这项研究旨在调查从生母羊牛奶制成的八种葡萄牙奶酪中回收的肠球菌,关于抗生素耐药性,毒力基因,苯扎氯铵(BAC)的最小抑制浓度(MIC),生物膜形成能力,和BAC根除生物膜(MBEC)。采用纸片扩散法评价5组7种抗生素的耐药性。编码抗生素青霉素(blaZ)抗性的基因的存在,红霉素(ermA,ermB,和ermC),万古霉素(vanA和vanB),氨基糖苷(aac(6')-Ie-aph(2″)-Ia),和β-内酰胺(pbp5)和编码毒力因子的基因,frsB,Cyla,gelE,esp,和agg,通过多重PCR进行了研究。通过分离物的MIC和MBC值评估浮游细胞对BAC的敏感性,使用肉汤微量稀释法。为了评估生物膜的形成能力和对BAC的抗性,生物膜是在不锈钢试样上产生的,其次是暴露于BAC。结果表明,对万古霉素的耐药性较高(87.5%),红霉素(75%),四环素(50%),和青霉素(37.5%)。在68.8%的分离物中观察到多药耐药性。在所有分离物中检测到编码毒力因子FrsB(frsB)和明胶酶E(gelE)的基因。在56.3%和37.5%的分离株中发现了esp和cylA基因,分别。所有分离物都表现出生物膜形成能力,无论孵育时间和温度测试。然而,在37°C下72小时后,屎肠球菌和粪肠球菌生物膜表现出显著差异(p≤0.05)。尽管大多数分离株(62.5%)对BAC(MIC≤10mg/L)敏感,相同分离株的生物膜,一般来说,对所测试的较高浓度的BAC(80mg/mL)具有抗性。这项研究使用从即食食品中分离出的肠球菌,比如奶酪,揭示了万古霉素耐药性和多药耐药性的高百分比,与毒力基因的存在有关,在能够产生抗BAC生物膜的分离物中,许多消毒剂的重要活性成分。这些结果强调需要采取有效的控制措施来确保乳制品的安全和质量。
This study aimed to investigate enterococci recovered from eight Portuguese cheeses made with raw ewe\'s milk, regarding antibiotic resistance, virulence genes, minimum inhibitory concentration (MIC) of benzalkonium chloride (BAC), biofilm formation capacity, and biofilm eradication (MBEC) by BAC. Antimicrobial resistance against seven antibiotics of five groups was evaluated using the disk diffusion method. The presence of the genes that encode resistance to the antibiotics penicillin (blaZ), erythromycin (ermA, ermB, and ermC), vancomycin (vanA and vanB), aminoglycoside (aac(6\')-Ie-aph(2″)-Ia), and β-lactam (pbp5) and the genes that encode virulence factors, frsB, cylA, gelE, esp, and agg, were investigated via multiplex PCR. The susceptibility of planktonic cells to BAC was evaluated by the MIC and MBC values of the isolates, using the broth microdilution method. To assess the biofilm-forming ability and resistance of biofilms to BAC, biofilms were produced on stainless steel coupons, followed by exposure to BAC. The results showed a high resistance to the antibiotics vancomycin (87.5%), erythromycin (75%), tetracycline (50%), and penicillin (37.5%). Multidrug resistance was observed in 68.8% of the isolates. Genes encoding the virulence factors FrsB (frsB) and gelatinase E (gelE) were detected in all isolates. The esp and cylA genes were found in 56.3% and 37.5% of the isolates, respectively. All isolates exhibited a biofilm-forming ability, regardless of incubation time and temperature tested. However, after 72 h at 37 °C, E. faecium and E. faecalis biofilms showed significant differences (p ≤ 0.05). Although most isolates (62.5%) were susceptible to BAC (MIC ≤ 10 mg/L), biofilms of the same isolates were, generally, resistant to the higher concentration of BAC (80 mg/mL) tested. This study using Enterococcus isolates from a ready-to-eat food, such as cheese, reveals the high percentages of vancomycin resistance and multidrug resistance, associated with the presence of virulence genes, in isolates also capable of producing biofilms resistant to BAC, an important active ingredient of many disinfectants. These results emphasize the need for effective control measures to ensure the safety and quality of dairy products.