Multi-drug resistant Vibrio cholerae O1 variant El Tor isolated in northern Vietnam between 2007 and 2010

Since 2007, there has been a re-emergence of cholera outbreaks in northern Vietnam. To understand the molecular epidemiological relatedness and determine the antibiotic susceptibility profiles of responsible V. cholerae O1 outbreak strains, a representative collection of 100 V. cholerae O1 strains was characterized. V. cholerae O1 strains isolated from diarrhoeal patients in northern Vietnam between 2007 and 2010 were investigated for antibiotic susceptibility and characterized by using phenotypic and genotypic tests, including PFGE analysis. Ten clinical V. cholerae O1 isolates from Bangladesh and Zimbabwe were included for comparison. The results revealed that all isolates were resistant to co-trimoxazole and nalidixic acid, 29 % were resistant to tetracycline and 1 % were resistant to azithromycin. All strains were susceptible to ampicillin–sulbactam, doxycycline, chloramphenicol and ciprofloxacin and 95 % were susceptible to azithromycin. MIC values did show reduced susceptibility to fluoroquinolones and 63 % of the strains were intermediately resistant to tetracycline. The isolates expressed phenotypic traits of both serogroup O1 Ogawa and El Tor and harboured an rstR El Tor and ctxB classical biotype. Among the outbreak isolates, only a single PFGE pattern was observed throughout the study period. This study shows that multi-drug resistant V. cholerae altered El Tor producing classical CT strains are now predominant in northern Vietnam.


INTRODUCTION
Cholera is an acute diarrhoeal disease, causing profuse watery stools, which, if untreated, leads to rapid dehydration and death (Kaper et al., 1995). Cholera is prevalent in developing countries with inadequate sanitation and poor access to safe drinking water (Zuckerman et al., 2007). From 1995 to 2005, approximately 100 000-300 000 cases of cholera causing 1500 deaths were reported annually to the WHO (Griffith et al., 2006). Cholera is caused by the Gram-negative bacterium Vibrio cholerae serogroups O1 and O139 (Kaper et al., 1995). Serogroup O1 can be categorized into two biotypes (classical or El Tor), and three serotypes (Ogawa, Inaba or Hikojima) . The seventh, and current, pandemic of cholera is being caused by the El Tor biotype while the fifth and sixth pandemics of cholera were caused by the classical biotype (Alam et al., 2010). Recently, new variants of V. cholerae O1 have emerged and spread throughout many countries of Asia, Africa and America that display a mixture of phenotypic and genotypic traits of both classical and El Tor biotypes (Alam et al., 2010;Ansaruzzaman et al., 2007).
Since the first variants of V. cholerae O1 El Tor were identified in Bangladesh, several other atypical El Tor strains have been identified and reported (Ansaruzzaman et al., 2007;Choi et al., 2010). Genotypic tests to differentiate V. cholerae O1 biotypes include ctxB (cholera toxin B), rstR (repeat sequence transcriptional) and tcpA (toxin-coregulated pili) gene typing (Safa et al., 2010). The El Tor strains that cause cholera in many countries have shifted from genotype 3 (found in El Tor strains from the seventh pandemic and the Latin American epidemic) to genotype 1 (found in strains of classical biotype worldwide and US Gulf Coast El Tor strains). In addition, atypical El Tor strains of V. cholerae O1 from Asia and Africa harboured an rstR allele known as rstR Classical , which corresponds with CTXW Classical and are reported as hybrid strains of the El Tor biotype (Ansaruzzaman et al., 2004). Moreover, the multi-drug resistant V. cholerae O1 altered El Tor has emerged as major problem in many countries in Asia and Africa (Ang et al., 2010;Jain et al., 2011;Kumar et al., 2010;Quilici et al., 2010).
In 2007, a cholera outbreak emerged in northern Vietnam, where only a few cases of cholera had previously been reported (Dalsgaard et al., 1999). The strains of V. cholerae O1 responsible for this outbreak were variants of the El Tor biotype displaying combinations of classical and El Tor features (Nguyen et al., 2009). New cholera cases continued to be detected in northern Vietnam until 2010, especially in Hanoi. The V. cholerae O1 strains associated with cholera outbreaks between 2007 and 2010 in northern Vietnam have not yet been characterized and their susceptibility to antimicrobials has not been reported. Moreover, there is lack of information about the genetic diversity among V. cholerae O1 strains isolated from patients in northern Vietnam between 2007 and 2010.
In the present study, we investigated the antimicrobial susceptibilities and the molecular epidemiological traits of V. cholerae O1 strains isolated in northern Vietnam from 2007 to 2010. V. cholerae O1 strains from Bangladesh and Zimbabwe were also included for comparison.

METHODS
Bacterial strains. In this study, we combined two collections of V. cholerae O1 strains that were selected randomly from diarrhoeal patients admitted to the National Hospital for Tropical Diseases (NHTD), a tertiary-care hospital in Hanoi, Vietnam. The NHTD served as a referral centre during the cholera outbreaks in Hanoi and the surrounding areas between 2007 and 2010. Antimicrobial susceptibility testing was performed on 100 isolates from patients admitted in 2008. A second collection contained 100 randomly selected strains isolated from patients with acute diarrhoea who were admitted to the NHTD between 2007 and 2010 (25 isolates for each year). V. cholerae was identified and confirmed by using standard biochemical methods (Islam et al., 2009). Serological identification of isolates was done by performing slide agglutination tests with commercially available polyvalent antiserum against V. cholerae O1 and monovalent antiserum against V. cholerae O1 serogroups Ogawa and Inaba (Bio-Rad) (Dalsgaard et al., 1999). The second collection was shipped in soft agar at ambient temperature to the ICDDR,B for phenotypic and genotypic testing and PFGE analysis. Reference strains for each of the V. cholerae O1 biotypes were used: classical (O395) and El Tor (N16961). Ten clinical isolates each of V. cholera O1 from Bangladesh and Zimbabwe were included for comparison.
Serotyping and biotyping. Vietnamese V. cholerae strains were serotyped by using the slide agglutination test with specific polyvalent antiserum to V. cholerae O1 and serotype specific antisera to Inaba and Ogawa from the ICDDR,B (Ansaruzzaman et al., 2004). For biotype analysis, we used chicken erythrocyte agglutination, sensitivity to polymycin B and Mukerjee classical phage IV and Mukerjee El Tor phage 5 tests (Kaper et al., 1995;Sack et al., 2004).
PCR assays. The characterization of biotype and genotype were performed with molecular methods. Species-specific identification of V. cholerae was carried out by using V. cholerae species-specific ompW PCR (Nandi et al., 2000). The serogroups of these strains were reconfirmed by multiplex PCR targeted to identify genes encoding O1 (rfb) and O139 (rfb), specific O biosynthetic genes and the cholera toxin (CTX) gene (ctxA) (Hoshino et al., 1998). Biotype specific characterizations were performed by using PCR assays targeted to detect tcpA (Classical and El Tor) (Keasler & Hall, 1993) and the type of the rstR (El Tor and Classical alleles) gene encoding the phage transcriptional regulator (Bhattacharya et al., 2006). Mismatch amplification mutation assay (MAMA)-PCR to detect sequence polymorphism between the classical and El Tor ctxB gene was performed to distinguish the type of CTXB of the V. cholerae O1 strains included in this study (Morita et al., 2008); primers and procedures were used as described previously (Alam et al., 2010).
PFGE. Intact agarose-embedded chromosomal DNA from 16 representative strains of V. cholerae O1 from Vietnam and four strains from Bangladesh and Zimbabwe was prepared and PFGE was performed using contour-clamped homogeneous electric field apparatus (Bio-Rad) according to the PulseNet procedure (Cooper et al., 2006). Genomic DNA was digested with the NotI restriction enzyme (Gibco-BRL). The electrophoresis was performed by using CHEF-DRII system apparatus and conditions. Images were captured and converted to TIF files for computer analysis (Nusrin et al., 2009). Salmonella enterica serotype Braenderup strain H9812 was used as a molecular size marker.

Serotyping and biotyping
Serogrouping and bio-typing of Vietnamese V. cholerae strains revealed that all the strains belonged to the serogroup O1 Ogawa and the biotype El Tor (Table 1). Slide agglutination tests with antisera showed that all strains were positive to polyvalent O1 and monovalent Ogawa. Biotyping showed that all strains were resistant to polymycin B 50 IU, agglutinated to chicken erythrocytes and were resistant to classical phage IV and sensitive to El Tor phage 5, which are typical traits of the El Tor biotype.

PCR assays
Examination of genotypic traits also confirmed that the Vietnamese V. cholerae were of the O1 serogroup and altered El Tor variant (Table 1). PCRs for the V. cholerae species-specific gene ompW and serogroup-specific gene rfbO1 were positive for all strains. All strains harboured rstR El and tcpA genes which are specific to strains of the El Tor biotype. Results of MAMA-PCR showed that all the V. cholerae isolates from Vietnam in this study harboured the ctxB1 allele which had been identified in strains of the classical biotype worldwide and in US Gulf Coast El Tor strains. All strains were positive for the ctxA gene.
There were four resistance patterns (Table 3) among the 100 strains tested. The most prevalent pattern was a resistance to nalidixic acid, co-trimoxazole and tetracycline (including cases of intermediate resistance).

PFGE
In the present study, PFGE analysis of the representative Vietnamese strains isolated between 2007 and 2010 showed an indistinguishable PFGE banding pattern that was typical of V. cholerae O1 El Tor (Fig. 1). The NotI restriction enzyme cleaved the chromosomal genome into 18 fragments with fragment sizes ranging from 20 to 398 kb. The PFGE patterns of isolates from Bangladesh and Zimbabwe differed slightly from the Vietnamese strains by a single band position, suggesting genetic divergence (Fig. 1). When cluster analysis was performed to produce dendrograms, prepared using the Dice similarity coefficient and unweighted pair group method with arithmetic mean (UPGMA) clustering, using the PFGE patterns, all the El Tor strains shared a major cluster (Fig. 2) in which all the Vietnamese strains shared 100 % similarity, whereas the Bangladesh and Zimbabwe strains formed another closely related cluster showing 98 % similarity to the Vietnamese strains.
The PFGE (Fig. 1) results also indicate that the altered strains from Vietnam are more closely related to the El Tor reference strain (N16961) than to the classical reference strain (O395).

DISCUSSION
According to the new biotyping scheme proposed for V. cholerae O1 (Raychoudhuri et al., 2008), the results show that the Vietnamese V. cholerae O1 strains are variant El Tor strains. The strains have phenotypic traits of the El Tor biotype and genotypic traits of both El Tor and classical biotypes (rstR El /ctxB1). These variant strains were first reported in Bangladesh (Nair et al., 2002. The emergence and spread of a new variant of V. cholerae O1 urges the redefinition of V. cholerae classification (Raychoudhuri et al., 2008;Safa et al., 2010). Recently, a new biotyping scheme was proposed, using the molecular marker genes ctxB, rtxC, tlc, tcpA and RS element, which added a further two biotypes, designated 'hybrid' and 'El Tor variant', to the classification of newly emerging V. cholerae O1 strains (Raychoudhuri et al., 2008).   It is currently known that all the V. cholerae O1 strains isolated in Bangladesh after 2001 were atypical or El Tor variant strains as they were phenotypically and genetically similar to El Tor but had the ctxB1 gene of the classical biotype (Nair et al., 2002. Subsequent studies reported such variant strains in Mozambique (Ansaruzzaman et al., 2004), Vietnam (Nguyen et al., 2009), Zimbabwe (this study) and Mexico (Alam et al., 2010). Although the Vietnamese V. cholerae strains isolated between 2007 and 2010 were of the El Tor variant biotype and had PFGE patterns closely related to those of V. cholerae strains isolated from contemporary cholera outbreaks in Zimbabwe and Bangladesh, they had minor but consistent divergences, suggesting that the Vietnamese strains had an independent lineage and occupied a local niche.
Using multi-locus variable number tandem repeat analysis, a recent study demonstrated that altered strains from Vietnam are distant from the El Tor prototype strain responsible for the seventh cholera pandemic (Choi et al., 2010). In spite of the minor divergences observed in the present study, the Vietnamese V. cholerae O1 strains with the RS1-CTX prophage (El Tor type rstR and classical ctxB) array on the large chromosome is likely to be more closely related to V. cholerae O1 El Tor variant strains currently circulating in Bangladesh , India  and Thailand (Okada et al., 2010). However, our data are insufficient to verify the source of the V. cholerae O1 strains currently circulating in Vietnam.
Most of the strains from Vietnam were multi-drug resistant with common resistance to nalidixic acid, co-trimoxazole  and tetracycline. All isolates were susceptible to doxycycline. Resistance to tetracycline, a common antimicrobial agent for the treatment of cholera (WHO, 2005), was also high (29 %). Both tetracycline and doxycycline belong to the tetracycline antibiotic group and cross-resistance between these agents exists (Fluit et al., 2005). The use of doxycycline for cholera treatment and the trend of V. cholerae O1 resistance to the tetracycline group of antibiotics should be monitored. All isolates were resistant to co-trimoxazole, a recommended first-line agent for children with diarrhoea. A high incidence of co-trimoxazole resistance in V. cholerae O1 strains has been reported in prior studies in Africa, Asia and South America Ibarra & Alvarado, 2007;Mandomando et al., 2007). In Vietnam, there is also an increase in resistance to co-trimoxazole among diarrhoeagenic strains of E. coli and Shigella isolated from children in Hanoi (Nguyen et al., 2005). This indicates that co-trimoxazole should not be the recommended treatment for children with diarrhoea in Vietnam.
All the O1 V. cholerae strains were susceptible to fluoroquinolones, including ciprofloxacin and levofloxacin. However, the MIC 90 of ciprofloxacin was close to the susceptibility breakpoint of 1 mg l 21 . All the V. cholerae O1 strains from Vietnam were completely resistant to nalidixic acid (MIC 256 mg ml 21 ). V. cholerae O1 strains resistant to fluoroquinolones have been reported from India (Krishna et al., 2006). The high incidence of nalidixic acid resistance in V. cholerae O1 and increased resistance of V. cholerae against ciprofloxacin is cause for concern (Islam et al., 2009). Although the MICs of ciprofloxacin for the Vietnamese V. cholerae O1 strains were still below the susceptibility breakpoint, they had increased compared to historical isolates; this may be an indication of the emergence of high-level fluoroquinolone resistance in the near future. The majority of the Vietnamese strains (95 %) were still susceptible to azithromycin and this antibiotic can still be used for treatment in Vietnam, though further development of resistance needs to be monitored. Azithromycin is used for treatment of diarrhoeal diseases worldwide (de Saussure, 2009) and has shown to be effective in treatment of cholera in children and adults (Saha et al., 2006). In general, the Vietnamese V. cholerae strains showed similar resistance patterns to Indian and Thai strains of the altered V. cholerae O1 El Tor biotype, carrying the classical ctxB allele; a new variant of V. cholerae O1 (Jain et al., 2011;Okada et al., 2010).

Conclusion
Between 2007 and 2010, cholera outbreaks in Vietnam have been caused by a single clone of a variant V. cholerae O1 biotype El Tor strain. This variant was closely linked to similar variants of V. cholerae O1 El Tor strains isolated in Bangladesh and Zimbabwe but had a slightly divergent lineage, suggesting that they have evolved in solitary and independent ecosystem. A significant proportion of strains were multi-drug resistant, although the majority were still borderline susceptible to fluoroquinolones and susceptible to azithromycin. Empirical treatment of cholera with fluoroquinolones needs to be reconsidered.