Vibrio cholerae classical biotype strains reveal distinct signatures in Mexico.

Abstract

Vibrio cholerae O1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, before the El Tor (ET) biotype initiated the seventh pandemic in Asia in the 1970s by completely displacing the CL biotype. Although the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. cholerae CL and ET biotypes, including a hybrid ET, were found associated with areas of cholera endemicity in Mexico between 1991 and 1997. In this study, CL biotype strains isolated from areas of cholera endemicity in Mexico between 1983 and 1997 were characterized in terms of major phenotypic and genetic traits and compared with CL biotype strains isolated in Bangladesh between 1962 and 1989. According to sero- and biotyping data, all V. cholerae strains tested had the major phenotypic and genotypic characteristics specific for the CL biotype. Antibiograms revealed the majority of the Bangladeshi strains to be resistant to trimethoprim-sulfamethoxazole, furazolidone, ampicillin, and gentamicin, while the Mexican strains were sensitive to all of these drugs, as well as to ciprofloxacin, erythromycin, and tetracycline. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA revealed characteristic banding patterns for all of the CL biotype strains although the Mexican strains differed from the Bangladeshi strains in 1 to 2 DNA bands. The difference was subtle but consistent, as confirmed by the subclustering patterns in the PFGE-based dendrogram, and can serve as a regional signature, suggesting the pre-1991 existence and evolution of the CL biotype strains in the Americas, independent from Asia.

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Published Ahead of Print 18 April 2012.
10.1128/JCM.00189-12.
2012, 50(7):2212. DOI:
J. Clin. Microbiol.
Alejandro Cravioto
Haruo Watanabe, Nur-A Hasan, Rita R. Colwell and
Rosario Morales, Jose Luis Mendez, Armando Navarro,
Fatema-tuz Johura, Nurul A. Bhuiyan, Gabriela Delgado,
Munirul Alam, M. Tarequl Islam, Shah Manzur Rashed,
Reveal Distinct Signatures in Mexico
Vibrio cholerae Classical Biotype Strains
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Vibrio cholerae Classical Biotype Strains Reveal Distinct Signatures in
Mexico
Munirul Alam,aM. Tarequl Islam,aShah Manzur Rashed,aFatema-tuz Johura,aNurul A. Bhuiyan,aGabriela Delgado,b
Rosario Morales,bJose Luis Mendez,bArmando Navarro,bHaruo Watanabe,cNur-A Hasan,dRita R. Colwell,d,e,fand
Alejandro Craviotoa
International Center for Diarrheal Disease Research, Bangladesh, Dhaka, Bangladesha; Department of Public Health, Faculty of Medicine, Universidad Nacional Autónoma
de México, Mexico, D.F., Mexicob; National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japanc; Maryland Pathogen Research Institute, University of Maryland,
College Park, Maryland, USAd; Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USAe; and Johns Hopkins Bloomberg
School of Public Health, Baltimore, Maryland, USAf
Vibrio cholerae O1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, be-
foretheElTor(ET)biotypeinitiatedtheseventhpandemicinAsiainthe1970sbycompletelydisplacingtheCLbiotype.Al-
though the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. chol-
eraeCLandETbiotypes,includingahybridET,werefoundassociatedwithareasofcholeraendemicityinMexicobetween1991
and1997.Inthisstudy,CLbiotypestrainsisolatedfromareasofcholeraendemicityinMexicobetween1983and1997were
characterizedintermsofmajorphenotypicandgenetictraitsandcomparedwithCLbiotypestrainsisolatedinBangladeshbe-
tween 1962 and 1989. According to sero- and biotyping data, all V. cholerae strains tested had the major phenotypic and geno-
typiccharacteristicsspecificfortheCLbiotype.AntibiogramsrevealedthemajorityoftheBangladeshistrainstoberesistantto
trimethoprim-sulfamethoxazole,furazolidone,ampicillin,andgentamicin,whiletheMexicanstrainsweresensitivetoallof
thesedrugs,aswellastociprofloxacin,erythromycin,andtetracycline.Pulsed-fieldgelelectrophoresis(PFGE)ofNotI-digested
genomicDNArevealedcharacteristicbandingpatternsforalloftheCLbiotypestrainsalthoughtheMexicanstrainsdiffered
fromtheBangladeshistrainsin1to2DNAbands.Thedifferencewassubtlebutconsistent,asconfirmedbythesubclustering
patternsinthePFGE-baseddendrogram,andcanserveasaregionalsignature,suggestingthepre-1991existenceandevolution
oftheCLbiotypestrainsintheAmericas,independentfromAsia.
V
humanandenvironmentalcomponentsinitslifecycle(23).There
are more than 200 distinct serogroups of V. cholerae, but only O1
and O139 are recognized as being responsible for epidemic and
pandemic cholera (17). V. cholerae O1, which is the predominant
pandemic serogroup to date, is further differentiated into two
well-established biotypes, namely, the classical (CL) and El Tor
(ET). This classification is based primarily on several phenotypic
properties, including hemolysis of sheep red blood cells, aggluti-
nation of chicken red blood cells, Voges-Proskauer reaction, and
susceptibility to polymyxin B (50 U) and biotype-specific phages
(17).Inadditiontophenotypictraits,majorgeneticmarkershave
also been used to determine the biotypes of V. cholerae strains,
such as the major toxin coregulated pilus gene (tcpA), the gene
encoding the cholera toxin subunit B (ctxB), and the repeat se-
quence transcriptional regulator (rstR) gene, all of which possess
CL- and ET-specific alleles, and the presence or absence of the
repeat in toxin (rtxC) gene (28). Recently, two genomic islands,
VibrioseventhpandemicislandI(VSP-I)andVSP-II,wereshown
tobeuniquetotheETstrainsoftheseventhpandemic,inthatthey
were absent from both the pre-seventh pandemic ET strains and
the CL biotype strains (11).
Since 1817, cholera has become pandemic, spreading into a
multitude of countries. The sixth pandemic and presumably ear-
lier cholera pandemics were caused by the V. cholerae CL biotype,
while the most extensive, and ongoing, seventh pandemic, which
started in 1961, is caused by the ET biotype (15). It was following
the sixth pandemic that the V. cholerae CL biotype was gradually
ibriocholerae,thecausativeagentofcholera,isoneofthemost
successful emerging and reemerging pathogens that has both
replaced by El Tor, and within a decade the CL biotype seemed to
have disappeared (3, 4). However, the CL biotype reemerged as
thepredominantepidemicbiotypeinsomeareasofBangladeshin
1982andcoexistedwithElTorstrainsforafewyearsuntilthelate
1980s (29). In spite of having a mysterious history of disappear-
anceandreappearanceinBangladesh,theCLbiotypewasthought
to have become extinct, with its last isolation being recorded in
1992 in southern Bangladesh (13, 17).
When the cholera epidemic struck Peru in January 1991 and
spread rapidly to other Latin American countries, reaching Mex-
ico in June of the same year, the causal agent, V. cholerae O1 El
Tor, was homogenous and considered to be an extension of the
seventh pandemic (30), presumably arriving via ships from areas
of cholera endemicity (20). This surmise was probably based on
the observation that there had been no sign of cholera in South,
Central, or North America for more than a century. Recently,
however, V. cholerae El Tor hybrid variant strains were shown to
be dominant among V. cholerae CL and ET biotype progenitor
strains associated with endemic cholera in Mexico between 1991
Received 24 January 2012 Returned for modification 23 March 2012
Accepted 9 April 2012
Published ahead of print 18 April 2012
Address correspondence to Alejandro Cravioto, acravioto@icddrb.org.
M.A. and M.T.I. contributed equally to this article.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00189-12
2212
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and 1997 (2). Although the pre- and post-1991 existence of V.
cholerae CL biotype strains along with ET hybrid and prototype
ET strains up until 1997 was suggestive of a local environmental
reservoirinMexico(2),littleisknownabouttheirphenotypicand
genetic traits and, more specifically, their ancestry to discern
whether the CL biotype strains isolated from endemic cholera in
Mexico were evolving locally or were of Asian origin. In the pres-
ent study, comparative phenotypic, molecular, and phylogenetic
data related to V. cholerae CL biotype strains isolated in Mexico
(n?6;1983to1997)andBangladesh(n?22;1962to1989)have
beenanalyzed,andtheresultsshowthattheV.choleraeCLbiotype
strainsmayhaveevolvedindependentlyinthetwogeographically
distant ecosystems.
MATERIALS AND METHODS
Bacterial strains. V. cholerae O1 classical (CL) biotype strains (n ? 28)
involvedinthisstudyincluded6strainsisolatedinMexico(1983to1997)
and 22 strains isolated in Bangladesh (1962 to 1989). The V. cholerae
reference CL strain O395 and reference El Tor (ET) strain N16961 were
also included for comparison. The Mexican isolates were obtained from
the Department of Public Health, Faculty of Medicine, National Autono-
mous University of Mexico (UNAM). These strains were isolated from
cholera patients (n ? 4) and surface water (n ? 2) as part of the nation-
wide cholera surveillance program between 1983 and 1997 (6). Bangla-
deshi V. cholerae CL biotype strains were obtained from the culture col-
lection at the International Centre for Diarrheal Disease Research,
Bangladesh (ICDDR,B). The bacterial strains were shipped in soft agar,
confirmed using standard culture methods, and identified by a combina-
tion of biochemical and molecular methods, as described previously (1).
Genomic DNA preparation. Genomic DNA extraction was carried
out following previously described methods (24). Briefly, cells were har-
vested by centrifugation, and the pellets were resuspended in TES buffer
(10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 10% SDS) and
incubated at 65°C for 10 min. Following proteinase K treatment at 50°C
for 18 h, DNA was extracted with phenol-chloroform-isoamyl alcohol
(25:24:1), precipitated in ethanol, and dissolved in TE buffer (10 mM
Tris-HCl,1mMEDTA,pH8.0).RNasetreatmentwasperformedat37°C
for 1 h, and ethanol-precipitated DNA was dissolved in TE buffer and
stored at ?20°C.
Serogrouping. The serogroups of the V. cholerae isolates that were
identified using biochemical and molecular methods were confirmed se-
rologicallybyaslideagglutinationtestusingspecificpolyvalentantiserato
V. cholerae O1 and O139, followed by a monoclonal antibody specific for
eachserogroup(1).Thesubtypesofallthestrainswerereconfirmedusing
a V. cholerae species-specific ompW PCR (22). The serogroups of these
strains were reconfirmed using polyvalent O1 and monovalent Inaba and
OgawaantiseraandbymultiplexPCRtargetedtoidentifygenesencoding
O1 (wbe)- and O139 (wbf)-specific O-biosynthetic genes and the cholera
toxin (CTX) gene (ctxA) (16).
Confirmationofbiotype.Biotypinginvolvedanumberofphenotypic
testsincludingsensitivitytopolymyxinBandMukherjeeCLphageIVand
MukherjeeETphageVtests(17).Classicalstrainsaretypicallysensitiveto
polymyxin B and type IV phage but resistant to type V phage.
In order to complement biotype characterization using phenotypic
traits, PCR assays were also carried out targeting various biotype-deter-
mining genes. The major subunit protein type of the toxin-coregulated
pilus (TCP) gene tcpA was determined using specific primers (26), which
can differentiate between CL and ET alleles using an amplified product
size of 620 bp and 451 bp, respectively. The repeat-sequence transcrip-
tional regulator gene (rstR), which contains CL- and ET-specific alleles,
wasdetectedusingprimersdescribedpreviously(18).ThertxCgeneofthe
RTX toxin gene cluster has been considered to be a significant marker for
differentiatingbiotypesandispresentinETbutnotinCLbiotypestrains.
In this study, the rtxC gene was amplified using primers described by
Chow et al. to produce a 203-bp PCR product (8). The cholera toxin
(CTX) subunit B gene (ctxB) allele was detected by a mismatch amplifi-
cation mutation assay (MAMA) PCR specific for the CL and ET biotypes
involving a conserved forward primer and two allele-specific polymor-
phism detection primers under PCR conditions as described previously
(21).
Antimicrobialsusceptibility.Bacterialsusceptibilitytoantimicrobial
agents was determined by standard disc diffusion methods (5, 9) using
commercially available discs (Oxoid International). A total of seven anti-
bioticswereused:erythromycin(E;15?g),tetracycline(TE;30?g),gen-
tamicin (CN; 10 ?g), ciprofloxacin (CIP; 5 ?g), trimethoprim-sulfa-
methoxazole (SXT; 30 ?g), ampicillin (AMP; 30 ?g), and furazolidone
(FR; 100 ?g).
PFGE. The whole agarose-embedded genomic DNA from V. cholerae
wasprepared,andpulsed-fieldgelelectrophoresis(PFGE)wascarriedout
with a contour-clamped homogeneous electrical field (CHEF-DRII) ap-
paratus (Bio-Rad) according to procedures described elsewhere (7). The
conditionsusedforseparationwereasfollows:2to10sfor13h,followed
by20to25sfor6h.Anelectricalfieldof6V/cmwasappliedatanincluded
field angle of 120°. Genomic DNA of the test strains was digested by the
NotIrestrictionenzyme(Gibco-BRL,Gaithersburg,MD),andSalmonella
enterica serovar Braenderup was digested by XbaI, with the fragments
beingusedasmolecularsizemarkers.Therestrictionfragmentsweresep-
arated in 1% pulsed-field-certified agarose in 0.5? TBE (Tris-borate-
EDTA) buffer. In the post-electrophoresis gel treatment step, the gel was
stained and destained. DNA was visualized using a UV transilluminator,
and images were digitized by a one-dimensional gel documentation sys-
tem (Bio-Rad).
Image analysis. The fingerprinting pattern in the PFGE gel was ana-
lyzedusingthecomputersoftwarepackageBioNumerics(AppliedMaths,
Belgium). After background subtraction and gel normalization, the fin-
gerprint patterns were subjected to typing on the basis of banding simi-
larityanddissimilarityusingtheDicesimilaritycoefficientandclustering
based on the unweighted-pair group method using average linkages
(UPGMA), as recommended by the software manufacturer, and results
are graphically represented as dendrograms.
Sequencing of the ctxB gene. The ctxB gene was amplified from the
strainsusingctxFandctxRprimersasdescribedpreviously(25).ThePCR
product was purified, and sequencing was carried out using the same
primersona96-capillarymodel3730xlsystemwithaBigDyeTerminator
kit (Applied Biosystems). The sequences of the ctxB gene for the CL ref-
erence strains were retrieved from GenBank. The deduced amino acid
sequences of the ctxB gene from all strains were aligned using Clustal W.
RESULTS AND DISCUSSION
The classical (CL) biotype, causing the sixth cholera pandemic,
was believed to be extinct even from the Bengal region of south
Asia, where cholera has been endemic for centuries and where V.
choleraehasbeenestablishedasautochthonousfloraoftheaquatic
ecosystem. Together with El Tor (ET) and variant ET strains, the
CLbiotypestrainswerefoundtobeassociatedwithendemicchol-
erainMexicobetween1991and1997(2).Sincetherehadbeenno
significantcholeraoutbreakforalmost100yearspriortothe1991
epidemic, little is known about the ancestry of these causative
pathogens,especiallyiftherewasalocalreservoirforpathogensin
theAmericasorifthepathogenswereintroducedfromAsia,ashas
been proposed for ET cholera in 1991 (20, 30). In this study, we
present comparative phenotypic and molecular data on V. chol-
erae CL biotype strains isolated in Mexico and Bangladesh and
showevidenceoftheindependentevolutionofCLbiotypestrains
in the two geographically distinct ecosystems.
TheV.choleraeCLbiotypestrainsisolatedinMexicoandBan-
gladesh showed biochemical and molecular properties typical of
the V. cholerae CL reference control O395. The strains were fur-
Classical Biotype Strains Evolved Locally in Mexico
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ther confirmed serologically by a slide agglutination test using
polyvalentantiserafollowedbyamonoclonalantibodyspecificfor
V. cholerae serogroup O1 (1). Strain origin, year of isolation, and
phenotypic results are shown in Table 1. All strains tested, irre-
spective of their origin, were sensitive to polymyxin B and phage
IV and resistant to phage V, which are typical phenotypic proper-
ties of CL biotype strains although V. cholerae O1 El Tor and CL
biotype strains have shown variable reactions to biotype-specific
phages and polymyxin B in previously reported data (28).
Antimicrobial resistance of V. cholerae to effective drugs can
hinder the treatment of the disease (27), affecting the recovery of
patients and increasing the chance of death. In the present study,
V. cholerae strains isolated in Mexico and Bangladesh were sub-
jectedtoantimicrobialsusceptibilityassays(5)againstciprofloxa-
cin(CIP),erythromycin(E),tetracycline(TE),trimethoprim-sul-
famethoxazole (SXT), furazolidone (FR), ampicillin (AMP), and
gentamicin (CN). As shown in Table 1, V. cholerae CL biotype
strains isolated in Mexico were found to be sensitive to all seven
antibiotics tested although the V. cholerae CL biotype strains iso-
lated in Bangladesh varied in their responses toward the various
drugs.Year-baseddatashowthatallV.choleraeCLbiotypestrains
isolatedinBangladeshbetween1962and1971weresensitivetoall
seven drugs, while the majority of the strains isolated between
1982 and 1989 showed multidrug resistance (MDR); six strains
wereresistanttoAMP,CN,FR,andSXT,andthreewereresistant
toAMP,FR,andSXT.InastudycarriedoutinBangladeshduring
the early 1980s, V. cholerae O1 strains were shown to be MDR,
havingtheresistancemarkersforAMP,kanamycin,streptomycin,
spectinomycin, TE, sulfonamides, and trimethoprim (14). Al-
though antibiotic resistance may not be a stable marker to be
maintained and transferred vertically and spatiotemporally, the
differences observed in the antibiotic resistance patterns of V.
cholerae CL biotype strains isolated in Bangladesh (1982 to 1989)
and Mexico (1983 to 1997) may be due to independent evolution
of V. cholerae in two geographically distant ecosystems.
Results of simplex and multiplex PCR screening (17) for V.
cholerae species-specific ompW and wbe genes encoding sero-
group O1-specific surface antigen further confirmed all of the
tested strains to be V. cholerae O1. These strains were toxigenic
sinceallofthemamplifiedprimersforctxAthatencodessubunitA
ofthecholeratoxin(CTX).TheabsenceoftheETbiotype-specific
rtxC gene and presence of the CL biotype-specific alleles of the
tcpAandrstRgenes(Table1)alsoconfirmedthattheseV.cholerae
O1 strains belonged to the CL biotype. The CL biotype-specific
ctxB allele was confirmed by MAMA PCR (21) followed by se-
quencing of the ctxB amplicons (2). Overall results of phenotypic
and genetic screening confirmed that all of the strains were the V.
cholerae CL biotype of the sixth pandemic prototype.
GenomiccharacterizationoftheV.choleraeCLbiotypestrains
isolated in Bangladesh was reported elsewhere using restriction
endonucleasecleavagepatternsoftherRNAgene(13).Inthecur-
rent study, PFGE of NotI restriction-digested genomic DNA was
performed (7) to determine the genetic fingerprint of V. cholerae
CL biotype strains isolated in two geographically distinct regions,
MexicoandBangladesh.TheNotIrestrictionenzymedigestedthe
genomic DNA of both the test and CL reference control strains
into 20 to 23 fragments, the sizes of which ranged from 20 to 350
kb. All of the test strains presented overall banding patterns that
were characteristic of V. cholerae CL biotype strains (O395), fur-
ther confirming their CL biotype attribute. Overall PFGE analysis
revealed the CL biotype strains to have two major banding pat-
terns (pulsotypes), I and II, and pattern II had three different
subtypes, IIa to IIc (Table 1). Four strains isolated in Mexico be-
tween 1991 and 1995, including the only strain isolated in 1983,
showed an identical banding pattern (pulsotype IIa) that was dif-
ferent from the PFGE patterns (pulsotypes I and IIb) of strains
isolated in Bangladesh. The predominant Mexican strains with
pulsotype IIa had a unique DNA band of around 115 kb that was
consistently absent from all of the V. cholerae CL biotype strains
isolated in Bangladesh, including the reference CL strain O395
from India (Fig. 1). These results reflect genetic divergence that
could serve as a distinct regional signature for the Mexican and
Bangladeshi CL biotype strains. The two remaining Mexican
strains isolated in 1997 produced slightly different patterns; one
belonged to pulsotype I, which is the predominant banding pat-
tern for Bangladeshi strains isolated between 1962 and 1989. The
other strain, with an extra band of around 80 kb (designated pul-
sotypeIIc),differedfromallteststrainsincludingtheCLreference
TABLE 1 Phenotypic and genotypic traits of V. cholerae O1 classical biotype strains isolated in Mexico and Bangladesh
Test strain
origin or
reference strain
name (n)a
Year(s) of
isolation
No. of
isolatesSourceb
Phenotypic response to:c
Genotypic traitd
Antibiotic
resistance profilef
PFGE type
(no. of
isolates)
Polymyxin
B (50 U)
Phage
IV
Phage
V
tcpA
type
ctxBe
type
rstR
typertxC
Bangladesh (22)1962–1971
1982–1989
7
6
3
6
2
2
2
Clin
Clin
Clin
Clin
Clin
Env
Clin
Clin
Clin
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
S
CL
CL
CL
CL
CL
CL
CL
CL
ET
CL
CL
CL
CL
CL
CL
CL
CL
ET
CL
CL
CL
CL
CL
CL
CL
CL
ET
?
?
?
?
?
?
?
?
?
Sensitive
AMP, CN, FR, SXT
AMP, FR, SXT
Sensitive
Sensitive
Sensitive
Sensitive
I
I (5), Ib (1)
I
I
IIa
IIa
IIc (1), I (1)
Mexico (6)1983–1995
1997
1965
1971
O395
N16961
an, number of strains.
bClin, clinical; Env, environmental.
cR, resistant; S, sensitive.
dET, El Tor; CL, classical.
eDetermined by MAMA PCR (21).
fAMP, ampicillin; CN, gentamicin; FR, furazolidone; SXT, trimethoprim-sulfamethoxazole; sensitive, sensitive to all of the drugs in this group (AMP, CN, FR, and SXT).
Alam et al.
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strain,suggestingauniquemolecularsignatureforthisstrain.The
Mexican strain that matched in the PFGE pattern with the Ban-
gladeshi strains cannot be clonal in the true sense because this
strain had a distinctly different spatiotemporal origin and likely
arose from genetically diverse V. cholerae populations involved
with the 1991 epidemic and the subsequent endemic cholera in
Mexico (2, 19).
Cluster analysis by dendrogram (UPGMA clustering method)
of the gel images separated the V. cholerae CL biotype strains into
two major clusters, A and B. Four strains isolated from Mexico
before 1997 showed similar banding patterns and belonged to
cluster B, whereas all but one of the Bangladeshi V. cholerae CL
biotype strains having a regional signature banding pattern be-
longed to a separate cluster A that also included the reference CL
strain O395 from India and one Mexican V. cholerae CL biotype
strainisolatedin1997.AlthoughdifferencesinthePFGEbanding
patterns were subtle, a clear dichotomy was observed in the clus-
tering patterns, with the majority of the Mexican V. cholerae CL
biotype strains forming a regional cluster separating them from
the V. cholerae CL biotype strains isolated in Bangladesh. This
furthersupportsthehypothesisofindependentstrainevolutionin
the two geographically distinct ecosystems found in Mexico and
Bangladesh.
Although cholera was said to be absent in Latin America for
nearlymorethanacenturybeforethe1991epidemicandalthough
thecausalagentV.choleraeETwasthoughttobeintroducedfrom
regions of cholera endemicity in Asia and Africa (20, 30), a few
sporadic cases reported in the Americas between 1973 and 1992,
including the U.S. Gulf Coast and one case reported in 1983 in
Cancun,Mexico(2,6),dosuggestalocalorigin.Endemiccholera
caused by a genetically divergent population of V. cholerae strains
inSouth(10,12,24)andCentralAmerica(2,19)stronglysuggests
thatV.choleraehaslongbeenadaptedtotheaquaticecosystemsof
these regions but remained unreported due to a lack of continu-
ous surveillance studies as cholera cases were sporadic and con-
sidered to be unremarkable. The epidemics in Peru and Mexico
seem to be independent events, as revealed by the pre-1991 pres-
ence of a V. cholerae CL biotype strain that was found to be asso-
ciated with endemic cholera, together with the ET and ET variant
strains in Mexico between 1991 and 1997 (2). It can be assumed
that the pre- and post-1991 CL biotype strains circulating in the
endemic cholera strains in Mexico may have contributed signifi-
cantly to the dynamics of V. cholerae populations in this region.
Even though the phenotypic and genetic differences observed be-
tween the CL biotype strains isolated in Mexico and Bangladesh
were perhaps subtle, the differences were consistent and thus can
serve as regional signatures, which supports the hypothesis of in-
dependent CL biotype strain evolution in Mexico.
ACKNOWLEDGMENTS
ThisresearchwassupportedinpartbyNIID,Tokyo,Japan,andNational
Institutes of Health grant 1RO1A13912901, under collaborative agree-
ments between the Johns Hopkins Bloomberg School of Public Health,
theUniversityofMaryland,CollegePark,MD,andtheInternationalCen-
terforDiarrhealDiseaseResearch,Bangladesh(ICDDR,B).ICDDR,Bac-
knowledges the following donors, which provide unrestricted support to
the Centre’s research efforts: Australian Agency for International Devel-
opment, Government of the People’s Republic of Bangladesh, Canadian
International Development Agency, Embassy of the Kingdom of the
Netherlands, Swedish International Development Cooperation Agency,
and the Department for International Development, United Kingdom.
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FIG 1 Dendrogram showing genomic fingerprints of patterns of V. cholerae O1 CL biotype strains isolated in Mexico (1983 to 1997) and Bangladesh (1962 to
1989).ThedendrogramwaspreparedbyDicesimilaritycoefficientandUPGMAclusteringmethodsbyusingPFGEimagesofNotI-digestedgenomicDNA;for
thistwoPFGEgelswererununderthesameconditions.Thescalebaratthetop(left)indicatesthecorrelationcoefficient(%).Thetwomajorclustersseparated
theMexicanV.choleraeCLbiotypestrains(clusterB)fromV.choleraeCLbiotypestrainsisolatedinBangladesh(clusterA),suggestingaregionalsignatureand
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Classical Biotype Strains Evolved Locally in Mexico
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