Удосконалення лабораторної діагностики V. cholerae О1/non О1 на основі молекулярно-генетичних досліджень

Abstract

Швидка етіологічна діагностика гострих кишкових інфекцій (ГКІ) залишається серйозною проблемою в системі охорони здоров’я. Для підвищення ефективності лабораторної діагностики вібріозів людини та визначення етіологічного агента проведені молекулярно- генетичні дослідження методом полімеразно-ланцюгової реакції (ПЛР). Використання видоспецифічних праймерів під гени холерних вібріонів, які розкривають їх патогенний потенціал та видову приналежність, дало можливість за короткий термін часу розшифрувати їх біологічні властивості. Порівняння генома штамів V. cholerae O1/nonО1 за основними генами патогенності показало їх спорідненість та визначило відмінність у видовій складовій вібріонів. Застосування ПЛР методу у лабораторній практиці забезпечує швидку та надійну ідентифікацію холерного вібріона і насамперед у визначенні його вірулентності.

Быстрая этиологическая диагностика острых кишечных инфекций (ОКИ) остается серьезной проблемой в системе охраны здоровья людей. Для повышения эффективности лабораторной диагностики вибриозов человека и определения этиологического агента проведены молекулярно-генетические исследования методом полимеразно-цепной реакции (ПЦР). Использование видоспецифичных праймеров под гены холерных вибрионов, которые раскрывают их патогенный потенциал и видовую принадлежность, дало возможность за короткий срок времени расшифровать их биологические свойства. Сравнение генома штаммов V. cholerae O1/nonО1 за основными генами патогенности показало их родство и определило отличие в видовой составляющей вибрионов. Применение ПЦР метода в лабораторной практике обеспечивает быструю и надежную идентификацию холерного вибриона и в первую очередь в определении его вирулентности.

Prompt etiological diagnosis of acute enteric infections (AEI) remains a major concern in public health. Clinical polymorphism of AEI emphasizes role of laboratory diagnostics in etiological agent identification. Bacteriological method of diagnostics is most common, but its high time and resources cost creates favorable conditions for new informative research methods. One of the state-of-the-art promising methods is molecular genetic, with polymerase chain reaction (PCR) using highly specific primers being most rapid, low cost and reliable. In order to enhance laboratory diagnostics of human vibrioses and to study biological properties of cholera vibrios, PCR studies of 35 strains of V.cholerae O1 and 100 strains of V.cholerae non O1 isolated from patients in Ukraine in various years were conducted. Research to identify species of cholera vibrios was performed by testing the genome of all studied V.cholerae strains for presence of 13 genes with different functions and origin. Study of 35 strains of V.cholerae O1 indicated that 3 (8,6%) strains V.cholerae O1 (one strain was isolated in 1992, and two strains in 1993) had 12 major pathogenic genes: ctxA, which produces cholera toxin; ace and zot, which produce Ace and Zot toxins; rstR, repressor of rstA gene from RS2 prophage, which is responsible for phage СТХφ replication; rstC, gene from RS1 prophage, antirepressor of rstR gene; tcpAE, responsible for coregulated pili toxin, which is an essential intestinal colonization factor; rtxC, regulatory gene from rtx cluster that encodes synthesis of RTX toxin; toxR, major gene of regulatory system that controls expression of key virulence and vital functions genes; mshA, which encodes mannose-sensitive hemagglutinin pilus IV adhesion type; wbeТ, which is responsible for biosynthesis of O1 serogroup; hapA, which encodes soluble hemagglutinin/protease; structural gene Hly, for Hly haemolysin synthesis. Genomes of these strains did not contain only the wbfR gene which indicates genetic locus of О139 serogroup. Presence of 12 major pathogenic genes in V.cholerae O1, especially genes ctxA and tcpAE, indicates their virulent properties, which allows them to cause cholera. It is worth mentioning that appearance of patients from which cholera vibrios with major pathogenic genes were isolated during a non-outbreak period in Ukraine is an indication of ecdemic cases of cholera. Under the circumstances, patients were timely diagnosed and etiological agent was identified, which offered the opportunity to conduct epidemic countermeasures and locate source of infection. Further genetic research of 32 V.cholerae O1 strains isolated from 1996 to 2012 (non-outbreak years) has shown that they had no major pathogenic genes in their genome – ctxA, ace, zot, rstR, rstC, wbfR. On the other hand, all researched strains contained vital functions and species-specific genes, notably hapA, toxR, Hly, rtxC which are 100% inherited. 5 strains of V.cholerae O1 isolated in 1999 had tcpAЕ gene which is responsible for coregulated pili toxin adhesion. There was no wbeT gene present in the genome of 9 (28,1%) strains, and 16 (50,0%) strains had no mshA gene. Genome of 9 (28,1%) V.cholerae O1 strains lacked both wbeT and mshA genes. Therefore, molecular genetic study of 32 V.cholerae O1 strains indicated absence of major pathogenic genes in their genome, which allows to classify them as avirulent variants of V.cholerae O1. Therefore, such cholera vibrios of O1 serogroup can’t cause cholera outbreak so they are safe from epidemiological point of view. The results of PCR testing of 100 V.cholerae non O1 strains isolated from patients in Ukraine in 2011-2013 indicated that genomes of all studied strains contained no сtxA, асе, zot, rstR, rstC, tcpAE, wbeT, wbfR genes. Only 15% strains of V.cholerae non O1 contained gene mshА, and 95% strains had genes hapA and toxR. Most conservative genes of V.cholerae non O1 strains are genes Hly and rtxC, which were found in 100% of studied strains. It is worth mentioning that presence of Hly and rtxC genes in the genomes of V.cholerae non O1 and virulent/avirulent V.cholerae O1 proves that they are critically important for functioning of cholera vibrios. The results obtained also indicate high importance of genes hapA and toxR, since they were found in 95 % of V.cholerae non O1 strains and in 100% of V.cholerae О1 strains. After comparing the genome structure of V.cholerae non O1 with the genomes of virulent/avirulent strains of V.cholerae O1 by major pathogenic genes, a significant difference from virulent strains and insignificant difference from avirulent strains was found. Both avirulent V.cholerae O1 strains and V.cholerae non О1 strains don’t have major virulence locuses CTXφ, RS2φ, RS1φ, TCP which are typical of virulent strains. Therefore such cholera vibrios can’t cause cholera outbreak so they are epidemiologically safe. At the same time, genomes of avirulent strains of V.cholerae O1 and V.cholerae non О1 were found very similar by presence of genes responsible for type specificity and persistency – rtxC, toxR, mshA, hapA, Нly. It is also important to mention that presence of wbe locus in the genome of cholera vibrios is a decisive factor between V.cholerae O1 and V.cholerae non О1, because it is present only in the vibrios of O1 serogroup. Therefore, research indicates that biological properties and pathogenic potential of studied cholera vibrio strains can be clearly determined using only PCR method. This allows PCR to be used as primary method in laboratory practice and increase the speed of cholera vibrio identification. ISSN 2078-2357. Наук. зап. Терн