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Review

Whole-genome sequence comparisons reveal the evolution of Vibrio cholerae O1 Eun Jin Kim1,2, Chan Hee Lee1,2, G. Balakrish Nair3, and Dong Wook Kim1,2 1

Department of Pharmacy, School of Pharmacy, Hanyang University, Ansan 426-791, Korea Institute of Pharmacological Research, Hanyang University, Ansan 426-791, Korea 3 Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Trivandrum 695 014, Kerala, India 2

The analysis of the whole-genome sequences of Vibrio cholerae strains from previous and current cholera pandemics has demonstrated that genomic changes and alterations in phage CTX (particularly in the gene encoding the B subunit of cholera toxin) were major features in the evolution of V. cholerae. Recent studies have revealed the genetic mechanisms in these bacteria by which new variants of V. cholerae are generated from type-specific strains; these mechanisms suggest that certain strains are selected by environmental or human factors over time. By understanding the mechanisms and driving forces of historical and current changes in the V. cholerae population, it would be possible to predict the direction of such changes and the evolution of new variants; this has implications for the battle against cholera. Changes in the global population of Vibrio cholerae In the field of infectious diseases, V. cholerae, the causative agent of the acute diarrheal disease cholera, has twice transformed methodological paradigms of study. Modern epidemiology began, in 1854, with the investigation of the London cholera epidemic by Dr John Snow [1]. More recently, the notorious cholera outbreak in Haiti in 2010 is a fine example of the application of the ultimate methods of ‘molecular epidemiology’ to trace the source of an outbreak by comparing the whole-genome sequences of causative bacterial strains from different locations [2–4]. Since then, comparisons of whole-genome sequences have been used as the standard tool of molecular epidemiology, as shown by the example of the European Escherichia coli outbreak in 2011 [5]. Comparison of the whole-genome sequences of historically and regionally important strains of V. cholerae also provided significant information for understanding the evolution of V. cholerae [6,7]. In addition to genomic changes in the bacteria, alterations in the cholera toxin phage (CTX) also became evident in the evolution of Corresponding author: Kim, D.W. ([email protected]). Keywords: Vibrio cholerae; cholera; cholera toxin; phage CTX. 0966-842X/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2015.03.010

V. cholerae [7,8]. Phage CTX is a 6.9-kb, lysogenic filamentous phage that harbors the cholera toxin genes (ctxAB) [9]. Phage CTX integrates into the chromosomes of nontoxigenic strains of V. cholerae, converting them to toxigenic strains [10]. The characteristic changes in CTX can be categorized by two key features: (i) variant CTX phages can be generated by recombination among two major types of CTX phage (CTXcla and CTX-1) and RS1 (a satellite phage of CTX phage), in addition to point mutations in ctxB; and (ii) V. cholerae can replace CTX phages they harbor. These genetic mechanisms are simple and, in fact, should be occurring at present and could happen at any time. Thus, during population changes in V. cholerae, certain variants appear to be selected naturally over time. By reviewing various strains, the history of population changes, and the underlying mechanism of genetic changes in CTX phages, perhaps we can obtain clues about the causes of population changes, or the evolution of V. cholerae. History of population changes in V. cholerae Seven cholera pandemics have been recognized since the early 19th century [11]. Although a recent bacterial genome reconstruction study, examining a preserved intestinal specimen of a cholera victim, demonstrated that the strains of the second cholera pandemic were similar to the classical biotype strains, little information is available about the V. cholerae strains that caused the first five pandemics [12]. The sixth and seventh (current) cholera pandemics are attributed to the classical and El Tor biotype strains, respectively [13]. A modest population change in the El Tor biotype strains (from Wave 1 strains to Wave 2 and 3 strains) was reported recently [7,14], and we are also witnessing another population change as it is happening [15]. Changes in the populations of V. cholerae O1 are highly dynamic and unusual among bacterial pathogens. A significant hallmark of V. cholerae is that the previously prevalent strains disappear globally when new variant strains emerge [14]. We describe the V. cholerae strains and the key events in each population change (Box 1). Trends in Microbiology xx (2015) 1–11

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The sixth cholera pandemic and V. cholerae O1 classical biotype strains The sixth cholera pandemic was caused by V. cholerae O1 serogroup classical biotype strains. Classical biotype strains are sensitive to polymyxin B, show negative results in the Voges–Proskauer test (which detects acetoin in a bacterial culture to determine neutral fermentation levels), and show differential lytic phage sensitivity – constituting the chief differences compared with El Tor biotype strains [11]. The sixth pandemic lasted from 1899 to 1923, followed by an interpandemic period of 40 years before the onset of the seventh pandemic in 1961 (Figure 1).

Classical biotype strains contain a biotype-specific CTX phage, CTXcla, which contains ctxB1 (Boxes 1 and 2). The typical CTX phage array in classical biotype strains comprises an array of toxin-linked cryptic (TLC) and a truncated CTXcla, followed by a CTXcla on chromosome 1 and a solitary CTXcla on chromosome 2, as shown in a representative strain, O395 (see Table I in Box 1). Nevertheless, various CTXcla phage arrays are also documented [16]. Classical biotype strains were prevalent until the 1960s, before the seventh pandemic. After the emergence of El Tor biotype strains in 1961, classical biotype strains declined in the next 10 years, disappearing by the 1980s; they are

Box 1. ctxB genotypes Representative strains that are discussed in this article are listed in Table I. According to the order of description in the literature, a numerical nomenclature system was suggested for various ctxB genotypes [28]. Twelve ctxB genotypes have been reported so far, based on SNPs in various Vibrio cholerae strains and CTX phages (Table II). ctxB1 is from V. cholerae O1 classical strains, such as O395 and 569B; however, it is also found in CTXUS Gulf, Wave 2 strains, early Wave 3 strains, and several O139 strains [6,48,64]. ctxB3 is found in Wave 1 El Tor strains, and ctxB7 is found in current Wave 3 strains, such as the 2010 Haiti cholera outbreak strain [2]. ctxB7 was first described in strains that caused cholera outbreaks in 2007 and 2006 in Orissa and Kolkata, India [25,65]. From these two reports and other retrospective studies, current Wave 3 strains containing ctxB7 were shown to have first emerged in 2006 in India [66]. Differences in key amino acids between ctxB1, ctxB3, and ctxB7 occur at the 20th, 39th, and 68th positions. ctxB2 was identified in El Tor strains isolated in Australia [28]. ctxB3 is found in Wave 1 El Tor strains. ctxB genotypes 4, 5, and 6 were

identified in O139 strains between 1999 and 2005 in Bangladesh [47]. ctxB8 and ctxB9 were reported earlier in the O27 V. cholerae strain in 2002 and in an O37 serogroup strain in 1995 [67,68]. Although ctxB8 and ctxB9 are not originally from the pandemic O1 or O139 V. cholera strains, they could be the reservoir of ctxB. Therefore, they should be included in the list of various ctxB genotypes. V. cholerae O1 strains from Zambia, collected between 1996 and 2004, were found to contain two new genotypes, ctxB10 and ctxB11 [69]. Three more novel ctxB genotypes were reported in a retrospective study in China in 2013 [70]. ctxB3b was identified in an El Tor strain collected in 1964 in China; it contains a repeat of 11 amino acids, although the rest of the sequence is the same as that in ctxB3. Two El Tor strains, collected in China in 1965 and 1977, and four strains collected in 1982, were reported to contain ctxB8; however, this genotype should be ctxB12. Three other environmental strains were reported to harbor ctxB9, but this ctxB contains the same SNPs as ctxB8 of the V. cholerae serogroup O27 strain (above).

Table I. Representative strains of Vibrio cholerae O1 described in this review Origin Sixth pandemic classical 1965, O395 US Gulf Coast strain 1986, IB4755 (2469-86) Australian strain 1986, BX330286 Seventh pandemic El Tor Wave 1 strain 1975, N16961 Wave 2 strains 2004, B33 1991, MG116926 2000, E1781 1994, MJ1236 1991, V212-1 Early Wave 3 strains 2002, CIRS101 2007, IB4122 (01.07.VP) 2004, IB4322 2007, IB4563 (CTX-4) 2003, IB4247 (CTX-5) 2007, IB4540 (CTX-6) Current Wave 3 strains 2006, IB4642 2010, Haiti strain 2010, Nepal strain a

NA, information not available.

2

Chromosome 1

Chromosome 2

Genome sequence information

Refs

India

TLC:TruncCTXcla:CTX cla

CTX cla

CP000626/CP000627

[6]

USA

(?)-CTXUS

None

ERS013193

[7,64]

Australia

TLC:CTXAus:CTXAus:RS1 env

None

ACIA00000000

[6,71]

Bangladesh

TLC:CTX-1:RS1

None

AE003852/AE003853

[6]

Mozambique Bangladesh Bangladesh Bangladesh India

None TLC:RS1:RS1 RS1:RS1 None TLC:RS1:CTX-1:RS1

CTX-2:CTX-2 CTX-2:CTX-2 CTX-2:CTX-2 CTX-2:CTX-2 CTX-2:CTX-2

ACHZ00000000 ERS013256 ERS016137 CP001485/CP001486 ERS013132

[6] [7,29] [7,29] [6,29] [24]

Bangladesh Vietnam India India India India

TLC:RS1:CTX-3 TLC:RS1:CTX-3 TLC:RS1:CTX-3 TLC:RS1:CTX-4 TLC:RS1:CTX-5 TLC:RS1:CTX-6

None None None None None None

ACVW00000000 ERS013264 ERS013254 NA a NA NA

[6] [7,32] [7,29] [24] [24] [24]

India Haiti Nepal

TLC:RS1:CTX-3b TLC:RS1:CTX-6b TLC:RS1:CTX-6b

None None None

ERS013255 AELH00000000.1 SRA039806.1

[7] [2] [3]

Gulf

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Table II. Various ctxB genotypes and related CTX and Vibrio cholerae strains ctxB Nucleotide positions and genotype 58 (20) 72 (24) 83 (28) C A A ctxB1, (His) (Gln) (Asp) ctxB cla

ctxB3, ctxBEl Tor

C (His)

A (Gln)

A (Asp)

amino acid positions a

CTX

V. cholerae strains

101 (34) 106 (36) 115 (39) 138 (46) 165 (55) 203 (68) A A C T A C CTX cla (His) (Thr) (His) (Phe) (Lys) (Thr)

A (His)

A (Thr)

T (Tyr)

T (Phe)

A (Lys)

T (Ile)

Sixth cholera pandemic strains (classical biotype) US Gulf Coast CTXUS Gulf strains Wave 2 strains of CTX-2 seventh cholera pandemic CTX-3, 4, 5, 6 Early Wave 3 strains of seventh cholera pandemic O139 strains in India CTX O139 CTX-1 Wave 1 strains of seventh cholera pandemic O139 CTXO139 (CTXCalcutta) CTX-3b Current Wave 3 strains of seventh and CTX-6b cholera pandemic Australia, El Tor CTX Aus

Refs [6]

[6] [29]

[24]

[48] [27]

[46]

ctxB7

A (Asn)

A (Gln)

A (Asp)

A (His)

A (Thr)

C (His)

T (Phe)

A (Lys)

C (Thr)

ctxB2

C (His) C (His)

A (Gln) A (Gln)

A (Asp) A (Asp)

A (His) A (His)

A (Thr) A (Thr)

C (His) T (Tyr)

G (Leu) T (Phe)

A (Lys) A (Lys)

C (Thr) T (Ile)

C (His) C (His) C (His) C (His)

A (Gln) A (Gln) A (Gln) C (His)

A (Asp) C (Ala) A (Asp) C (Ala)

A (His) A (His) C (Pro) A (His)

A (Thr) A (Thr) A (Thr) A (Thr)

T (Tyr) C (His) T (Tyr) C (His)

T (Phe) T (Phe) T (Phe) T (Phe)

A (Lys) A (Lys) A (Lys) A (Lys)

C (Thr) C (Thr) C (Thr) C (Thr)

NA

O1, China, 11 amino [70] acids repeat (LAGKREMAIIT, position 52-62) O139 [47]

NA

O139

[47]

NA

O139

[47]

NA

C (His) C (His) C (His) C (His)

A (Gln) A (Gln) A (Gln) A (Gln)

A (Asp) A (Asp) A (Asp) A (Asp)

A (His) C (Pro) C (Pro) A (His)

A (Thr) A (Thr) A (Thr) G (Ala)

C (His) T (Tyr) C (His) T (Tyr)

G (Leu) T (Phe) T (Phe) G (Leu)

C (Asn) A (Lys) A (Lys) C (Asn)

C (Thr) T (Ile) C (Thr) C (Thr)

NA

O27, and O1, China, [67,70] previously described as ctxB9 O37 [68]

NA

O1, Zambia

[57]

NA

O1, Zambia

[57]

NA

O1, China, [70] previously described as ctxB8

ctxB3b

ctxB4 ctxB5 ctxB6 ctxB8

ctxB9 ctxB10 ctxB11 ctxB12

NA b

[24]

[6]

a

Key positions of ctxB1, ctxB3, and ctxB7 are shown in bold.

b

NA, information not available.

now believed to be extinct [17]. A recrudescence of the classical biotype strains was seen from 1982 to the 1990s in Bangladesh, after 10 years in which no clinical isolates of classical biotype strains had been reported in this area [18,19]. These strains were shown to be genetically related to the sixth pandemic classical biotype strains; however, they did not spread beyond Bangladesh [19]. There have been several pre-seventh pandemic strains that predated the seventh pandemic – such as Indonesian strains, Australian strains, and US Gulf Coast strains; these strains were collected during the interpandemic period and the early seventh pandemic period (Figure 1). Based on their genomic analysis, these strains are considered to lie between the classical and El Tor strains and are unique to the local population [6,7,20].

The seventh cholera pandemic and V. cholerae O1 El Tor biotype strains The first documented major V. cholerae population change observed was the shift from classical biotype strains to El Tor biotype strains, as observed in the 1960s, with the emergence of El Tor biotype strains in 1961 as causative agents of sporadic outbreaks in Sulawesi in Indonesia [21]. El Tor biotype strains spread globally from Asia to the African and European continents in the 1970s and finally reached South America in the 1990s [11,22]. The current cholera pandemic, due to El Tor strains, is considered to be the seventh pandemic. El Tor biotype strains differ from classical biotype strains by 20 000 SNPs and the acquisition of a set of genomic islands, such as Vibrio seventh pandemic (VSP) -1 and VSP-2 [6,23]. 3

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6th pandemic 1899–1923

Bacterial strains

7th pandemic 1961 to present

1920

1930

1940

1950

1960

1970

1980

1990

2000

2010

ctxB1

Classical strains with CTXcla

Classical

Toxin

ctxB1, ctxB2

Pre-seventh El Tor Wave 1

ctxB3

Wave 1 El Tor with CTX-1

ctxB3, ctxB1, various

O139 El Tor Wave 2 and early Wave 3 El Tor current Wave 3

Atypical El Tor with CTX-2 or CTX-3 ∼ CTX6

ctxB1

Atypical El Tor with CTX-3b or CTX-6b

ctxB7 TRENDS in Microbiology

Figure 1. History of changes in populations of Vibrio cholerae from the end of the sixth cholera pandemic. The sixth pandemic by classical biotype strains lasted from 1899 to 1923, whereas the seventh pandemic began in 1961 and continues. During the interpandemic period, from 1924 to 1961, classical biotype strains and various types of pre-seventh pandemic strains (US Gulf Coast strains and Australian strains) were isolated. Wave 1 strains of the seventh pandemic were prevalent between 1961 and the early 1990s, and classical biotype strains have disappeared. Surprisingly, classical biotype strains, which are genetically related to the sixth pandemic classical strains, reappeared and coexisted with El Tor biotype strains between 1982 and 1993 in Bangladesh [19]. However, the emergence and persistence of these classical biotype strains were limited to Bangladesh. O139 strains emerged in 1992, and Wave 2 and early Wave 3 strains emerged at the same time. Globally, Wave 1 strains were completely replaced by Wave 2 and Wave 3 strains by the early 2000s. Whereas Wave 2 strains have waned since 2000, Wave 3 strains have altered their ctxB from ctxB1 to ctxB7. O139 strains survived until recently, following their emergence in 1990; however, the ctxB gene of O139 strains has also changed from ctxB3 to ctxB1 or to other types.

Box 2. Various CTX phages CTX phages are categorized primarily by rstR and ctxB genotype and further subdivided by SNPs throughout the genome (Tables I and II). Whereas rstREl Tor and rstRcla are discussed in this article, rstRCalcutta identified in CTXO139 and rstRenv identified in environmental strains have also been reported in Vibrio cholerae strains [72]. CTXcla contains rstRcla and ctxB1, and the rest of the phage genome differs from that in the CTX phages of El Tor strains by several SNPs in each gene, as shown in Table I [6]. CTXUS Gulf is found in strains unique to the US Gulf Coast and is similar to CTXcla since it contains rstRcla and ctxB1. However, the rest of the genome differs from CTXcla and CTX phages of El Tor strains; therefore, CTXUS Gulf is unique to US Gulf Coast strains [64]. Not much information is available for CTXAUS, CTX phage in the pre-seventh pandemic strain, or Australian strains, except that rstRcla and ctxB2 were identified in this CTX phage.

Wave 1 strains of the seventh pandemic contain CTX-1 that harbors rstREl Tor and ctxB3 on chromosome 1. Wave 2 strains primarily contain a tandem repeat of CTX-2 on chromosome 2, whereas some members of Wave 2 strains contain CTX-1 and/or RS1 on chromosome 1. CTX-2 is also similar to CTXcla in that it contains rstRcla and ctxB1; therefore, it has been called CTXcla in some reports [31]. However, the rest of the genome of CTX-2 is more like that of CTX-1, indicating that it is a mosaic of CTXcla and CTX-1. CTX-3, -4, -5, and -6 have been found in early Wave 3 strains, and they contain rstREl Tor and ctxB1. CTX-3b and CTX-6b have been identified among current Wave 3 strains, and they contain rstREl Tor and ctxB7. Genomic analysis of early and current Wave 3 strains suggests that current Wave 3 strains were generated by a point mutation at amino acid 20 of ctxB1 in early Wave 3 strains [7].

Table I. Characteristics of various CTX phages

CTX cla CTXUS

Gulf

CTX Aus CTX-1, CTXEl CTX-2 CTX-3 CTX-3b CTX-4 CTX-5 CTX-6 CTX-6b 4

Tor

Strains

rstR type

Other regions of CTX phage

ctxB type

O395 IB4755 (A217, or 2469-86) BX330286 N16961 B33 IB4122 (01.07VP) IB4642 IB4563 IB4247 IB4540 IB5230

Classical Classical

Classical US Gulf Coast CTX

Classical El Tor Classical El Tor El El El El El

Tor Tor Tor Tor Tor

Refs

ctxB1 ctxB1

CTX phage sequence information CP000626 KJ619459

No information available El Tor El Tor, variations in rstA and rstB (Table II) El Tor, variations in rstA and rstB (Table II)

ctxB2 ctxB3 ctxB1 ctxB1

ACIA00000000 AE003852 GQ485644 GQ485652

[6,71] [64] [6,64] [32,64]

El El El El El

ctxB7 ctxB1 ctxB1 ctxB1 ctxB7

GQ485651 KJ540260 KJ540261 KJ540263 KJ540264

[64] [24] [24] [24] [2,24]

Tor, Tor, Tor, Tor, Tor,

variations variations variations variations variations

in in in in in

rstA rstA rstA rstA rstA

and and and and and

rstB rstB rstB rstB rstB

(Table (Table (Table (Table (Table

II) II) II) II) II)

[64] [64]

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Table II. Genetic variations of rstA and rstB of CTX prophages in seventh pandemic El Tor strainsa CTX type

Strain name

Origin

RS1

N16961

CTX-1

rstA 27

162

183

258

927

933

942

rstB 74-76

87

93

105

189

360

364

366-368

371-372

379

381

Sequence information

Bangladesh, 1975













C

T

T

D

T

C

A

G

G

A

D

D

A

D

AE003852

N16961

Bangladesh, 1975

C

C

C

G

T

C

G

GTA

A

T

G

A

A

C

ACC

TT

T

A

AE003852

CTX-2

B33

Mozambique, 2004

T

T

A

C










D

T

C




























GQ485644

CTX-3

IB4122

Vietnam, 2007













C

T

T







































GQ485652

CTX-3b CTX-4 CTX-5 CTX-6 CTX-6b

IB4642

India, 2006













C

T

T







































GQ485651

IB4563

India, 2007













C

T

T

D


































KJ540260

IB4247

India, 2003













C

T

T

D

T

C

A

























KJ540261

IB4540

India, 2007













C

T

T

D

T

C

A

G






















KJ540263

IB5230

Haiti, 2010













C

T

T

D

T

C

A

G






















KJ540264

a

Dots indicate sequences identical to those in CTX-1; D indicates deletion of nucleotide(s).

Further, the two biotype strains harbor disparate CTX prophages in their genome [14,24]. Classical biotype strains contain CTXcla, and El Tor has CTX-1. These two phages have similar genomic structure and sequences (Box 1), differing in the phage type-specific gene rstR and many SNPs throughout the genome [25]. Because rstR is phagetype-specific, it has been used to distinguish the two types of phage. Whereas other genes differ by several SNPs, ctxA (the gene encoding the active subunit of cholera toxin) is identical in the two phages. ctxB differs by two nucleotides (two amino acids) at positions 106 (39) and 203 (68) [26]. El Tor strains primarily contain a satellite phage, RS1, at the CTX phage integration site of chromosome 1 and thus contain various arrays of RS1 and CTX, whereas no elements are integrated on chromosome 2 [27]. The seventh cholera pandemic remains ongoing; it has lasted for more than 50 years, which is much longer than earlier cholera pandemics. However, the El Tor strains in the seventh pandemic have undergone several changes that are subtle but which are considered to be significant with regard to the worldwide spread of the disease [28]. Based on their genome analysis and the CTX phage, the seventh pandemic strains have been categorized into three waves of global dissemination [7]. Wave 1 strains. Wave 1 El Tor strains were previously termed prototype El Tor strains compared to the atypical Wave 2 and Wave 3 El Tor strains [14]. Wave 1 strains are defined as El Tor biotype strains that produce biotypespecific cholera toxin, ctxB3 (Box 1) [28,29]. Wave 1 strains were prevalent in the Indian subcontinent until the early 1990s, when they were replaced by Wave 2 and Wave 3 strains (Figure 1) [30]. In 2000, a representative Wave 1 strain, N16961, was the first V. cholerae isolate to be analyzed by whole-genome sequencing [27]. Whereas many CTX phages within Wave 2 and Wave 3 strains have been analyzed by phage genome sequencing (Box 2), few phages in Wave 1 strains have been examined in detail. Studies of additional Wave 1 strains and their CTX phages might reveal diversity that could be comparable with that of CTX phages in Wave 2 and Wave 3 strains. Wave 2 strains. Wave 2 strains are defined as El Tor biotype strains that contain a tandem repeat of CTX-2, which harbors ctxB1, on chromosome 2 (Box 2) and various

arrays, such as no element, RS1:RS1, TLC:RS1:RS1, and TLC:RS1:CTX-1:RS1, on chromosome 1 [25,29,31]. Wave 2 strains were first isolated from the Indian subcontinent in 1991 and have since spread to East Asian countries (Thailand, China, and Vietnam) and African countries (Mozambique and Zambia) [32–35]. Certain Wave 2 strains contain two types of CTX phage in a single bacterial cell, in which inter-phage recombination can occur. Wave 2 strains are grouped in a single subclade within all seventh pandemic strains, implying that they originated from a single ancestral strain. Wave 2 strains constitute an independent lineage between Wave 1 and Wave 3 strains and were critical in the generation of Wave 3 strains [24]. Currently, Wave 2 strains are not isolated widely, except in a few regions such as Papua New Guinea [36]. Wave 3 strains. Wave 3 strains are El Tor biotype strains that harbor any of CTX-3, 4, 5, 6, CTX-3b, or CTX-6b on chromosome 1 and lack elements on chromosome 2 [7,24,25]. They usually contain TLC:RS1:CTX on chromosome 1, similar to Wave 1 strains, except that Wave 3 strains possess ctxB1 or ctxB7 instead of ctxB3 [15]. After the emergence of Wave 2 and Wave 3 strains in 1991 on the Indian subcontinent, Wave 1 strains began to decline and were considered to have been replaced completely by Wave 2 and Wave 3 strains in the mid-1990s [37]. This same phenomenon spread to other countries and continents in the mid-1990s, and the global population change from Wave 1 to Wave 2 and Wave 3 strains was completed by 2000 [38–41]. Wave 3 strains are subcategorized into three or more subclades by genomic analysis, implying that they originated from multiple ancestors [7,8]. Wave 3 strains can also be subclassified by ctxB genotype: Wave 3 strains that were collected between 1991 and 2010 contained ctxB1 (hereafter called ‘early Wave 3 strains’), and the current Wave 3 strains that first appeared in 2006 in India harbor ctxB7 [42]. Wave 2 and early Wave 3 strains have now been nearly entirely replaced by current Wave 3 strains in India, and current Wave 3 strains continue to spread globally, as seen in the 2010 Haiti cholera epidemic [43,44]. O139 serogroup. Within El Tor strains, a new serogroup, O139, emerged in 1992. Serogroup O139 strains have caused outbreaks, primarily in Asian countries [45]. 5

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Review By genomic analysis, O139 strains were shown to be derived from El Tor strains by obtaining a new O-antigen biosynthesis gene cluster [11]. O139 strains used to contain CTX-1, and subsequently some strains acquired CTXO139 – which contains the genes rstRCalcutta and ctxB3 [46]. However, recent isolates of O139 strains contain ctxB1, ctxB4, ctxB5, or ctxB6, indicating that O139 strains were also involved in establishing the gene pool of V. cholerae strains for genetic change (Box 1) [47,48]. Comparative genomics of V. cholerae Phylogenetic analyses of V. cholerae strains of the sixth and seventh cholera pandemics have shown disparities between genomes [2,6,7]. In addition to the accumulation of SNPs throughout the genome, dynamic lateral genetransfer events contributed to the evolution of V. cholerae. Whereas the acquisition of VSP-1 and VSP-2 islands is considered to be a key difference between classical and El Tor biotype strains, several gene-transfer events have also been observed among El Tor strains [6]. Acquisition of SXT (a 100-kb integrative mobile genetic element that carries resistance genes to sulfamethoxazole and trimethoprim – and to other antibiotics such as chloramphenicol and tetracycline) might have been a factor that influenced the population shift from Wave 1 to Wave 2/3 strains [7]. However, slight variations in SXT exist between strains [49,50]. GI-12–14 are specific among Wave 2 strains, and a truncated version of VSP-2 has been identified among Wave 3 strains [49]. In addition to the genomic changes, a substitution of CTX phage occurred in population-shift events. By whole-genome sequencing analysis of Wave 1, 2, and 3 strains within the seventh pandemic, we learned that the seventh pandemic strains originated from a single ancestor [7]. Gradual genome changes (3.3 nucleotides/year) have brought about the accumulation of 250 SNPs throughout the genome of El Tor strains during the last 50 years [7], suggesting that V. cholerae strains have developed a replacement mechanism for CTX phage to generate Wave 2 and 3 strains from Wave 1 strains. Recent studies have examined how CTX phage replacement occurs [14,24,45,51]. Genetic mechanisms leading to the generation of Wave 2 and Wave 3 strains An earlier report proposed an evolutionary pathway to explain the emergence of toxigenic classical and El Tor biotype strains by independent genomic evolution and the acquisition of biotype-specific CTX phages [52]. Yet, the infection of a classical biotype strain by CTX-1 phage demonstrated that CTX phage integration is not a biotype-specific event and that genetic exchange between two phages is possible in host bacteria to generate mosaic CTX phages in Wave 2 and Wave 3 strains [9]. Studies that have proposed several genetic mechanisms for the development of Wave 2 and Wave 3 strains are described below. Excision of CTX, RS1, and TLC Wave 1 El Tor strains are believed to have been generated from nontoxigenic strains by integration of the TLC element, followed by integration of CTX-1 and the RS1 element [53]. The CTX phage and RS1 integrate into the 6

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chromosomes of V. cholerae via the dif sequence (deletioninduced filamentation, a chromosomal deconcatenation site) and the Xer recombination system (Figure 2) [30]. The replication of phage CTX from prophage status does not require the excision of the phage genome [54]; thus, the excision of phage CTX from toxigenic strains has not been well documented. However, because phage CTX and the RS1 element share common genes (rstREl Tor, rstA, and rstB), excision of RS1 or CTX-1 can be mediated by homologous recombination. Two recent studies demonstrated the excision of CTX-1 and/or RS1 from chromosome 1 of Wave 1 El Tor strains by a recA-dependent mechanism in vivo and in vitro [24,55] – this excision could even be promoted by rstC [55]. Moreover, the entire array, as well as TLC, can be removed, perhaps via the 18-nucleotide dif sequence [30]. The elimination of RS1, CTX, and TLC is critical in the generation of Wave 2 and Wave 3 strains, as shown below, and might be a significant trait for evolution of new variants in the future. Generation of Wave 2 strains Wave 2 strains are clustered within the seventh pandemic strains, suggesting that they originated from a single ancestral strain [7]. This hypothetical strain was perhaps a Wave 1 strain that obtained CTX-2 on chromosome 2. The CTX-1, RS1, and TLC have been removed piecemeal from the hypothetical strain by the mechanism described above, resulting in the generation of various Wave 2 strains [49,56,57]. Such events were demonstrated in strain V212-1, which contained the TLC:RS1:CTX-1:RS1 array on chromosome 1 and CTX-2:CTX-2 on chromosome 2 (Figure 3A) [24]. The manner in which CTX-2 and CTXcla were generated and disseminated remains undetermined. Replicative CTX-2 or CTXcla has not been reported [58]. Classical strains are unable to produce CTXcla phage because they do not possess a tandem repeat of CTX phage or a CTX:RS1 array. Wave 2 strains harbored a tandem repeat of CTX-2 on chromosome 2 but failed to produce CTXcla phage [31]. This is perhaps due to limitations in the experimental setup for the in vitro transduction of CTXcla and CTX-2 [due to the phage immunity to transduce CTXcla or CTX-2 into classical strains, or because El Tor strains do not express the CTX phage receptor TCP (toxin co-regulated pilus) in laboratory settings] [9]. Therefore, the presence of the replicative-form pCTXcla or CTXcla phages must be demonstrated to determine the entire evolutionary pathway of V. cholerae. Generation of various CTX phages of Wave 3 strains CTX-3CTX-6 in Wave 3 strains were shown to be generated in an ancestor strain of Wave 2 strains that contained CTX-1 on chromosome 1 and CTX-2 on chromosome 2 [24]. These CTX phage genomes could have arisen by consecutive recombination events (Figure 3B) – first, recombination between CTX-1 and CTX-2 (through a region that flanked ctxB) could have generated a new CTX prophage genome on chromosome 1, then, intrastrand recombination between an RS1 and the newly generated CTX prophage could have produced the CTX-3, 4, 5, or 6, depending on the recombination position. RS1 can provide

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Figure 2. El Tor strains can replace the CTX phage in chromosome 1. The cycle of integration and removal of phages CTX and RS1 in El Tor strains. (1) The integration of the TLC (toxin-linked cryptic) element provides a functional dif sequence for the integration of CTX phage, or an RS1 element, into nontoxigenic Vibrio cholerae strains. (2) RS1 and/or CTX-1 is integrated next to TLC. (3) Additional RS1 and/or CTX is integrated to generate various arrays. (4) RS1 and/or CTX is removed by homologous recombination. Depending on the array, a solitary CTX or RS1 can remain. (5A) A new CTX phage can integrate next to RS1, as shown in the generation of Wave 3 strains, or (5B) RS1 is further removed, in addition to TLC, which could be used for the integration of new elements, allowing the integration cycle to begin again. This process might be also occuring in chromosome 2 and in classical biotype strains.

diversity of CTX phages, as well as promoting the excision of phage CTX [55]. The SNPs of rstA and rstB of CTX-1, 3, 4, 5, and 6 indicate that CTX-3CTX-6 are mosaics of CTX-1 and RS1 (see Table II in Box 2). Generation of Wave 3 strains Wave 1 strains contain various arrays of CTX-1 and RS1 on chromosome 1. When the array ends with RS1, the excision of CTX-1/RS1 results in a strain that contains TLC:RS1. CTX-3CTX-6, generated as above, can be transduced into this strain (Figure 4). This replacement of phage CTX was demonstrated in N16961, which harbors TLC:CTX-1:RS1. The CTX-1 was eliminated by recombination between CTX-1 and RS1, then the CTX-3 or CTX-6 could be transferred to this strain [24]. As discussed, strains of V. cholerae can replace their ‘old’ CTX phage with ‘new’ CTX phage, which can explain why there are several subclades among Wave 3 strains. Current Wave 3 strains that harbor CTX-3b or CTX6b could have been generated by a point mutation in

strains that contained CTX-3, or CTX-6, respectively, because they lie at the tip of each subclade of Wave 3 strains in a genome-based phylogenetic tree [7]. If this model holds true, this genetic change occurred in at least two subclades simultaneously, indicating that this alteration was inevitable in the evolution of V. cholerae. Wave 2 strains and early Wave 3 strains appeared simultaneously in the early 1990s on the Indian subcontinent, and the genetic mechanism above indicates that Wave 3 strains could have been generated immediately from intermediary strains. Wave 2 and the early Wave 3 strains somehow overlap with respect to the time in which they coexisted and the cholera toxin they produce. However, Wave 2 strains have vanished, whereas Wave 3 strains evolved to the current Wave 3 strains. Thus, population shifts of V. cholerae strains can be envisioned, according to the changes in ctxB – i.e., from (i) ctxB1 of sixth pandemic strains to (ii) ctxB3 of Wave 1 El Tor strains in the seventh pandemic strains to (iii) ctxB1 again in Wave 7

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Figure 3. Wave 2 strains and CTX phages of Wave 3 strains are generated from an intermediary strain between Wave 1 and Wave 2 strains. (A) An intermediary strain (for example, V212-1) contains RS1:CTX-1:RS1 on chromosome 1 and a tandem repeat of CTX-2 on chromosome 2 [24]. RS1 and CTX-1 are removed from chromosome 1, allowing the generation of various Wave 2 strains. (B) A similar intermediary strain can act as the source of CTX phages for Wave 3 strains. Here, we show an example of a CTX-3 phage generated in V212-1. (1) A double-crossover recombination event between CTX-1 and CTX-2 through the region flanking ctxB can generate CTX-1* (CTX-1 containing ctxB1) on chromosome 1. (2) Additional recombination between RS1 and CTX-1* via rstA or rstB generates CTX-3CTX-6, depending on the recombination position. (3) The new CTX phage can be transduced to other recipient strains.

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Various Wave 2 strains by eliminaon of CTX-1, RS1 and TLC from chromosome 1

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Figure 4. Generation of Wave 2 and Wave 3 strains from Wave 1 El Tor strains. Wave 1 strains diverged into many subclades during the early seventh pandemic period. (A) One of the subclades was infected by CTX-2, becoming an intermediary strain between Wave 1 and Wave 2 strains. Stepwise removal of CTX-1, RS1, and, eventually, the TLC (toxin-linked cryptic) element from chromosome 1 generated various Wave 2 strains. CTX-3CTX-6 were also generated from the intermediary strain and transduced to Wave 3 strains. (B) Some subclades of Wave 1 strains lost CTX-1 and thus contain only RS1. These strains were infected by CTX-3CTX-6, generated in (A), and became early Wave 3 strains. Some early Wave 3 strains turned into current Wave 3 strains through a point mutation in ctxB. Block arrows indicate changes in bacterial strains, and arrows denote changes in CTX phage and the RS1 element.

8

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Review 2 and early Wave 3 strains, and finally to (iv) ctxB7 of current Wave 3 strains (Figure 1). Why is this population change taking place? We are beginning to understand the molecular mechanisms underlying the population shifts of V. cholerae from the elegant analysis and comparisons of the complete genome sequences. However, we are still unable to understand why such changes occur or what drives them. The fact that these shifts in V. cholerae populations keep occurring in cholera endemic areas seems to suggest that existing human immunity in endemic areas may be driving these changes, although we cannot exclude the possibility that it could be the environmental factors. A recent study suggested that V. cholerae exerted strong selective pressure on the affected human population in cholera-endemic area [59]. However, this long-term host– pathogen interaction cannot be unidirectional, and we believe that humans also influenced the evolution of V. cholerae. Studies have suggested that El Tor strains have environmental advantages over classical strains, but the reasons for the population change from classical biotype to El Tor biotype strains are still unknown [60,61]. The evolutionary pathway of Wave 2 and Wave 3 strains from Wave 1 strains, as suggested in recent studies, explains the genetic mechanisms of generation of new variants. However, the genetic mechanisms do not sufficiently address how and why bacterial population changes are occurring and do not identify the driving forces behind such changes. Determining the evolution of pathogens can be approached by examining (i) the genetic mechanisms within the pathogens, (ii) environmental factors that favor new variants, and (iii) host factors. Little information about V. cholerae strains in their natural habitat is available because it is an estuarine organism and because the V. cholerae strains that have been described so far are primarily clinical isolates. Two extreme scenarios of population changes of V. cholerae in the ocean and among humans could be a starting point in identifying the evolution of V. cholerae. In the first case, the entire population in the ocean changes – and, consequently, so do the clinical isolates. The pattern of population change is simple in this scenario. During the sixth cholera pandemic, the V. cholerae strains in the ocean were classical biotype strains, as were the clinical isolates. From 1961 to the early 1990s, the V. cholerae population in the ocean changed entirely to Wave 1 El Tor biotype strains. Similar population changes occurred during 1990–2006 and 2006–2014 when the population shifted from Wave 1 El Tor strains to Wave 2 and early Wave 3 strains (containing ctxB1) and then eventually to current Wave 3 strains that contained ctxB7, respectively. In the other scenario, the pathogenic O1 strains in the ocean do not change dramatically, but the clinical isolates are modulating, perhaps due to environmental or human factors over time. There could be other unidentified or unselected variants. Here, the V. cholerae population has remained steady, although certain members might

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be enriched locally or temporarily when an outbreak or epidemic is ongoing. The actual situation might be a compromise between these two scenarios. Recent studies suggest that earlier prevalent strains that were not collected among the clinical isolates continue to exist in nature. A single classical biotype isolate was gathered during a cholera outbreak in Iran in 2012 [62]. Also, several V. cholerae O1 prototype El Tor strains were harvested in 2011 and 2012 in Bangladesh, where prototype strains have not been recorded among clinical isolates for over 10 years [63]. These findings indicate that prototype El Tor strains, classical strains, and certain unidentified variants remain in a niche in the ocean, waiting for selective forces. The population shift from Wave 1 El Tor strains to Wave 2 and early Wave 3 strains occurred approximately 30 years after the emergence of Wave 1 strains, but the population change from Wave 2 and early Wave 3 strains to current Wave 3 strains that contain ctxB7 took roughly 20 years. Perhaps the population change is occurring more rapidly. Conclusions and future directions Various V. cholerae strains in the sixth and seventh pandemics, and the CTX phages that they harbor, are described in this review. Although several classical and Wave 1 El Tor strains are discussed, there are limited genomic data available on these strains compared with recent Wave 2 and Wave 3 strains. We assume that the genetic changes among Wave 2 and Wave 3 strains (gain and loss of genomic islands, accumulation of SNPs) also occurred in classical and Wave 1 El Tor strains. Nevertheless, current studies on genomics indicate that the replacement of phage CTX is part of the continuous evolution of V. cholerae. The causes of population shift from classical biotype of the sixth cholera pandemic to El Tor biotype strains of the seventh cholera pandemic are still not clear owing to the massive changes in the genome in addition to the replacement of CTX. Although subtle genomic changes could contribute to the fitness of new strains, examining the changes of CTX, or ctxB, might be a more reasonable starting point in determining the root cause of the population changes within the seventh pandemic strains (Box 3). The results of this approach may also help us to reveal the causes of population changes from the sixth cholera pandemic to the seventh cholera pandemic. The cause of population changes in V. cholerae is important to

Box 3. Outstanding questions  The replicative form of CTXcla and CTX-2 has never been experimentally demonstrated; are these phages defective in replication? Or, would it be possible to set up an in vitro replication system for CTXcla and CTX-2?  Cholera toxins encoded by ctxB1 and ctxB7 differ by a single amino acid in a signal peptide; are they different in secretion? How does this difference cause the strain shift?  The population changes of Vibrio cholerae occur in 30 years; is this related to the generation time of human populations in cholera-endemic regions?  What would be the factors if the environment causes population shifts in V. cholerae? 9

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Review identify because it can guide our strategies to combat the disease successfully. Acknowledgments This work was supported by grant 2012R1A2A2A01009741 from the National Research Foundation (NRF) of Korea.

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