vedomostiregmedРегуляторные исследования и экспертиза лекарственных средствRegulatory Research and Medicine Evaluation3034-30623034-3453Federal State Budgetary Institution ‘Scientific Centre for Expert Evaluation of Medicinal Products’ of the Ministry of Health of the Russian Federation (FSBI ‘SCEEMP’)10.30895/1991-2919-2024-14-4-483-492vedomostiregmed-669Research ArticleМИКРОБИОЛОГИЧЕСКИЙ АНАЛИЗMICROBIOLOGICAL TESTINGСравнение условий выделения микроорганизмов-контаминантов при микробиологическом мониторингеComparison of Incubation Conditions for Microbial Contaminant Isolation in Microbiological Environmental Monitoringhttps://orcid.org/0000-0002-4825-8356ГунарО. В.GunarO. V.

Гунар Ольга Викторовна, д-р фарм. наук

Петровский б-р, д. 8, стр. 2, Москва, 127051

Olga V. Gunar, Dr. Sci. (Pharm.)

8/2 Petrovsky Blvd, Moscow 127051, Russian Federation

gunar@expmed.ruhttps://orcid.org/0000-0003-1773-7423СахноН. Г.SakhnoN. G.

Сахно Надежда Геннадьевна, канд. фарм. наук

Петровский б-р, д. 8, стр. 2, Москва, 127051

Nadezhda G. Sakhno, Cand. Sci. (Pharm.)

8/2 Petrovsky Blvd, Moscow 127051, Russian Federation

sakhno@expmed.ruhttps://orcid.org/0009-0008-7494-3403ТынчероваО. С.TyncherovaO. S.

Тынчерова Ольга Сергеевна

Петровский б-р, д. 8, стр. 2, Москва, 127051

Olga S. Tyncherova

8/2 Petrovsky Blvd, Moscow 127051, Russian Federation

tyncherovaos@expmed.ruФедеральное государственное бюджетное учреждение «Научный центр экспертизы средств медицинского применения» Министерства здравоохранения Российской ФедерацииРоссияScientific Centre for Expert Evaluation of Medicinal ProductsRussian Federation202428082024144483492Copyright © Гунар О.В., Сахно Н.Г., Тынчерова О.С., 20242024Гунар О.В., Сахно Н.Г., Тынчерова О.С.Gunar O.V., Sakhno N.G., Tyncherova O.S.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.vedomostincesmp.ru/jour/article/view/669ВВЕДЕНИЕ

ВВЕДЕНИЕ. Программы микробиологического мониторинга состояния помещений для фармацевтического производства, включенные в нормативные документы разного уровня, различаются. В частности, это касается условий проведения эксперимента: используемых питательных сред, температуры и времени инкубации. Для унификации процедур контроля качества необходимо разработать единую стратегию обработки проб микробиологического анализа.

ЦЕЛЬ

ЦЕЛЬ. Определение оптимальных условий инкубации посевов при микробиологическом мониторинге «чистых» помещений.

МАТЕРИАЛЫ И МЕТОДЫ

МАТЕРИАЛЫ И МЕТОДЫ. Проведено сравнение условий культивирования тест-штаммов Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Aspergillus fumigatus ВКПМ F-62, Aspergillus terreus ВКПМ F-1269, Penicillium chrysogenum ВКПМ F-3, а также изолятов из окружающей среды Staphylococcus epidermidis, Kocuria rosea, Micrococcus luteus, Bacillus spp., Sphingomonas paucimobilis. Питательные среды: триптиказо-соевый агар (TSA), агар Сабуро с декстрозой и хлорамфениколом (SDCA), агар Ризонера (R2A). Режимы инкубации: 2 сут при 30–35 ºС, затем 3 сут при 20–25 ºС; 3 сут при 20–25 ºС, затем 2 сут при 30–35 ºС; 48–72 ч при 30–35 ºС (культивирование аэробных бактерий); 5–7 сут при 20–25 ºС (культивирование дрожжевых и плесневых грибов).

РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ

РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ. При культивации бактерий в среде TSA и R2A статистически значимых различий результатов, полученных при разных температурных условиях, выявлено не было. Коэффициент прорастания изолятов из окружающей среды был существенно ниже (на 19–37%) в случае их выращивания на TSA при двухтемпературной схеме инкубации (первоначальное выдерживание посевов при более низкой температуре). Выявлены группы микроорганизмов (бактерии с угнетенными физиологическими функциями и замедленным ростом, а также плесневые грибы), требующие при микробиологическом мониторинге подбора условий культивирования.

ВЫВОДЫ

ВЫВОДЫ. Установлена необходимость стандартизации выполнения микробиологического анализа при мониторинге и его регламентация в установленном порядке в виде общей фармакопейной статьи. Показана возможность использования при микробиологическом мониторинге в качестве питательной среды как триптиказо-соевого агара, так и агара Ризонера. Применение двухтемпературной схемы инкубации с использованием одной неселективной питательной среды требует валидации в каждом конкретном случае. При культивировании тест-штаммов последовательность изменения температуры не оказывает влияния на коэффициент их прорастания, однако в случае бактерий, выделенных из окружающей среды, предпочтительным следует считать режим инкубации с более высокой температурой (30–35 ºС) на начальном этапе инкубации.

 

 

INTRODUCTION. Microbiological environmental monitoring programmes for clean rooms for pharmaceutical production vary depending on the regulatory document. This is particularly evident in the experimental conditions, including the culture media used for sampling, as well as the temperature and time of incubation. To harmonise quality control procedures, it is necessary to develop a unified strategy for processing microbiological samples.AIM. This study aimed to investigate the optimal conditions for sample incubation during microbiological monitoring of clean rooms.MATERIALS AND METHODS. The study compared several culture conditions for indicator microorganisms, including Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Aspergillus fumigatus F-62, Aspergillus terreus F-1269, and Penicillium chrysogenum F-3 (the latter three strains were obtained from the Russian National Collection of Industrial Microorganisms), as well as for environmental isolates, including Staphylococcus epidermidis, Kocuria rosea, Micrococcus luteus, Bacillus spp., and Sphingomonas paucimobilis. The culture media used were trypticase soy agar (TSA), Sabouraud's dextrose chloramphenicol agar (SDCA), and Reasoner’s 2A agar (R2A). The incubation regimes used were as follows: 2 days at 30–35 ºC and then 3 days at 20–25 ºC; 3 days at 20–25 ºC and then 2 days at 30–35 ºC; 48–72 hours at 30–35 ºC (for aerobic bacteria); 5–7 days at 20–25 ºC (for yeasts and moulds).RESULTS. The comparison showed no statistically significant differences between the results obtained with TSA and R2A under different temperatureconditions. The germination rates of environmental isolates grown on TSA were significantly lower (by 19–37%) in the two-tiered incubation scheme that started at a lower temperature. The study identified groups of microorganisms requiring special culture conditions for microbiological environmental monitoring (bacteria with suppressed physiological functions and moulds).CONCLUSIONS. The study confirmed the need to standardise microbiological testing used in environmental monitoring and to provide for its proper regulation by drafting a general monograph on the matter. The authors demonstrated the applicability of both TSA and R2A as culture media for microbiological environmental monitoring. Currently, the use of a two-tiered incubation scheme with one non-selective culture medium requires validation on a case-by-case basis. Although the sequence of temperature levels did not affect the germination rates of microorganisms significantly, the incubation regime starting at a higher temperature (30–35 ºС) was determined as preferable for bacterial environmental isolates.

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The authors declare that there are no conflicts of interest present.