Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw 02-096, Poland.
Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw 02-096, Poland.
J Dairy Sci. 2017 Sep;100(9):7435-7445. doi: 10.3168/jds.2017-12597. Epub 2017 Jul 12.
The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC and MIC, respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to both compounds. The MIC and MIC of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae, respectively. The MIC and MIC of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae, respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC, whereas an 8-fold increase of the MIC was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti-Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for protothecal mastitis in dairy cows.
本研究的目的是评估碘丙炔基丁基氨基甲酸酯（IPBC）和两性霉素 B（AMB）对从比利时、法国、意大利、德国和波兰的奶牛场回收的 2 型 Prototheca zopfii 基因型和 Prototheca blaschkeae 分离株的体外作用。还评估了 IPBC 与 AMB 对 Prototheca 分离株的组合作用以及 IPBC 对牛乳腺上皮细胞的毒性。对 96 株 2 型 P. zopfii 基因型和 42 株 P. blaschkeae 分离株进行了 IPBC 和 AMB 对 Prototheca 分离株的体外活性检测。测定了 IPBC 和 AMB 的最小抑菌浓度（MIC）和最小杀藻浓度（MAC）。为了确定 IPBC 和 AMB 联合使用是否具有协同、相加或拮抗作用，还进行了二维棋盘组合试验来计算部分抑制浓度。使用 3-(4,5-二甲基-2-噻唑-2-基)-2,5-二苯基四唑溴化物（MTT）测定法对 IPBC 对牛乳腺上皮细胞系的细胞毒性进行了分析。IPBC 的 50%和 90%分离株的 MIC（MIC 和 MIC，分别）分别为 4 和 8 mg/L，而 AMB 的 MIC 分别为 0.5 和 1 mg/L。P. zopfii 基因型 2 和 P. blaschkeae 的 MIC 谱不同，后者对这两种化合物更敏感。IPBC 的 MIC 和 MIC 分别为 4 和 8 mg/L 用于 P. zopfii 基因型 2 和 1 和 2 mg/L 用于 P. blaschkeae，分别。AMB 的 MIC 和 MIC 均为 1 mg/L 用于 P. zopfii 基因型 2 和 0.25 和 1 mg/L 用于 P. blaschkeae，分别。IPBC 和 AMB 均具有杀死 Prototheca spp.的能力。90%的 IPBC 分离株的 MAC 是 MIC 的两倍，而在 AMB 的情况下，MIC 增加 8 倍即可杀藻。总体而言，尽管部分抑制浓度指数仅对 3 株 P. zopfii 基因型 2 分离株显示出协同活性，但 IPBC 和 AMB 的联合使用表现出增强的杀藻作用。对于所有其余的分离株（87.5%），这种组合仅产生相加作用。MTT 测定结果表明，在所研究的浓度下，IPBC 和 AMB 对乳腺上皮细胞均无毒性（细胞活力>90%）。值得注意的是，只有最高浓度的 IPBC（即 8 mg/L）对测试的细胞系产生轻微的细胞毒性作用（分别在 2 和 4 小时的 MTT 处理后，细胞活力分别为 88.54 ± 3.88 和 90.66 ± 3.0）。这是首次证明 IPBC 对 Prototheca 的抗活性。此外，IPBC 与 AMB 的联合使用增强了彼此的作用，产生了相加而不是协同的相互作用。两种药物在与 Prototheca spp.的 MIC 值相对应的浓度下使用，对乳腺上皮细胞均无毒性作用。总之，IPBC 单独或与 AMB 联合使用均可被认为是奶牛 Prototheca 乳腺炎治疗方案中的一种有前途的选择。
