Synergistic Effect of Enzyme Preparations and Gentamycin on Biofilms of Bordetella pertussis

E. M. Zaitsev, M. V. Britsina, M. N. Ozeretskovskaya, I. G. Bazhanova
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Abstract

Relevance. An increase in the incidence of whooping cough, a high proportion of severe forms of the disease, and a decrease in the sensitivity of circulating strains of B. pertussis to antibiotics require the development of more effective etiotropic therapies, including those capable of influencing biofilm forms of the whooping cough pathogen, which differ from planktonic cells by increased resistance to the host immune system and antibacterial drugs.Аim of the work is to study the effect of trypsin and lidase in combination with gentamycin on the growth of biofilms of Bordetella pertussis strains on an abiotic substrate.Materials and methods. In the experiments B. pertussis strains isolated in the Russian Federation from whooping cough patients in 2001‒2010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin (10 mcg/ml), lidase (20 IU/ml), gentamycin (2.0 mg/ml, 0.4 mg/ml and 0.08 mg/ml) and their combinations in roundbottomed polystyrene 96­well plates was evaluated by staining with 0.1% gentian­violet solution.Results. Gentamycin partially suppressed the formation of biofilms and caused partial destruction of the formed biofilms in the absence of growth of microbial colonies when sowing supernatants from biofilm cultures on a dense nutrient medium. The minimum suppressive concentration of gentamycin (MSC) was 2 mg/ml. Trypsin completely suppressed the growth of biofilms and caused the complete destruction of the formed biofilms. Lidase also suppressed the growth of biofilms, but less effectively affected the formed biofilms. The growth of colonies typical of B. pertussis was noted when sowing supernatants from biofilm cultures in the presence of trypsin and lidasе on a dense nutrient medium. Trypsin in combination with all the studied concentrations of gentamycin completely suppressed the growth of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused complete destruction of biofilms in the absence of microbial growth on a dense nutrient medium. Lidase in combination with all the studied concentrations of gentamycin also suppressed the formation of biofilms (MSC 0.08 mg/ml), and in combination with gentamycin at a concentration of 2.0 mg/ml caused partial destruction of the formed biofilms in the absence of microbial growth on a dense nutrient medium.Conclusion. The synergistic effect of the combination of trypsin and lidase with gentamycin on growing and formed biofilms of B. pertussis strains was revealed. The combined use of trypsin or lidase with gentamicin reduced its MSC for growing biofilms by 25 times. The most pronounced effect on the formed biofilms was the combination of trypsin with gentamycin at a concentration of 2 mg/ml, which caused their complete destruction and death of planktonic cells. The effect of the combination of lidase with gentamycin on the formed biofilms was less pronounced.
酶制剂与庆大霉素对百日咳杆菌生物膜的协同作用
的相关性。百日咳发病率的增加、严重形式百日咳的比例很高以及循环百日咳菌株对抗生素的敏感性下降,都需要开发更有效的致病因疗法,包括能够影响百日咳病原体生物膜形式的疗法,百日咳病原体生物膜形式与浮游细胞不同,对宿主免疫系统和抗菌药物的抵抗力增强。Аim的工作是研究胰蛋白酶和利酶联合庆大霉素对百日咳杆菌菌株在非生物底物上生长的影响。材料和方法。实验采用2001-2010年在俄罗斯联邦从百日咳患者身上分离的百日咳b型菌株:178号(血清型1.2.0)、287号(血清型1.0.3)和317号(血清型1.2.3),在密集的营养培养基上生长。采用0.1%龙胆紫染色法,考察了胰酶(10 mcg/ml)、利酶(20 IU/ml)、庆大霉素(2.0 mg/ml、0.4 mg/ml和0.08 mg/ml)及其组合在圆底聚苯乙烯96孔板中生物膜形成的强度。庆大霉素部分抑制了生物膜的形成,并在微生物菌落没有生长的情况下造成形成的生物膜的部分破坏,当从生物膜培养的上清液在密集的营养培养基上播种时。庆大霉素(MSC)最小抑制浓度为2 mg/ml。胰蛋白酶完全抑制生物膜的生长,使形成的生物膜完全破坏。Lidase对生物膜的生长也有抑制作用,但对生物膜的形成影响较小。当在密集的营养培养基上播种胰蛋白酶和利达西存在的生物膜培养的上清时,注意到百日咳菌落的典型生长。胰蛋白酶与所有研究浓度的庆大霉素联合使用(MSC 0.08 mg/ml)可完全抑制生物膜的生长,而与2.0 mg/ml浓度的庆大霉素联合使用在密集的营养培养基上,在没有微生物生长的情况下,可完全破坏生物膜。Lidase与所有研究浓度的庆大霉素联合使用(MSC 0.08 mg/ml)也能抑制生物膜的形成,而与浓度为2.0 mg/ml的庆大霉素联合使用在密集的营养培养基上,在没有微生物生长的情况下,形成的生物膜被部分破坏。揭示了胰酶和利酶与庆大霉素联合对百日咳菌株生长和形成生物膜的协同作用。胰蛋白酶或利德酶与庆大霉素联合使用使其生长生物膜的间充质干细胞减少了25倍。对形成的生物膜最显著的影响是胰蛋白酶与庆大霉素以2 mg/ml的浓度联合使用,导致生物膜完全破坏和浮游细胞死亡。利德酶与庆大霉素联用对生物膜形成的影响不明显。
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