Abriouel Hikmate, Lucas Rosario, Ben Omar Nabil, Valdivia Eva, Maqueda Mercedes, Martínez-Cañamero Magdalena, Gálvez Antonio
Area de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaén, 23071-Jaén, Spain.
Syst Appl Microbiol. 2005 Jul;28(5):383-97. doi: 10.1016/j.syapm.2005.01.007.
The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcusfaecium by species-specific PCR, DNA-rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48. gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin (MIC > 2 mgl(-1)) and levofloxacin (MIC > 4 mgl(-1)) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.
从山羊奶酪中分离出的产细菌素菌株RJ16，经种特异性PCR、DNA-rRNA杂交和rDNA测序鉴定为粪肠球菌。从菌株RJ16纯化得到的细菌素是一种耐羧肽酶A的肽，分子量（7125 Da）与环肽肠球菌素AS-48非常接近。来自菌株RJ16的细菌素和AS-48具有相同的抗菌谱，尽管前者对单核细胞增生李斯特菌和蜡样芽孢杆菌菌株的活性略低。产菌菌株表现出交叉免疫性。用AS-48结构基因的引物对菌株RJ16的总DNA进行PCR扩增，并对扩增片段进行测序，结果显示除了一个预测AS-48第20位Glu残基变为Val的单突变外，序列几乎相同（99.5%）。因此，粪肠球菌RJ16产生的细菌素应被视为AS-48的变体，我们将其称为AS-48RJ。PCR扩增表明菌株RJ16含有完整的as-48基因簇。用as-48基因簇的探针进行杂交，揭示了菌株RJ16中as-48基因的染色体定位，这是该细菌素性状染色体定位的首个实例。菌株RJ16产生了食品加工中感兴趣的酶（酯酶、酯酶脂肪酶和植酸酶活性），并且不会使生物胺的氨基酸前体脱羧。菌株RJ16不表现出血清溶血或明胶酶活性，PCR扩增显示缺乏编码已知毒力决定因素（聚集物质、胶原黏附素、肠球菌表面蛋白、心内膜炎抗原以及溶血素和明胶酶产生）的基因。菌株RJ16对环丙沙星（MIC>2 mg l-1）和左氧氟沙星（MIC>4 mg l-1）耐药，对呋喃妥因和红霉素表现出中度耐药，但对氨苄西林、青霉素、链霉素、庆大霉素、利福平、氯霉素、四环素、奎奴普丁/达福普汀、万古霉素和替考拉宁敏感。总之，本研究结果表明，这种广谱产细菌素菌株可能在食品保鲜中有潜在用途。
