistance (Zeng et al., 2004). so, the re- 23 kb. L. pentosus cIcc 22157 showed two plas- sistant lactic acid bacteria of natural or isolat- mid dnA bands of 10 kb and 5 kb, respective- ed from human intestinal can indirectly reflect ly. L. rhamnosus cIcc 22175 and L. plantarum the habitat of used antibiotic.
cIcc 23124 contained respectively 2 and 4 of It can be seen  from table 5, the  29  strains plasmid dnA bands and both of the two strains contained a 10 kb plasmid. L. helveticus cIcc Ital. J. Food Sci., vol. 27 - 2015 285

table 5 - the percentage of the antibiotic resistance of 29 Antibiotics
of resistant strains
of resistance (%)
penicillin G 11 multi-polymyxin B 29 chloramphenicol 3 L. casei
22154 showed only one plasmid dnA band of about 10 kb.
L. acidophilus
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- the tolerance of the selected six strains to low strains to 13 antibiotics.
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).
286 Ital. J. Food Sci., vol. 27 - 2015

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.
pH, bile salt, pepsin and trypsin is presented in the viable counts of the tested six strains still re- fig. 2. As shown in fig. 2A, the viable counts of mained at 106 cfu/mL or more after 6 h exposure L. pentosus cIcc 22161 strain reduced to be- to 10 mg/mL trypsin solution (pH 8.0).
low 106 cfu/mL after 4 h and 1.32 × 104 cfu/ mL  after  6 h. However, the viable numbers of 3.4 Detection of Resistance genes L. helveticus cIcc 22154,  L. pentosus cIcc 22157,  L. plantarum cIcc 23124,  23180 and According to the above results, except strain L. rhamnosus cIcc 22175 were still more than L. pentosus cIcc 22161 and L. helveticus cIcc 106 cfu/mL after 6 h in the gastric acid of pH 22154, the tested strains may be able to sur- 2.5. thus, these five strains showed higher tol- vive in the simulated gastrointestinal environ- ment. However, if the above strains contain an- for bile tolerance, except the L. pentosus cIcc tibiotics-resistant plasmids, there is the possi- 22161, the viable counts of the other five strains bility of resistance transfer to other bacteria, es- were still more than 106 cfu/mL after 6 h in the pecially pathogenic bacteria. It will be a poten- medium containing bile salt (fig. 2b). However, tial hazard to human health and be a serious so- the viable cells of L. pentosus cIcc 22161 had cial problem. so, the plasmid-determined resist- decreased to 2.0×106 cfu/mL within 3 h. And ant gene should be checked firstly before sub- it declined to only 1.8 × 104 after 6 h.
for pepsin tolerance, among of six strains, After 0.02% sds combined with heat treat- the viable cells of L. pentosus cIcc 22161 and ment of the four strains (cIcc 22175, 22157, L. helveticus cIcc 22154 decreased significant- 23124, 23180), only the plasmids of L. plantar- ly in 6 h and it is less than 104 cfu/mL and 106 um cIcc 23180 were removed and the resist- cfu/mL after 6 h exposure to 5 mg/mL pepsin ance to cephalothin and chloromycetin disap- solution (pH 2.5), respectively (fig. 2c). peared simultaneously (unpublished results). for trypsin tolerance, as can be seen in fig. 2d, so, the primers of β-lactam resistance-relat- Ital. J. Food Sci., vol. 27 - 2015 287

ed genes including blr, ECP-1569 and nps-1 as uct types. And studies have shown that more well as chloromycetin resistance-related genes genes associated with antibiotic resistance are including cmlA, cat and cmlA1 were designed. located in plasmids and transposons (doucEt the plasmid-determined resistant genes of L. et al., 1992; MAyyA et al., 2011). but unlike the plantarum cIcc 23180 were detected by Pcr. chromosome dnA, both plasmids and transpo- As shown in fig. 3, the plasmid of L. plantar- sons can provide the possibility of transferabili- um cIcc 23180 contained β - lactam resist- ty for resistance genes between bacteria. PIEr et ance gene blr, excluding other resistant genes. al. (2003) proved the high transferability of plas- blr gene encodes beta-lactamase, which can mid pcf10 that encodes tetracycline resistance hydrolyze β-lactam ring and then make the from Enterococcus faecalis oG1rf to Enterococ- β-lactam antibiotic inactivation. this is proba- cus faecalis bf3098c during cheese and sausage bly the main reason of the bacteria resistant to fermentation. JoAnnA et al. (2008) reported the β-lactam antibiotics. In the present study, the transferability of erythromycin resistant plasmid successful amplification of blr gene in L. plan- (pAMβ1) from Lactococcus lactis sH4174 to Lac- tarum cIcc 23180 indicated that its cefalotin 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.
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- fig. 3 - the Pcr result in the genome and plasmid of cIcc ther studied. this study provides a reference in investigating the relationships between antibi- M. Marker; 1. blr; 2. EcP-1569; 3. nps; 4. cmlA; 5. cat; 6. cmlA1; 7.control.
otic resistance spectrum and the plasmids and evaluating the safety of probiotics.
resistance may be due to the effect of the beta- lactamase to β-lactam antibiotics. While in the study, the genes of cat, cmlA and this work was supported by the science and technolo- gy research youth fund Project (2010240) and the natu- cmlA1 were not detected in the plasmids of L. ral science foundation of Hebei Province (c2013208161, plantarum cIcc 23180. However, L. plantarum c2010000863). Authors also thank national High-tech Pro- cIcc 23180 strain was resistant to chloram- ject ("863 Plan", no. 2011AA100902) from chinese Ministry of science and technology for part fund. phenicol. At the same time, plasmid elimination and Escherichia coli transformant test showed that chloramphenicol resistance-related genes should be present in plasmid dnA of L. plantar- um cIcc 23180 (unpublished results). there- charteris W.P., Kelly P.M., Morelli L., collins J.K. 1998a. An- fore, the plasmid of the cIcc 23180 strain may tibiotic susceptibility of potentially probiotic Lactobacillus contain other genes encoding chloramphenicol species. Journal of food Protection. 61: 1636. charteris W.P., nelly P.M., Morelli L., collins J.K. 1998b. development of an in vitro methodology to determine the In recent years, more studies have been done transit tolerance of potentially probiotic Lactobacillus and on antibiotic resistance of probiotics. It was bifidobacterium species in the upper human gastroin- shown that the antibiotic resistance was varia- testinal tract. Journal of Applied bacteriology. 84: 759. ble, species-dependent and related to the prod- doucet P.f., trieu c.P., Andremont A., courvalin P. 1992. 288 Ital. J. Food Sci., vol. 27 - 2015
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Paper Received January 16, 2014 Accepted August 25, 2014 Ital. J. Food Sci., vol. 27 - 2015 289


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