1961-2010年中国O1群El Tor型霍乱弧菌产毒株ctxB和rstR基因变迁

The study of ctxB and rstR variations of toxigenic Vibrio cholerae O1 El Tor strains isolated from 1961 to 2010 in China

目的 分析1961-2010年中国不同地区分离的O1群El Tor型霍乱弧菌产毒株的ctxB变异特征和rstR基因类型.方法 选取中国1961-2010年间分离的O1群霍乱弧菌菌株作为研究对象,共385株.应用PCR方法扩增ctxB基因并进行序列测定和分析;使用rstR型特异性引物扩增rstR基因,确定其基因型别.结果 ctxB基因序列分析表明,385株被检菌株中52.5％（202/385）携带ctxBET,47.5％（183/385）携带ctxBc1ass,即第39位的酪氨酸（Y）变为组氨酸（H）,68位的异亮氨酸（Ⅰ）变为苏氨酸（T）,呈现古典生物型菌株（O395）的序列特征.1961-1992年以携带ctxBET的菌株占绝对优势,比例高达98.4％（182/185）;1993年后,携带ctxBclass的菌株增加,其中1993-2005年间携带ctxBc1ass菌株的比例达到97.2％ （174/179）;自2006年开始,重新出现了ctxBET为主导或二者共存的局面.rstR基因的检测表明约62.9％（242/385）的菌株携带rstRET,6.8％ （26/385）的菌株携带rstRclass,其余菌株中存在不同的rstR组合形式,其中,以rstRET＋ rstRc1as的组合最多,占75.7％（75/99）.与ctxB相似,不同型别rstR的分布呈现时间特异性,1961-1992年期间以携带rstRET的菌株占主导（87％,161/185）,1993年以后出现多态性变异,携带其他类型rstR如rstRclass,rstRenv及不同rstR组合的菌株明显增多,与rstRET共同存在.96％（25/26）、84％（63/75）和18/18的分别携带rstRclass、rstRET＋ rstRclass组合和rstRET＋ rstRc1ass＋ rstRenv组合的菌株均分离于1993年之后.结论 中国霍乱弧菌菌株中ctxB和rstR的型别分布有明显的时间特异性,rstR存在多种组合形式,反映了菌株多样性的遗传和进化特征.

Objective To understand the ctxB and rstR variations of toxigenic Vibrio cholerae （ V. cholerae）O1 E1 Tot strains isolated from different provinces in China from 1961 to 2010. Methods All 385 toxigenic V. cholerae O1 E1 Tor strains were selected, which were isolated in China between year 1961 and 2010. ctxB gene was amplified by PCR method and sequenced for further analysis, rstR was detected with PCR by using the genotype specific primers. Results ctxB sequence analysis revealed that 52. 5% （202/385）isolates carried ctxBET and 47.5% （183/385）carried ctxBclass, namely y39 to H and 168 to T substitutions which were specific to the classical biotype CT-B sequence. From 1961 to 1992, strains carrying ctxBET were predominant and the proportion was as high as 98.4% （182/185）. After 1993, strains carrying ctxBET were sharply increased. Especially during year 1993 to year 2005, 97.2% （174/179） of the isolated strains carried ctxBclass. Since 2006, resurgence of dominant strains carrying ctxBET or co-existing of strains with ctxBET or ctxBclass was noticed, rstR genotype detection showed that 62. 9% （ 242/385 ） of the tested strains carried the rstRET, while 6. 8% （26/385） with rstRclass, and the remainings contained at least two types of rstR in different combination forms, among which rstRET ＋ rstRclass combination were the most, accounting for 75.7% （75/99）. Similar to the ctxB, the distribution of rstR genotypes showed time specificity. From 1961 to 1992, strains carrying rstRET predominated （87.0% , 161/185）. After 1993, the diversity of rstR genotypes was observed accompanying by a sharp increase of strains containing other rstR genotype, such as rstRclass,rstRenv and different combinations. There were separately 96% （25/26）, 84% （63/75） and 18/18 strains containing rstRclass, rstRET ＋ rstRclass and rstRET ＋ rstRclass ＋ rstRenv isolate（l after 1993. Conclusion The distribution of different genotypes of ctxB and rstR showed obvious time-specificity, and there were various combining forms of rstR, reflecting the diversity of the genetic and evolutionary characteristics of Chinese K cholerae isolates.