RU2306563C1 - Method for detecting the sensitivity of microorganisms to liquid combined bacteriophage - Google Patents

Abstract

FIELD: medicine, surgery.

SUBSTANCE: a whole wound detritus should be diluted with physiological solution to be homogenized and centrifuged for depositing large-scale particles of wound detritus. Then it is necessary to determine the sensitivity of the obtained supernatant to liquid combined bacteriophage for 18-24 h due to interacting with liquid combined bacteriophage upon nutritive medium. The innovation provides the chance for earlier onset of a purposeful bacteriophagotherapy and enables to shorten the terms of investigation.

EFFECT: higher accuracy and efficiency of detection.

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Description

The invention relates to medicine, namely to surgery, and can be used in the treatment of suppurative complications of the wound process.

There is a method of determining phagosensitivity by plating the selected cultures of pathogens on solid nutrient media and then applying the appropriate bacteriophage (Perepanova TS, Bacteriophage therapy of urological infections. Guidelines No. 96/53. // TS Perepanova, O.S. Darbeeva, G .A. Kotlyarova et al. - M., 1996, - 7 pp.).

This method involves the preliminary isolation and identification of pure cultures from the wound, which extends the study time to 3-5 days.

A significant drawback of the method is also the high consumption of nutrient media, sterile laboratory glassware and its complexity.

In addition, this method for determining the phage sensitivity of pure cultures does not take into account the interaction of bacteria of different species in the wound community.

The invention is aimed at solving the problem: reducing research time with high efficiency of the method.

Effect: the possibility of an early start of targeted bacteriophage therapy.

These problems are solved by interacting with a whole wound site taken from a pathological focus on a solid nutrient medium with a liquid combined bacteriophage, incubating the culture and taking into account the results of the absence of bacterial growth without isolation of pure cultures and separately determining their sensitivity to the specified bacteriophage.

The method is as follows.

After collecting the whole wound discharge, 3-5 ml of physiological saline is added to it, homogenized, centrifuged for 5-7 minutes at 1500 rpm on an OPN-3 centrifuge to precipitate large particles of wound detritus. Then the supernatant is inoculated with a spatula in an amount of 0.1-0.2 ml per solid nutrient medium, such as blood agar. After 10 minutes, 0.01-0.05 ml of a liquid combined bacteriophage is applied to the medium, for example, Sextafag, manufactured by the FSUE NPO Mikrogen of the Ministry of Health of the Russian Federation, a branch of the Perm NPO Biomed. Crops are incubated at a temperature of 37 ° C for 18-24 hours. The absence of bacterial growth in the area of application of the bacteriophage (the presence of a sterile spot) indicates the sensitivity of the present microflora to the liquid combined bacteriophage.

Examples of specific performance.

Example 1. Patient Bs, 25 years old, was admitted to the hospital with a diagnosis of acute pancreatitis, severe, infected pancreatic necrosis. Retroperitoneal phlegmon on the left. Prior to admission, he was operated on in a district hospital where a laparotomy, debridement and drainage of an omental bursa were performed, and a burso-omentostomy was formed. In the postoperative period, abundant purulent discharge from the bursomentostomy was noted. 3 ml of physiological saline was added to the taken whole wound discharge from the burso-omentostomy (0.2 ml), homogenization and centrifugation were performed at 1500 rpm for 5 minutes to precipitate large particles of detritus. Then the supernatant was seeded with a spatula in an amount of 0.1 ml per solid nutrient medium. After 10 minutes, 0.05 ml of liquid combined bacteriophage was applied to the medium. Inoculation was incubated at a temperature of 37 ° C for 18 hours. When evaluating the results, there was a lack of growth in the area of application of the bacteriophage, which indicated the sensitivity of microflora to the liquid combined bacteriophage. After that, the liquid combined bacteriophage was immediately included in the treatment complex in the form of daily washing of the burso-omentostomy and application of dressings with it. On the 5th day, normalization of body temperature and a decrease in leukocytosis were noted. During bacteriological examination of the wound discharge, cultures of Pseudomonas aeruginosa and Klebsiella spp., Sensitive to the corresponding species bacteriophages and to a liquid combined bacteriophage, were isolated. Microbiological monitoring (day 7) indicated a 10-fold decrease in the level of microbial contamination of the bursomentostomy. By the 15th day, signs of purification of the burso-omentostomy appeared and a further decrease in the concentration of microorganisms in the purulent discharge was noted.

Example 2. Patient S. on, 37 years old, diagnosis: infected wound of the left leg. To pus from the wound in an amount of 0.15 ml was added 5 ml of physiological saline. Homogenization and centrifugation (7 minutes, 1500 rpm) were carried out to precipitate large particles of detritus. Subsequently, the supernatant was inoculated with a spatula in an amount of 0.2 ml per solid nutrient medium. After 10 minutes, 0.01 ml of liquid combined bacteriophage was applied to the medium. Inoculation was incubated 24 hours at a temperature of 37 ° C. In the area of application of the liquid combined bacteriophage, the absence of bacterial growth (the presence of a sterile spot) was noted. Immediately in the treatment complex included a liquid combined bacteriophage in the form of a daily wound treatment and dressing with it. According to the results of a classical bacteriological study, a Staphylococcus epidermidis culture sensitive to staphylococcal bacteriophage and liquid combined bacteriophage was isolated from the wound. By 3 days, according to clinical and bacteriological criteria, the wound cleared. Granulation growth and marginal epithelization were noted.

Positive effect: the proposed method is economical, reduces the consumption of nutrient media by 2-6 times, does not require a difficult preparatory stage, less time-consuming. The method can be used as an express method in any medical institution. The application of the method gives a time gain for the start of targeted bacteriophage therapy, since the study takes no more than 18-24 hours compared to 72-96 hours in the prototype method.

Claims (1)

The method of determining the sensitivity of microorganisms to a liquid combined bacteriophage by interacting wound microflora in a nutrient medium with a liquid combined bacteriophage, incubating the mixture and taking into account the results of the absence of bacterial growth, characterized in that the whole wound is taken as the test material, diluted with saline, homogenized and centrifuged for the deposition of large particles of wound detritus, and then determine the sensitivity of the obtained supernatant to liquid combined bacteriophage for 18-24 hours