山东省分离的霍乱弧菌毒力基因的研究

目的：了解山东省不同年代、不同地区收集的霍乱菌株携带的毒力基因情况、分布特点和变迁。方法：应用分子杂交、PCR技术对霍乱弧菌的CTX基因元件中的ctxA、zot基因，VPI毒力岛中的tcpA、tcpH、aldA、toxT、acfB基因，TLC因子中的cri、orf2—3基因，RTX基因簇中的rtxA、rtxC，以及toxR基因进行了检测。结果：用原位杂交检测，180株霍乱弧菌中有168株携带CTX遗传单元中的ctxA、zot、RS1基因，阳性率均为93．33％，表现出高度的一致性。用打点杂交检测不同时期的28株01群霍乱弧菌，所有被检菌都具有ToxR、RTX相关基因，在1980～1987年间分离的O1群霍乱弧菌流行株中，有92．86％携带编码TLC因子的基因，而1994～2000年间分离的流行株菌株中编码TLC因子的基因携带率仅有7．14％。Southern杂交结果显示，48株O1群霍乱弧菌可产生大小为9．4kb和6．5kb左右的两条ctxA杂交带，用zot基因探针进行杂交，11株O1群霍乱弧菌可产生与ctxA杂交带同样大小的条带。1株O139霍乱弧菌具有检测的所有毒力基因，但其ctxA（zot）杂交带与O1群霍乱弧菌有差异。结论：目前已知的霍乱弧菌毒力基因在山东省分离到的菌株中有广泛的分布，并且随时间的推移而发生变迁。     

杭州市O139群霍乱弧菌耐药变迁及毒力基因携带

目的 研究杭州市1994～2003年分离的O139群霍乱弧菌耐药性变迁以及携带ctxA、tcpA毒力基因的情况。方法 运用K—B法和PCR，检测90株O139群霍乱弧菌对抗生索的敏感性以及是否携带ctxA、tcpA毒力基因。结果 90株O139群霍乱弧菌中无丁胺卡那耐药的菌株，13株对诺氟沙星和环丙沙星耐药；对氨苄青霉索、复方新诺明分别有5年和6年的耐药率为100％；2002、2003年耐药谱增加到7种。87．87％的O139群霍乱弧菌同时携带ctxA、tepA毒力基因。结论10年来分离自杭州的O139群霍乱弧菌的耐药情况日益严重；O139群霍乱弧菌是否携带ctxA、tcpA毒力基因在对抗生素敏感性方面差异有统计学意义。     

武汉市霍乱弧菌中毒力相关基因及分类鉴定

目的为了解武汉地区历年收集的霍乱弧菌毒力相关基因分布情况,并对这些菌株进行分类鉴定。方法分别对霍乱弧菌中的ctxA、zot、ace、ompU、tcpA、toxR、tcpI与hlyA等基因进行PCR检测,并对所有霍乱弧菌进行PCR分类鉴定。结果分离的大部分霍乱弧菌菌株携带有所有的毒力基因,有3株细菌仅有个别非核心毒力基因扩增阳性;该PCR方法分类对O1型所有菌株都能很好地进行分类鉴别,对O139型则与血清型鉴定存在不一致情况。结论PCR方法对产毒型和非产毒型O1/O139霍乱弧菌以及O1/O139和非O1/O139霍乱弧菌鉴定效率非常好。     

福建省非典型埃尔托霍乱弧菌的遗传多样性研究

目的探索福建省非典型埃尔托霍乱弧菌(aEVC)的进化变迁规律,了解aEVC的变异株种类、基因特征,并分析不同类别变异株对霍乱流行趋势的影响。方法运用单基因片段分析技术,选择1962-2005年代表性O1群埃尔托霍乱菌株49株,对ctxB基因扩增产物进行序列测定和比对分析,确定ctxB基因型;同时对毒力因子tcpA和rstR基因分别进行古典型(Cl)和埃尔托型(El)特异的PCR扩增,确定其基因型别。结合ctxB、tcpA、rstR3种基因不同型别的组合形式,确定福建省aEVC菌株的多样性。结果1986年前菌株ctxB基因以埃尔托型B3型为主占92.00%(23/25),而1994年后菌株以古典型B1型为主占95.83%(23/24)。根据ctxB、rstR、tcpA3种基因不同型别的组合形式,1986年前菌株88.00%(22/25)表现为埃尔托型基因特征B3、El、El,1994年后菌株100.00%(24/24)表现为杂合型(Hybrid)变异株基因特征B1、El和(或)Cl、El。结论福建省霍乱菌株存在aEVC进化事件。ctxB、rstR、tcpA基因表现为B1、El和(或)Cl、El的杂合型变异株是福建省aEVC的主要特征型别,此类菌株于1994年后全面取代原来流行的埃尔托霍乱弧菌。不同血清型的aEVC变异株对菌株流行能力的影响不同,稻叶型埃尔托型菌株比杂合型流行时间要长,而小川型杂合型菌株比埃尔托型流行时间要持久。     

嘉兴市霍乱弧菌分离株检测分析

目的比较分析嘉兴市霍乱弧菌菌株的血清型、毒力基因和分子分型特征。方法采用血清学和分子生物学方法对近年来分离到13株霍乱弧菌菌株进行血清型分布、毒力基因(ctxA、ace、zot、tcpA、cri和rtxA)携带和ERIC-PCR分型研究。结果 13株霍乱弧菌菌株,2株为O139群霍乱弧菌,11株为O1群霍乱弧菌,其中小川型9株,稻叶型2株。2株O139群霍乱弧菌菌株携带3种毒力基因;11株O1群霍乱弧菌菌株,除1株携带4种毒力基因外,其余10株携带全部6种毒力基因。ERIC-PCR分型分析显示,不同的霍乱弧菌菌株之间ERIC-PCR型别表现出一定的遗传多样性。结论嘉兴市霍乱弧菌菌株大多携带多种毒力基因,而且来源多样,防控工作任重而道远。     

5株O139群霍乱弧菌生物学性状研究

目的研究从病人和水产品中分离到的5株O139群霍乱弧菌的生物学性状。方法参照《霍乱防治手册》对5株菌的生长特征、生化特征、血清学反应及药物敏感性进行鉴定；应用多重PCR方法对所试菌株进行毒力相关基因检测，用Vero细胞测定方法检测ctxA阳性菌株霍乱肠毒素的表达。结果5株O139群霍乱弧菌生物学性状和血清学反应与阳性对照菌体O139群霍乱弧菌MO45基本相同；2株来自重型病人和1株来自水产品的菌株ctxA、zot、ace、tcpA、tcpI、hlyA、rtxA、rtxC、ompU、tarR 11种毒力相关基因全部阳性。经Vero细胞测定，其霍乱肠毒素效价达到1：160；在2株来自轻型病人的菌株中仅hlyA、rtxA、rtxC、toxR 4种毒力相关基因为阳性。结论O139群霍乱弧菌致病(泻)的决定基因簇并非仅为ctxA、zot、ace、cep等毒力相关基因。     

霍乱弧菌4种新类型tcpA基因发现及序列分析

目的研究中国霍乱弧菌（VC）毒力协同调节菌毛A亚单位基因（tcpA）的多态性.方法分别进行聚合酶链反应及限制性片段长度多态性分析、型特异引物PCR分型、基因测序及序列分析.结果 200株O1群埃尔托型（EVC）产毒株和100株O139群VC产毒株的tcpA基因均与EVC国际标准株N16961株为同一类型（t2型）.50株EVC非产毒株中只有3株携带tcpA基因,1株为t2型,另2株为2种新类型.20株O139群VC非产毒株均不携带tcpA基因.20株非O1非O139群VC中只有2株携带tcpA基因,且为2种新类型.4种新类型tcpA基因与国外发现的4种类型比较,基因序列及推测的氨基酸序列同源性分别为58.3%～77.5%和61.0%～85.5%;5′端约102bp基因序列基本一致,推测的氨基酸序列完全一致;3′端约210bp基因序列变异最大,推测的氨基酸序列变异也最大.抗原表位分析,C末端差异最大.结论建立快速准确区分不同类型tcpA基因的方法,并发现4种新类型.     

广东省外环境O1/O139群霍乱弧菌毒力基因分型

目的 分析广东省外环境来源O1/O139群霍乱弧菌毒力基因的携带及基因分型特征,为霍乱防控提供依据.方法 选取2008 -2009年广东省O1/O139群霍乱弧菌水体分离株69株,水产品分离株16株和同期病例分离株5株,应用多重聚合酶链反应对ctxA、ace、zot、tcpA、tcpl、hlyA、ompU、toxR等8种毒力基因进行检测和分型分析.结果 90株O1/O139群霍乱弧菌均携带hlyA和toxR基因;5株病例菌株中有3株携带8种毒力基因,另外2株小川型菌株为非产毒株,基因型为hlyA+ toxR+ ompU+ zot+ tcpA+ tcpl+型和hlyA+ toxR+ tcpA+型;水体菌株中,稻叶型菌株以hlyA+ toxR+ ompU+ ace+ zot+ tcpl+型(34.15％)为主,小川型(66.67％)和O139群(70％)以hlyA+ toxR+型为主;水产品菌株中,稻叶型菌株以hlyA+ toxR+ ompU+ tcpl+型(75.00％)为主,小川型菌株各种基因型别均有分布,无明显优势基因型别.结论 广东省外环境来源O1/O139群霍乱弧菌以非产毒株广泛存在,毒力基因型别多样.     

霍乱弧菌主要毒力和管家基因序列分析

目的分析广东霍乱弧菌（VC）代表菌株主要毒力基因和管家基因序列。方法PCR扩增霍乱弧菌的主要毒力基因（ctxAB和tcpA）和管家基因（dnaE、hlyA、mdh和recA）、基因测序、对序列进行生物信息学分析。结果不同年代分离的2株埃尔托型（EVC）产毒株和1株0139群VC产毒株的主要毒力基因序列与国内外相关报告比较，ctxA基因及推测的氨基酸序列的同源性分别为99．7％～100％和98．8％～100％．ctxB基因及推测的氨基酸序列的同源性分别为98．8％～100％和96．8％～100％，tcpA基因序列与EVC国际标准株N16961 100％同源。3株产毒株的dnaE、hlyA、mdh和recA基因序列同源性分别为99．8％～100％，100％，99．5％～99．8％和100％，氨基酸序列同源性分别为100％．100％，98．5％～99．3％和100％；3株产毒株和O139群VC非产毒株的管家基因序列同源性分别为97．0％～97．2％，91．8％，94．1％～94．4％，96．9％，氨基酸序列同源性分别为100％，94．6％，94．3％～98．5％和99．7％。结论广东省不同年代EVC产毒株和O139群VC产毒株的群体遗传学关系高度密切，而与O139群VC非产毒株较远，O139群VC产毒株可能起源于EVC产毒株。     

霍乱弧菌产毒株的形成

在细菌进化过程中,非致病菌向致病菌进化的一个主要策略是由携带毒力基因的噬菌体进入宿主菌,并在宿主细菌中进行基因交流,改变宿主菌的表型或增加宿主菌的毒力,使无致病力的菌株转变为致病菌。本文就致病性霍乱弧菌的分子进化、CTXФ霍乱弧菌|产毒株|毒力相关基因和VPI基因家族的结构、功能和基因组的多样性,以及CTXФ基因组整合的分子生物学机制进行综述。     

多重PCR检测霍乱弧菌的毒素相关基因

目的 探索建立一种快速、敏感地检测霍乱弧菌O1群、O139群、非O1／非O139群的毒素相关基因的方法。方法 针对霍乱弧菌霍乱肠毒素A亚单位基因(ctxA)、小带联结毒素基因(zot)、辅助霍乱肠毒素基因(oce)、霍乱弧菌毒素共调菌毛A基因(tcpA)、毒素表达调控蛋白基因(toxR)分别设计引物，建立多基因PCR方法；通过一次扩增反应之产物经琼脂糖凝胶电泳，可检测出菌株的毒素相关基因的携带情况。结果 阳性对照菌株：MO45(霍乱弧菌O139群)检出5种毒素相关基因，结果符合设计要求；其他不同来源的霍乱弧菌(O1群、O139群、非O1／非O139群)检出1—5种不等的毒素相关基因；根据毒素相关基因携带情况，可将菌株分为5个基因型，并可区分为产毒株和非产毒株；多重PCR敏感度可达10^2cfu／ml。结论 该方法快速、特异、敏感，具有较大的应用价值。     

Phenotypic and genetic analyses of 111 clinical and environmental O1, O139, and non-O1/O139 Vibrio cholerae strains from different geographical areas

A total of 111 clinical and environmental O1, O139 and non-O1/O139 Vibrio cholerae strains isolated between 1978 and 2008 from different geographical areas were typed using a combination of methods: antibiotic susceptibility, biochemical test, serogroup, serotype, biotype, sequences containing variable numbers of tandem repeats (VNTRs) and virulence genes ctxA and tcpA amplification. As a result of the performed typing work, the strains were organized into four clusters: cluster A1 included clinical O1 Ogawa and O139 serogroup strains (ctxA(+) and tcpA(+)); cluster A2 included clinical non-O1/O139 strains (ctxA(-) and tcpA(-)), as well as environmental O1 Inaba and non-O1/O139 strains (ctxA(-) and tcpA(-)/tcpA(+)); cluster B1 contained two clinical O1 strains and environmental non-O1/O139 strains (ctxA(-) and tcpA(+)/tcpA(-)); cluster B2 contained clinical O1 Inaba and Ogawa strains (ctxA(+) and tcpA(+)). The results of this work illustrate the advantage of combining several typing methods to discriminate between clinical and environmental V. cholerae strains.     

Molecular analysis of vibrio cholerae strains isolated in Malaysia: public health implications

The genetic diversity or clonality among Vibrio cholerae O1, O139 and non-O1/ non-O139 of clinical and environmental origin using ribotyping and PFGE was performed in order to ascertain the public health implications of the different genotypes circulating within the Malaysian environment. Using an in-house typing scheme, of the 214 strains included, 202 strains were isolated locally between 1992 and 1998, seven were obtained from Bangladesh and five were reference strains. Amongst the 176 El Tor O1 strains, 152 clinical strains demonstrated five ribotypes--E1a, E1b, E2a, E3 and E1c. E1b was the most predominant ribotype demonstrated by 84% of the El Tor O1 strains and was present in all years demonstrating that this strain was intrinsic to Malaysia. PFGE analysis of these strains demonstrated minimal variation amongst the 15 PFGE profiles obtained. Ribotpye E2a amongst five clinical and two environmental O1 strains, were from one location and had previously been reported in Indonesia and the Philippines, thus demonstrating strong evidence that these strains may have been imported into Malaysia. Among Vibrio cholerae O139 strains, 91.7% were of ribotype A1a similar to the original O139, while two others were of ribotype A1b and one of A1e, corresponding to ribotypes 1, 2 and 3 of Dalsgaard and colleagues' scheme for O139 strains. PFGE analysis demonstrated that 89% of ribotype A1a could be differentiated into three PFGE genotypes which were very closely related. The eight non-O1/non-O139 serogroup strains were heterogeneous in both ribotype and PFGE patterns.     

Characterization of Vibrio cholerae isolated from the aquatic basins of the State of Pernambuco, Brazil

Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.     

Association of a disease approximating cholera caused by Vibrio cholerae of serogroups other than O1 and O139.

One hundred and six patients suffering from severe dehydrating diarrhoea were studied of whom 36 patients were positive for Vibrio cholerae. Out of 36, 15 were positive for V. cholerae O1, 10 for V. cholerae O139 and 11 for V. cholerae non-O1 non-O139. O1 and O139 were positive for the 301-bp ctxA amplicon and 471-bp tcpA amplicon indicating that the strains possessed toxigenic capability whereas no non-O1 non-O139 strain possessed ctxA or tcpA genes. Post-admission severity of purging and amount of ORS required were less in the V. cholerae non-O1 non-O139 group (P < 0.05) compared to the V. cholerae O1 and O139 groups. It appears from this study that a cholera-like clinical condition can be caused in the absence of CT as exemplified by strains of non-O1 non-O139.     

Detection of virulence associated genes, haemolysin and protease amongst Vibrio cholerae isolated in Malaysia

Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.     

Characterization of Vibrio cholerae from 1986 to 2012 in Yunnan Province,southwest China bordering Myanmar

Vibrio cholerae is an important infectious pathogen causing serious human diarrhea. We analyzed 568 V. cholerae strains isolated from 1986 to 2012 in Yunnan province, southwest China bordering Myanmar. Polymerase chain reactions for detecting virulence genes, antibiotic susceptibility tests and pulse-field gel electrophoresis (PFGE) were performed. The results showed all the strains were El Tor biotype from 1986. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. All of the strains were sensitive to aminoglycosides and quinolone antibiotics while resistant to β-lactamase and carbapenem antibiotics increased gradually. 568 V. cholerae were divided into 218 PFGE-NotI patterns, and the isolates before 2001 and after 2011 were separated into two groups according to PFGE results. The strains isolated before 2001 were mainly referred to native cholera in Yunnan, and after 2011 were primarily referred to as imported strains from Myanmar, which showed the variation of V. cholerae in this area. The molecular characteristics of V. cholerae indicated regularity in bacterial variation and evolution in Yunnan province.     

Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup

Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.     

Ｏ１群霍乱弧菌毒力基因和菌株流行能力关系研究

摘要：目的　调查福建省１９６２－２００５ 年不同流行时期、不同流行血清型的Ｏ１ 群埃尔托型霍乱弧菌
（ＥＶＣ）毒力基因分布情况;探讨霍乱弧菌（ＶＣ）不同毒力基因特征与霍乱流行能力的关系。方法　运用
ＰＣＲ 扩增技术,检测１９６２－２００５ 年６９ 株（稻叶２１、小川４８） 霍乱患者分离株的犮狋狓犃基因, 同时进行
犮狋狓犅、狋犮狆犃、狉狊狋犚、犺犾狔犃毒力因子的古典型（犆犾）和埃尔托型（犈犾）基因亚型的扩增;结合流行病学资料
分析不同毒力特征菌株的流行能力。结果　６９株Ｏ１群霍乱弧菌犮狋狓犃基因阳性６２株,犮狋狓犃基因阴性的菌
株均不同程度地携带有其他各相关毒力基因;各毒力因子古典型和埃尔托型的阳检率分别为犮狋狓犅６０．９％
（４２株）和７２．５％ （５０株）,狉狊狋犚３７．７％ （２６株）和７２．５％ （５０株）,狋犮狆犃４．３５％ （３株）和６９．５７％ （４８
株）,犺犾狔犃６５．２２％ （４５株）和７９．７１％ （５５株）。犮狋狓犅 犆犾、狉狊狋犚 犆犾基因以及菌株同时携带狉狊狋犚 犆犾和狉狊狋犚
犈犾基因的阳性率在稻叶型（阳性率分别为３８．１％ 、０ 和０） 和小川型（阳性率分别为７０．８％ 、５４．２％ 和
３５．４％ ）菌株中的差异有统计学意义（犘均＜０．０５）。犮狋狓犅 犈犾、狉狊狋犚 犆犾、狉狊狋犚 犈犾基因的阳性率在１９６２－
１９６４年、１９７８ 年、１９９４－２０００ 年的小川型３ 个不同流行时期（阳性率分别为犮狋狓犅 犈犾：１００．０％ 、
１００．０％ 、４４．８％ ;狉狊狋犚 犆犾：０、０、８９．７％ ;狉狊狋犚 犈犾：１００．０％ 、８７．５％ 、５８．６％ ） 的差异有统计学意义
（犘均＜０．０５）。犮狋狓犅 犆犾、犮狋狓犅 犈犾、狉狊狋犚 犈犾基因的阳性率在１９８０－１９８６ 年、２００５ 年稻叶型两个不同流行
时期（阳性率分别为犮狋狓犅 犆犾：１３．３％ 、１００．０％ ;犮狋狓犅 犈犾：１００．０％ 、６６．７％ ;狉狊狋犚 犈犾：９３．３％ 、５０．０％ ）
的差异有统计学意义（犘均＜０．０５）。结论　福建省１９６２－２００５年Ｏ１群霍乱菌株不同程度地携带有相关的
毒力因子;各毒力因子埃尔托型基因片段的检出率均要高于古典型,以狉狊狋犚和狋犮狆犃基因的差异最明显;
相对于稻叶血清型,小川血清型更容易整合古典型的犮狋狓犅和狉狊狋犚基因片段;犮狋狓犅和狉狊狋犚基因的类型和不
同血清型霍乱菌株的流行能力有相关性。
关键词：霍乱弧菌;毒力基因;ＰＣＲ;等位基因
中图分类号：Ｒ１８３．４　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１４）０１ ００４１ ０５     

Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996-2004 possessing the unique VSP-II region of El Tor variant

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.