了解环境变量如何影响生物膜的形成对于管理虾生产中与弧菌生物膜相关的感染具有重要意义。因此,我们评估了温度的影响,时间,以及使用多因素实验设计在这两种弧菌的生物膜发育中的初始接种物。弧菌生物膜的浮游生长抑制和抑制/根除,更准确的副溶血弧菌(VP87和VP275)和霍乱弧菌(VC112)从虾养殖场分离通过桉树和番石榴水叶提取物进行评估,并与四环素和头孢曲松进行比较。初步结果表明,副溶血性弧菌生物膜发育的最佳生长条件为24h和24°C(p<0.001),而霍乱弧菌生物膜为72小时和30°C(p<0.001)。使用集落形成单位(CFU)计数测定作为参考,应用多元线性回归ANOVA。和R平方值用作生物膜分析的拟合优度测量。然后,两种植物提取物均采用高效液相色谱法分析,使用二极管阵列检测器(DAD)和质谱(MS)进行双重在线检测,以评估其化学成分,其中桉树提取物的主要鉴定化合物是cypelloginA,cypelloginB,还有cypellocarpinC,而GuavinosideA,B,和C化合物是番石榴提取物的主要化合物。对于浮游生物生长抑制,桉树提取物在200μg/mL时显示出对所有弧菌菌株的最大抑制作用为75%(p<0.0001),而番石榴提取物在1600μg/mL时表现出最大抑制,抑制为70%(p<0.0001)。根据合意性分析,通过两种条件(24℃24小时和30℃72小时)对弧菌菌株进行生物膜抑制和根除测定。关于在24°C下24小时,在抗生素和植物提取物之间的CFU计数中观察到差异,与两种抗生素相比,两种植物提取物在8x时显示出更高的活细胞减少率,16x,和32xMIC值(桉树提取物:1600、3200和6400μg/mL;而番石榴提取物:12800、25600和52000μg/mL)。关于在30°C下72小时,结果表明,番石榴叶提取物和四环素对生物量的抑制作用较差。然而,与相同的头孢曲松MIC值(5-80μg/mL)相比,桉树提取物将弧菌生物膜内的活细胞总数从2x降低到32xMIC值(400-6400μg/mL),不能减少活细胞。桉树提取物在两种生长条件下表现出相似的结果,显示所有弧菌分离物在400μg/mL浓度下的平均抑制约80%(p<0.0001)。此外,根除生物膜试验表明,在两种生长条件下,对所有弧菌菌株均有明显的根除作用。但生物膜根除值明显较低。与两种抗生素相比,两种提取物植物在8x时都显示出更高的活细胞减少率,16x,和32xMIC值在两个增长集,其中800μg/mL的桉树提取物减少了所有弧菌菌株的70%的生物量和90%的活细胞(p<0.0001)。总体结果表明,在厄瓜多尔的虾业中,一种针对弧菌病的可行替代方法。
Understanding how environmental variables influence biofilm formation becomes relevant for managing Vibrio biofilm-related infections in shrimp production. Therefore, we evaluated the impact of temperature, time, and initial inoculum in the biofilm development of these two Vibrio species using a multifactorial experimental design. Planktonic growth inhibition and inhibition/eradication of Vibrio biofilms, more exactly V. parahaemolyticus (VP87 and VP275) and V. cholerae (VC112) isolated from shrimp farms were evaluated by
Eucalyptus and Guava aqueous leaf extracts and compared to tetracycline and ceftriaxone. Preliminary results showed that the best growth conditions of biofilm development for V. parahaemolyticus were 24 h and 24°C (p <0.001), while V. cholerae biofilms were 72 h and 30°C (p <0.001). Multivariate linear regression ANOVA was applied using colony-forming unit (CFU) counting assays as a reference, and R-squared values were applied as goodness-of-fit measurements for biofilm analysis. Then, both plant extracts were analyzed with HPLC using double online detection by diode array detector (DAD) and mass spectrometry (MS) for the evaluation of their chemical composition, where the main identified compounds for Eucalyptus extract were cypellogin A, cypellogin B, and cypellocarpin C, while guavinoside A, B, and C compounds were the main compounds for Guava extract. For planktonic growth inhibition,
Eucalyptus extract showed its maximum effect at 200 μg/mL with an inhibition of 75% (p < 0.0001) against all Vibrio strains, while Guava extract exhibited its maximum inhibition at 1600 μg/mL with an inhibition of 70% (p < 0.0001). Both biofilm inhibition and eradication assays were performed by the two conditions (24 h at 24°C and 72 h at 30°C) on Vibrio strains according to desirability analysis. Regarding 24 h at 24°C, differences were observed in the CFU counting between antibiotics and plant extracts, where both plant extracts demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values (
Eucalyptus extract: 1600, 3200, and 6400 μg/mL; while Guava extract: 12800, 25600, and 52000 μg/mL). Concerning 72 h at 30°C, results showed a less notorious biomass inhibition by Guava leaf extract and tetracycline. However,
Eucalyptus extract significantly reduced the total number of viable cells within Vibrio biofilms from 2x to 32x MIC values (400-6400 μg/mL) when compared to the same MIC values of ceftriaxone (5-80 μg/mL), which was not able to reduce viable cells.
Eucalyptus extract demonstrated similar results at both growth conditions, showing an average inhibition of approximately 80% at 400 μg/mL concentration for all Vibrio isolates (p < 0.0001). Moreover, eradication biofilm assays demonstrated significant eradication against all Vibrio strains at both growth conditions, but biofilm eradication values were substantially lower. Both extract plants demonstrated a higher reduction of viable cells when compared with both antibiotics at 8x, 16x, and 32x MIC values at both growth sets, where Eucalyptus extract at 800 μg/mL reduced 70% of biomass and 90% of viable cells for all Vibrio strains (p < 0.0001). Overall results suggested a viable alternative against vibriosis in the shrimp industry in Ecuador.