Postgraduate Program in Health Sciences, Medicine, Federal University of Uberlândia (UFU), Brazil.
Institute of Biology, Federal University of Uberlândia (UFU), Brazil.
J Microbiol Methods. 2021 Jun;185:106231. doi: 10.1016/j.mimet.2021.106231. Epub 2021 Apr 28.
Some species of Klebsiella, such as Klebsiella pneumoniae and Klebsiella oxytoca, are important nosocomial pathogens frequently involved in outbreaks in Neonatal Intensive Care Units (NICU) and have the ability to form a biofilm. This study aims to evaluate the biofilm production of K. pneumoniae and K. oxytoca isolates collected from the hands of health professionals, neonates' blood and the environment of a Brazilian NICU, using three colorimetric methods and a classical method of counting the colony-forming units and compare the analysis among these techniques. The biofilm formation was carried out by the microplate technique, using three colorimetric assays: crystal violet, safranin and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5 [(phenylamino) arbonyl] - 2H-tetrazolium hydroxide (XTT). Also, colony-forming units were determined. Twenty-eight isolates of K. pneumoniae were collected from the blood, hands and environment and five of K. oxytoca from the hands and environment. All of them were strong biofilm producers, but K. pneumoniae isolates produced more biofilm than K. oxytoca when compared to the American Type Culture Collection (ATCC) strains used as positive controls. The number of viable cells in the biofilm produced by K. pneumoniae isolated from blood was significantly higher than in the control sample. Regarding the three colorimetric tests used in the study, the violet crystal obtained a higher absorbance average. The use of crystal-violet and XTT in the evaluation of biofilm in vitro make possible a complete analysis, since that it can quantify the total biomass (including the extracellular matrix) and evaluate the metabolic activity. In conclusion, this study identified isolates of K. pneumoniae and K. oxytoca that produce biofilms in the NICU and the bloodstream of neonates. This fact deserves attention since these patients are immunocompromised. The best methods will be chosen to answer research questions by always adopting more than one method so that more than one parameter or component of the biofilm is analyzed.
一些克雷伯氏菌属的物种，如肺炎克雷伯氏菌和产酸克雷伯氏菌，是重要的医院获得性病原体，常与新生儿重症监护病房（NICU）的爆发有关，并有形成生物膜的能力。本研究旨在使用三种比色法和一种经典的菌落形成单位计数法评估从巴西 NICU 的医护人员手部、新生儿血液和环境中收集的肺炎克雷伯氏菌和产酸克雷伯氏菌分离株的生物膜产生情况，并比较这些技术之间的分析。生物膜形成是通过微孔板技术进行的，使用三种比色测定法：结晶紫、沙黄和 2,3-双（2-甲氧基-4-硝基-5-磺苯基）-5-[(苯氨基)羰基]-2H-四唑氢氧化铵（XTT）。同时，还测定了菌落形成单位。从血液、手部和环境中采集了 28 株肺炎克雷伯氏菌和 5 株产酸克雷伯氏菌，均为强生物膜生产者，但与用作阳性对照的美国典型培养物保藏中心（ATCC）菌株相比，肺炎克雷伯氏菌分离株产生的生物膜更多。从血液中分离的肺炎克雷伯氏菌产生的生物膜中的活细胞数量明显高于对照样品。关于研究中使用的三种比色试验，结晶紫获得了更高的平均吸光度。结晶紫和 XTT 在体外生物膜评价中的使用可以进行完整的分析，因为它可以定量总生物量（包括细胞外基质）并评估代谢活性。总之，本研究鉴定了在 NICU 和新生儿血液中产生生物膜的肺炎克雷伯氏菌和产酸克雷伯氏菌分离株。由于这些患者免疫力低下，这一事实值得关注。将选择最佳方法来回答研究问题，始终采用多种方法，以便分析生物膜的多个参数或成分。
