Chin Sieo Chin
(Department of Microbiology, Institute of Bioscience, University Putra Malaysia)
,
Abdullah Norhani
(Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Institute of Bioscience, University Putra Malaysia)
,
Siang Tan Wen
(Department of Microbiology, Institute of Bioscience, University Putra Malaysia)
,
Wan Ho Yin
(Institute of Bioscience, University Putra Malaysia)
In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bac...
In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, $17\%,\;58\%,\;and\;25\%$ were found to exhibit a high degree of resistance to $200\;{\mu}g/ml$ of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least $50\;{\mu}g/ml$ of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least $50\;{\mu}g/ml$ of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.
In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, $17\%,\;58\%,\;and\;25\%$ were found to exhibit a high degree of resistance to $200\;{\mu}g/ml$ of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least $50\;{\mu}g/ml$ of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least $50\;{\mu}g/ml$ of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.
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문제 정의
Hence, a preliminary study was conducted in order to determine the potential of these Lactobacillus strains as transformation hosts. The objectives of this study were to investigate the resistances of the 12 Lactobacillus probiotic strains to three antibiotics used commonly as selective markers in transformation studies, to eliminate any plasmid-mediated antibiotic resistance in the resistant strains, and, after the curing process, to compare the plasmid profiles of the cured derivatives with those of their respective wild-type strains.
We assayed 12 Lactobacillus strains with regard to their susceptibility to three antibiotics, namely chlorampheni col, erythromycin, and tetracycline. This study focused primarily on these three antibiotics, because they serve as selective markers in transformation studies of lactic acid bacteria (Kok et al.. 1984; Dao and Ferretti, 1985; Heng et al., 1997). These antibiotics have also been listed as antibiotics which are authorized for veterinary medicine in Europe for the treatment of food-producing animals, including avian, bovine, piscine, and porcine species (Schiwarz and Chaslus-Dancla, 2001).
제안 방법
In addition to visual observations, we also evaluated bacterial growth with a spectrophotometer (DU-65, Beckman, USA). Turbidity of less than 0.15 at an OD of 600 nm was considered "no growth", and turbidity of 0.15 or greater was considered "growth" All experiments were conducted three times, each with triplicate.
After 48 h of incubation under anaerobic conditions at 39℃, the emergent colonies were duplicated onto fresh MRS agar and MRS agar containing 10 ㎍/ml of either chloramphenicol, erythromycin, or tetracycline. Colonies that failed to grow on the MRS anti biotic plates after incubation were considered to have been cured, and their duplicates on the MRS agar were extracted and maintained in MRS broth for further analysis. The curing rate (%) was calculated according to the following formula:
이론/모형
The susceptibility of the wild-type Lactobacillus strains to antibiotics was evaluated using a slightly modified version of the macrodilution broth method developed by Jones et al. (1985). Chloramphenicol, erythromycin, and tetracy cline were the antibiotics 니sed in this procedure.
성능/효과
According to the results of the present study, we are able to concede that seven wild-type Lactobacillus strains (L. crispatus 112, L. brevis 123, 1211 and 1218, L. fermentum 125, 124 and C17) and five cured derivatives (L. acidophilus I16C and I26C, L. fermentum I24C and C17C, and L. brevis C10C), all of which were found to be susceptible to <50 ㎍/ml of tetracycline, erythromycin, and/or chloramphenicol (the normal concentration used for selection of Lactobacillus transformants) appear to harbor potential as transformation hosts.
, 1982). Among the four selected curing agents, novobiocin was the most effective, as it proved capable of curing erythromycin resistance in four Lactobacillus strains; evidencing the highest curing rate for L fermentum 124 (64%). It was also noted that the cured derivatives were susceptible to 10 ㎍/ml of erythromycin, whereas the wild-type strains exhibited resistance at > 200 ㎍/ml of erythromycin.
However, these chemicals proved to be fairly ineffective with regard to the curing of plasmids from the Lactobacillus strains. Of the seven Lactobacillus strains (L. acidophilus 116 and 126, L fermentum 124, C16 and C17, L. brevis Cl and CIO) subjected to plasmid curing, five strains (L. acidophilus 116 and 126, L fermentum 124 and C17, and L. brevis CIO) were successfully cured of their erythromycin resistance. The chemical agents used failed to eliminate chloramphenicol and tetracycline resistance in the tested strains.
참고문헌 (38)
Adwan, K., N. Abu-Hasan, and H. Al-Asmar. 1998. Analysis of neomycin, kanamycin, tobramycin and amikacin resistance mechanisms in gentamicin-resistant isolates of Enterobacteriaceae. J. Med. Microbiol. 47, 1019-1021
Bringel, F., L. Frey, and J. C. Hubert. 1989. Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction. Plasmid 22, 193-202
Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins. 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61, 1636-1643
Danielsen, M. 2002. Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure. Plasmid, 48, 98-103
Dao, M. L. and J. J. Ferretti. 1985. Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl.Environ. Microbiol, 49, 115-119
Endrz, H. P., N. van den Braak, H. A. Verburg, and A. van Bolkum. 1999. Vancomycin resistance: Status quo and quo vadis. Eur. J. Clin. Microbiol. Infect. Dis. 18, 683-690
Fons, M., T. Hege, M. Landire, P. Raibaud, R. Ducluzeau, and E. Maguin. 1997. Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance. Plasmid 37, 199-203
Gasson, M. J. and G. F. Fitzgerald. 1994. Gene transfer systems and transposition, p. 1-51. In M. J. Gasson and W. M. de Vos (eds.), Genetics and Biotechnology of Lactic Acid Bacteria, Chapman and Hall Press, London, U.K
Ghosh, S., N. R. Mahapatra, T. Ramamurthy, and P. C. Banerjee. 2000. Plasmid curing from an acidophilic bacterium of the genus Acidocella. FEMS Microbiol. Lett. 183, 271-274
Giles, J. S., H. Hariharan, and S. B. Heaney. 1995. The plasmid profiles of fish pathogenic isolates of Aeromonas salmonicida, Vibrio anguillarum, and Vibrio ordalii from the atlantic and pacific coasts of Canada. Can. J. Microbiol. 41, 209-216
Heng, N. C., H. F. Jenkinson, and G. W. Tannock. 1997. Cloning and expression of an endo-1,3-1,4- $\beta$ -glucanase gene from Bacillus macerans in Lactobacillus reuteri. Appl. Environ. Microbiol. 63, 3336-3340
Jin, L. Z., Y. W. Ho, M. A. Ali, N. Abdullah, K. B. Ong, and S. Jalaludin. 1996. Adhesion of Lactobacillus isolates to intestinal epithelial cells of chicken. Lett. Appl. Microbiol. 22, 229-232
Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin. 1998a. Effects of adherent Lactobacillus cultures on growth, weight of organs and intestinal microflora and volatile fatty acids in broilers. Anim. Feed Sci. Technol. 70, 197-209
Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin. 1998b. Growth performance, intestinal microbial populations, and serum cholesterol of broilers fed diets containing Lactobacillus cultures. Poult. Sci. 77, 1259-1265
Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin. 2000. Digestive and bacterial enzyme activities in broilers fed diets supplemented with Lactobacillus cultures. Poult. Sci. 79, 886-891
Jones, R. N., A. L. Barry, T. L. Gavan, and J. A. Washington. 1985. Susceptibility tests: Microdilution and macrodilution broth procedures, p. 972-977. In E. H. Lennette, A. Balows, W. J. Hausler and H. J. Shadomy (eds.), Manual of Clinical Microbiology, American Society for Microbiology, Washington D.C
Kalavathy, R., N. Abdullah, S. Jalaludin, and Y. W. Ho. 2003. Effects of Lactobacillus cultures on growth performance, abdominal fat deposition, serum lipids and weight of organs of broiler chickens. Bri. Poult. Sci. 44, 139-14
Kok, J., J. M. van der Vossen, and G. Venema. 1984. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl. Environ. Microbiol. 48, 726-731
Kullen, M. J. and T. R. Klaenhammer. 1999. Genetic modification of intestinal lactobacilli and bifidobacteria, p. 65-83. In G. Tannock (ed.), Probiotics: A Critical Review, Horizon Scientific Press, Wymondham, U.K
Leer, R. J., N. van Luijk, M. Posno, and P. H. Pouwels. 1992.. Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol. Gen. Genet. 234, 265-274
Lin, C. F., Z. F. Fung, C. L. Wu, and T. C. Chung. 1996. Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4. Plasmid 36, 116-124
O'Sullivan, D. J. and T. R. Klaenhammer. 1993. Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl. Environ. Microbiol. 59, 2730-2733
Posno, M., R.J. Leer, N. van Luijk, M.J.f. van Giezen, P.T.H.M. B. C. Lokman, and P.H. Pouwels. 1991. Incompatibility of Lactobacillus vectors with replicons derived from small cryptic Lactobacillus plasmids and segregational instability of the introduced vectors. Appl. Environ. Microbiol. 57, 1822-1828
Ruiz-Barba, J. L., J. C. Piard, and R. Jimenez-Diaz. (1991) Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations. J. Appl. Bacteriol. 71, 417-421
Son, R., G. Rusul, and M. I. A. Karim. 1996. Plasmid and antimicrobial resistance transfer from poultry strains to aquatic strains of Escherichia coli. J. Vet. Mal. 8, 7-13
Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie van Leeuwenhoek 76, 115-137
Vescovo, M., L. Morelli, and V. Bottazzi. 1982. Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri. Appl. Environ. Microbiol. 43, 50-56
Wegener, H. C. and S. Schwarz. 1993. Antibiotic-resistance and plasmids in Staphylococcus hyicus isolated from pigs with exudative epidermitis and from healthy pigs. Vet. Microbiol. 34, 363-372
Whitehead, T. R. and M. A. Cotta. 2001. Sequence analyses of a broad host-range plasmid containing ermT from a tylosin-resistant Lactobacillus sp. isolated from swine feces. Curr. Microbiol. 43, 17-20
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