Literature DB >> 29374205

Vibrio cholerae embraces two major evolutionary traits as revealed by targeted gene sequencing.

Kazuhisa Okada1,2, Warawan Wongboot3, Siriporn Chantaroj4, Wirongrong Natakuathung3, Amonrattana Roobthaisong3, Watcharaporn Kamjumphol3, Fumito Maruyama5, Taichiro Takemura6, Ichiro Nakagawa5, Makoto Ohnishi7, Shigeyuki Hamada3,8.   

Abstract

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species.

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Year:  2018        PMID： 29374205      PMCID： PMC5785995          DOI： 10.1038/s41598-018-19995-7

Source DB:  PubMed          Journal:  Sci Rep        ISSN： 2045-2322            Impact factor:   4.379


Introduction

Vibrio cholerae is a gram-negative bacterial pathogen responsible for cholera, and several million cholera cases including 21,000–143,000 deaths occur worldwide each year[1]. Serological grouping of V. cholerae has identified up to 206 O-serogroups[2]. Epidemic/pandemic cholera is typically ascribed to serogroup O1; however, in 1992, a novel serogroup O139 V. cholerae caused outbreaks in Asian countries[3]. V. cholerae carries several virulence-related genes to provoke pathogenic processes in the infected hosts. The key virulence factors of serogroups O1 and O139 include cholera toxin (CT), which is responsible for profuse watery diarrhea, and a pilus colonization factor known as toxin-coregulated pilus (TCP). Although most non-O1/non-O139 or environmental isolates of V. cholerae do not produce CT and lack the cholera toxin genes, some strains possess heat-stable enterotoxin (Stn)[4], hemolysin (HlyA)[5,6], repeat in toxin (RTX)[7], Cholix toxin (ChxA)[8,9], hemagglutinin protease (HAP)[10], type 6 secretion system (T6SS)[11], or type III secretion system (TTSS)[12]. However, the pathogenic mechanisms of these isolates remain to be elucidated. High throughput sequencing facilitates the rapid and accurate identification of virulence factors of pathogenic bacteria, and can be used to identify the pathways of infectious disease transmission[13-15]. Although genomic technologies are rapidly evolving, their widespread implementation in clinical microbiology laboratories and for monitoring public health is limited owing to the need for effective semi-automated pipelines, standardized quality control and data interpretation, bioinformatics expertise, and infrastructure[16]. Relatedness and differences among V. cholerae isolates have been investigated by several molecular fingerprinting methods for a prolonged duration[17]. Pulsed field gel electrophoresis (PFGE) has been used frequently for typing of the O1 and O139 serogroups of V. cholerae[18,19]. Although PFGE is highly reproducible and its discriminatory power is sufficiently high, it is laborious, and is limited with regard to intra- and inter- laboratory comparison compared to sequence-based methods[17,20]. Multilocus sequence typing (MLST) overcomes the poor portability of traditional and older molecular typing approaches[20]. It is a technique whereby several internal control genes (loci) are sequenced, and relatedness among isolates is displayed as a dendrogram constructed using the matrix of pairwise differences between the allelic profiles. This approach provides high discriminatory power and is informative for the study of V. cholerae[21,22]. Single locus sequence typing (SLST) also has been widely used to determine the relationships in other organisms[23-25]. We found that V. cholerae O1 genomes possess either metY or hchA/luxR on the specific gene locus MS6_A0927 on a conserved syntenic region of the chromosome II[26]. The locus was evidence that a unique O1 strain MS6 isolated from a diarrheal patient was distinguished from pandemic O1 strains and was most closely related to US Gulf Coast strains. MS6 and US Gulf Coast strains carried the metY gene in the locus, whereas the seventh cholera pandemic O1 strains carried luxR-hchA genes. In this paper, we report the prevalence, distribution, and sequence diversity of alternative genes located in MS6_A0927 among a large population of diverse V. cholerae and discuss their future evolutionary aspects.

Results and Discussion

V. cholerae separates into two clusters based on the locus MS6_A0927

We investigated the distribution of the metY (M) gene and luxR-hchA (LH) genes among vibrios. First, BLAST searches were performed using M (1,269 bp) from V. cholerae strain MS6 and LH from V. cholerae strain O395 (1,600 bp) as query sequences against 186 genomes including 178 V. cholerae, 6 V. mimicus, 1 V. metecus, and 1 V. parilis obtained from the NCBI database (www.ncbi.nlm.nih.gov). All strains carried either M (n = 57) or LH (n = 128), except for strain 87395 that was revealed to carry both M and LH (Table S1). We then designed a multiplex PCR system for detection of M, LH, toxR, VC2346, tcpA, and ctxAB, and determined the sequences of M and LH in the locus MS6_A0927 of 153 strains of non-O1 V. cholerae and 2 strains of V. mimicus (Table 1). Eleven genotypes were obtained by the multiplex PCR assay, including toxR/M (53.5%), toxR/LH (30.3%), ctxAB/tcpA/toxR/VC2346/LH (3.2%), ctxAB/tcpA/toxR/M (2.6%), ctxAB/tcpA/toxR/LH (2.6%), toxR/VC2346/M (2.6%), tcpA/toxR/M (1.9%), tcpA/toxR/LH (1.3%), tcpA/toxR/VC2346/M (0.6%), tcpA/toxR/VC2346/LH (0.6%), and M (0.6%). Two major virulence genes, ctxAB and tcpA, were detected in 13 strains from 9 different serogroups (O8, O26, O37, O44, O48, O49, O75, O141, and O191). Moreover, the M gene was detected in 94 V. cholerae and 2 V. mimicus (62%, 96/155), whereas 59 V. cholerae (38%, 59/155) carried LH. PCR using the primers MS6_A0926F and MS6_A0928R prior to sequencing of M or LH amplified target regions with expected molecular sizes, 1.96 kb or 2.13 kb, in all test strains except for V. cholerae O35 N2_17 (Fig. 1). The DNA sequences of the PCR products were determined and the existence of M and LH was confirmed. Strain N2_17 carried both M and LH in the locus, although the multiplex PCR failed to detect its M gene. L and H were always detected together.
Table 1

Characteristics of non-O1 Vibrio cholerae and V. mimicus as revealed by multiplex-PCR assays and M/LH profiling.

SerogroupID #CategoryYearLocationMultiplex-PCRSubcladeSerogroupID #CategoryYearLocationMultiplex-PCRSubclade
ctxAB tcpA toxR VC2346 LH M ctxAB tcpA toxR VC2346 LH M
O2N1CUU−++−−+M64O75N2_31C1979India−−+−+−M61
O3N2CUU−−+−+−LH6O76N50UUU−−+−+−LH31
O5N3C1964Philippines−−+−−+M53O77N51UUU−−+−−+M47
O6N4C1962India−++−−+M30O78N52UUU−−+−+−LH12
O6N2_2CUJapan−−+−−+M68O79N53UUU−−+−−+M37
O8N2_3C1993Argentina−−+−−+M69O80N54UUU−++−+−LH37
O8N2_4C1962Philippine−−+−−+M58O81N55UUU−−+−−+M6
O8N2_5C1994Thailand+++−−+M50O82N56UUU−−+−+−LH4
O9N5C1968Philippines−−+−+−LH41O83N57UUU−−+−+−LH10
O10N6C1968India−−+−−+M70O84N2_32C1983India−−+−−+M26
O11N7C1962India−−+−−+M67O85N58UUU−−+−+−LH40
O12N8C1972India−−+−+−LH1O86N59UUU−−+−−+M6
O13N9C1962Philippines−−+−−+M35O87N60UUU−−+−−+M51
O14N2_6CUJapan−−+−+−LH1O88N61UUU−−+−−+M71
O14N2_7C1964India−−+−+−LH1O89N62UUU−−+−−+M41
O14N2_8C1993Argentina−−+−+−LH1O90N63UUU−−+−+−LH17
O14N2_9C1993Indonesia−−+−+−LH1O91N64UUU−−+−−+M63
O15N10C1979India−−+−−+M4O92N65UUU−−+−+−LH12
O16N11C1971India−−+−+−LH23O93N66UUU−−+−−+M42
O17N12C1968India−−+−−+M13O94N67UUU−−+−+−LH42
O18N13C1964India−−+−+−LH24O95N68UUU−−+−−+M29
O19N14C1968India−−+−+−LH43O96N69UUU−−+−−+M6
O20N2_10C1980USA−−+−−+M23O97N70UUU−−+−+−LH34
O20N2_11C1962India−−+−−+M23O98N2_34C1993Indonesia−−+−+−LH1
O21N15C1968India−−+−+−LH24O98N2_35C1976India−−+−−+M57
O23N16C1971India−−−−−+M18O99N71UUU−−+−−+M72
O24N17C1962Philippines−−+−−+M34O100N72UUU−−+−+−LH1
O25N18C1962India−−+−+−LH30O102N74UUU−−+−+−LH24
O26N2_12C1991Japan+++++−LH38O103N75UUU−−+−+−LH22
O26N2_14C1993Sri Lanka+++++−LH38O104N76UUU−−+−−+M45
O26N2_15C1994Brazil+++++−LH38O105N77UUU−++−−+M44
O26N2_16C1996Japan+++++−LH38O106N78UUU−−+−−+M43
O27N19C1962Philippines−−+−−+M65O107N79UUU−−+−+−LH4
O28N20C1962Philippines−−+−−+M46O108N80UUU−−+−+−LH4
O30N21 (VM)C1962Philippines−−+−−+M16O109N81UUU−−+−+−LH28
O110N82UUU−−+−+−LH36
O32N22 (VM)C1968India−−+−−+M75O111N83UUU−−+−+−LH4
O113N84UUU−−+−+−LH46
O34N23C1968India−−+−+−LH29O114N85UUU−−++−+M77
O35N24C1969India−−+−−+M37O115N86UUU−+++−+M6
O35N2_17C1994Korea−−+−++LH19&M5O116N87UUU−−+−−+M74
aO117N88UUU−−++−+M76
O36N25C1969Philippines−−+−−+M32O118N89UUU−−+−−+M15
O37N26C1969India+++−+−LH1O119N90UUU−−+−−+M51
O37N2_19E1996Korea+++−+−LH1O120N91UUU−−+−+−LH27
O39N2_20E1994Korea−−+−+−LH1O121N92UUU−−+−−+M54
O39N2_21C1968India−−+−+−LH33O122N93UUU−−+−+−LH28
O40N27C1972India−−+−−+M26O123N94UUU−−+−−+M30
O42N28C1973India−−+−−+M4O124N95UUU−−+−−+M26
O43N29C1973India−−+−+−LH20O125N96UUU−−+−−+M4
O44N2_22C1994Thailand+++++−LH32O126N97UUU−−+−−+M31
O44N2_23C1973India−−+−−+M66O127N98UUU−−+−+−LH12
O45N30C1973India−−+−−+M38O128N99UUU−−+−+−LH39
O46N31C1973India−−+−−+M18O129N100UUU−−+−−+M26
O47N32C1973India−−+−−+M4O130N101UUU−−+−−+M49
O48N33C1973India−−+−−+M58O131N102UUU−−+−−+M60
O48N2_24E1988Taiwan+++−+−LH18O132N103UUU−−+−−+M4
O49N2_25C1994Thailand+++−+−LH45O133N104UUU−−+−−+M4
O49N2_26C1974India−−+−−+M49O134N105UUU−−+−−+M6
O51N34C1973India−−+−−+M4O135N106UUU−−++−+M78
O52N35C1973India−−+−−+M79O136N107UUU−−+−+−LH44
O54N36C1974India−−+−−+M55O137N108UUU−−+−−+M36
O57N37C1976Denmark−−+−−+M27O139N2_38U1993Sri Lanka−−+−−+M28
O58N38C1974India−−+−+−LH47O139N2_39U1993Pakistan−−+−−+M28
O59N39C1974India−−+−−+M33O141N2_40C1993India+++−−+M14
O60N40C1975India−−+−+−LH25O141N2_41E1995Brazil−−+−−+M5
O61N41C1974India−−+−−+M4O142N109UUU−−+−−+M41
O64N42C1975India−−+−−+M30O144N2_42E1994Denmark−−+−−+M46
O65N43C1975India−++−+−LH2O144N2_43C1993India−−+−−+M59
O66N2_27E1993Argentina−−+−−+M56O145N111UUU−−+−−+M62
O66N2_28C1975India−−+−+−LH16O146N112UUU−−+−−+M41
O67N44C1979India−−+−+−LH26O147N113UUU−−+−−+M41
O68N45E1978Japan−−+−−+M11O148N114UUU−−+−−+M39
O69N46C1979India−−+−+−LH21O149N2_44E1994Germany−−+−+−LH35
O69N2_29E1994Denmark−−+−−+M52O149N2_45C1993Indonesia−−+−−+M6
O71N47UUU−−++−+M73O150N115UUU−−+−−+M40
O72N48UUU−−+−−+M11O151N116UUU−−+−−+M6
O74N49UUU−++++−LH34O152N117UUU−−+−−+M4
O75N2_30C2006USA+++−−+M44O191N2_46C1996Japan+++−−+M48

Abbreviations: C, human isolate; E, environmental isolate; U, unknown; VM, V. mimicus.

a, Only LH was detected by the multiplex PCR assay but M was determined by targeted sequencing.

Figure 1

Strategy for determination of the M and LH sequences in the locus MS6_A0927. (A) Schematic diagram of M (upper) and LH (lower) regions between MS6_A0926 and MS6_A0928 and positions of sequencing primers. (B) PCR products obtained using the universal primers MS6_A0926 and MS6_A0928. Lane M, 1 kb DNA ladder; lane 1, Vibrio cholerae O1 El Tor MS6; lane 2, V. cholerae O1 El Tor N16961; lane 3, V. cholerae O35 N2_17. The expected sizes of PCR products for MS6 (M) and N16961 (LH) were 1.96 and 2.13 kb, respectively. Arrowhead indicates the amplicon that contained both sequences M and LH. All serotyped strains of non-O1 V. cholerae (n = 153) and V. mimicus (n = 2) from our bacterial stock were examined by this strategy and the results are presented in Table 1.

Characteristics of non-O1 Vibrio cholerae and V. mimicus as revealed by multiplex-PCR assays and M/LH profiling. Abbreviations: C, human isolate; E, environmental isolate; U, unknown; VM, V. mimicus. a, Only LH was detected by the multiplex PCR assay but M was determined by targeted sequencing. Strategy for determination of the M and LH sequences in the locus MS6_A0927. (A) Schematic diagram of M (upper) and LH (lower) regions between MS6_A0926 and MS6_A0928 and positions of sequencing primers. (B) PCR products obtained using the universal primers MS6_A0926 and MS6_A0928. Lane M, 1 kb DNA ladder; lane 1, Vibrio cholerae O1 El Tor MS6; lane 2, V. cholerae O1 El Tor N16961; lane 3, V. cholerae O35 N2_17. The expected sizes of PCR products for MS6 (M) and N16961 (LH) were 1.96 and 2.13 kb, respectively. Arrowhead indicates the amplicon that contained both sequences M and LH. All serotyped strains of non-O1 V. cholerae (n = 153) and V. mimicus (n = 2) from our bacterial stock were examined by this strategy and the results are presented in Table 1. Overall, 339 of 341 strains of vibrios, mostly V. cholerae, carried either M or LH in the locus MS6_A0927, and consequently those vibrios were separated into two clades: M (45.4%) and LH (54.6%) (Fig. 2). The remaining two strains of V. cholerae carried both M and LH in the same locus; thus, all V. cholerae strains carried either M or LH, or both. The M/LH sequence profiling further classified the 341 strains of vibrios into 127 subclades.
Figure 2

Vibrio cholerae organisms were classified into two clusters, M and LH. Dendrograms were constructed based on the genes M or LH from 341 Vibrio strains by the neighbor-joining method using MEGA v.6.0. Scale bars indicate nucleotide substitutions per site. Color coding is based on the presence of genes for cholera toxin (ctxAB) and toxin-coregulated pilus (tcpA): red, ctxAB+, tcpA+; blue, ctxAB−, tcpA+; green, ctxAB+, tcpA−; black, ctxAB−, tcpA−. The M and LH genes exhibited 79 and 46 sequence variations, respectively. The predominant subclades of LH and M were LH1, LH2, M4, and M6 in the order of description.

Vibrio cholerae organisms were classified into two clusters, M and LH. Dendrograms were constructed based on the genes M or LH from 341 Vibrio strains by the neighbor-joining method using MEGA v.6.0. Scale bars indicate nucleotide substitutions per site. Color coding is based on the presence of genes for cholera toxin (ctxAB) and toxin-coregulated pilus (tcpA): red, ctxAB+, tcpA+; blue, ctxAB−, tcpA+; green, ctxAB+, tcpA−; black, ctxAB−, tcpA−. The M and LH genes exhibited 79 and 46 sequence variations, respectively. The predominant subclades of LH and M were LH1, LH2, M4, and M6 in the order of description.

Evolutionary selection of M or LH in V. cholerae

We detected LH only in V. cholerae and not in other vibrios. In contrast, M was present in strains of V. cholerae, V. mimicus, V. parilis, V. metecus, V. splendidus, V. cyclitrophicus, V. tasmaniensis, V. tubiashii, and V. jasicida, but not in V. fluvialis, V. furnissii, V. parahaemolyticus, V. vulnificus, and V. anguillarum in publicly available database. The nucleotide sequence of M showed higher similarity within a single species than between different species (Fig. S1). Exceptionally, M from V. cholerae CP1037(10) showed higher similarity to that from in V. mimicus than to those of most V. cholerae. V. mimicus, V. parilis, and V. metecus carry M on the MS6_A0927 in chromosome II, similarly to V. cholerae, whereas V. splendidus, V. tasmaniensis, and V. cyclitrophicus carry M near the homologue of the glutamine-fructose-6-phosphate transaminase gene (MS6_0339), which is located on chromosome I. The latter three species and V. tubiashii and V. jasicida harbored homologous of MS6_A0926 and MS6_A0928 at various distances (0.2 kb to 10 kb) from the two loci. V. fluvialis and V. furnissii, which are more closely related to V. cholerae and V. mimicus[27], carried homologues of the aldehyde dehydrogenase (MS6_1585) and sigma 54-dependent transcriptional regulator (MS6_1586) genes between the two loci, although they did not carry M and LH. The alternative selection of M/LH at MS6_A0927 would have occurred in ancestral populations of V. cholerae. The distributions of M and LH in the strains of V. cholerae were generally associated with genome-based phylogeny (Fig. 3). The V. cholerae O1 lineage carried LH, except for the four strains in phylogenetic groups C and D. All 112 strains of groups A and B except for CP1046 exhibited subclade LH2, and the four strains of the group E showed LH1. The difference in these two subclades, LH1 and LH2, was ascribed to the absence of thymine in the 8-bp poly-T region of the H gene, which caused a frameshift to generate a modified protein that was shorter by 83 amino acids. The four strains in groups C and D showed subclades M2 and M1, respectively. Subclade M1 was represented by a Thai strain, MS6. This strain was very similar to the Russian strain P-18785[28]. The US gulf coast strains 2740–80 and 3569-08 were designated M2, which differed from M1 by one nucleotide. The sequences of the neighboring genes kbl (MS6_A0926) and lysR (MS6_A0928) were mostly identical among the strains of groups AB and E, but they were different from those in groups C and D, corresponding to the subclustering results for the targeted gene sequence of MS6_A0927 (Table 2). LH was likely replaced with M in the strains of groups C and D after they diverged from a common ancestor of classical and El Tor-biotype organisms.
Figure 3

Phylogenetic relationships among Vibrio cholerae and other Vibrio spp. and distribution of genes M and LH. A maximum likelihood tree showed phylogenetic relationships among 178 strains of V. cholerae, 6 V. mimicus, 1 V. metecus, and 1 V. parilis. Color coding is based on the presence of M: red; LH: blue; and M and LH: green. Bootstrap supports (%) are indicated at branching points. Branch lengths are proportional to sequence differences. Pathogenic O1 strains were classified into five phylogenic groups, A to E. Asterisk indicates a possible recombination event for the M gene through horizontal gene transfer.

Table 2

Sequence variations among the 186 vibrios as revealed by the M/LH sequence profiling and other 9 single locus sequence typing assays.

Strain(s)MS6_A0927A0926A0928Genes used for MLSTGroup
Subclade kbl lysR adk gyrB mdh metE pntA purM pyrC
N16961/others (n = 97)LH21a11(7)1(11)1(4)1(37)1(12)1(1)1(20)Ab
EDC−022LH2211(7)1(11)1(4)1(37)1(12)1(1)1(20)
EM-1536LH2211(7)1(11)1(4)1(37)1(12)1(1)1(20)
EM_1626LH2211(7)1(11)1(4)1(37)1(12)1(1)1(20)
VC1761LH2211(7)1(11)1(4)1(37)1(12)1(1)1(20)
CP1042(15)LH2211(7)1(11)1(4)1(37)1(12)1(1)1(20)
2011EL-1133LH2 S 11(7)1(11)1(4)S(37)S(NA)S(NA)S(NA)
2010EL-1749LH2111(7)1(11)1(4)NA(NA)1(12)1(1)1(20)
INDRE_91_1LH2111(7)1(11)1(4)1(37)1(12)1(1)S(NA)
CP1041(14)LH2111(7)1(11)1(4)2(206)1(12)1(1)1(20)
CP1040(13)LH2111(7)1(11)1(4)2(206)1(12)1(1)1(20)
CP1046 LH3 111(7)1(11)1(4)1(37)1(12)1(1)1(20)
8457LH2111(7)1(11)1(4)1(37)1(12)1(1)2(38)B
M66-2LH2121(7)1(11)1(4)1(37)1(12)1(1)2(38)
MAK757LH2121(7)1(11)1(4)1(37)1(12)S(NA)2(38)
BX330286LH2111(7)1(11)1(4)1(37)1(12)1(1)S(37)
MS6M1 S 31(7)2(2)1(4)1(37)1(12)1(1)2(38)C
P18785M1331(7)2(2)1(4)1(37)1(12)1(1)2(38)
3569-08M2331(7)S(NA)1(4)1(37)1(12)2(NA)2(38)D
2740-80M2331(7)2(2)1(4)1(37)1(12)1(1)2(38)
O395LH1111(7)1(11)1(4)3(9)1(12)1(1)2(38)E
RC27LH1111(7)1(11)1(4)3(9)1(12)1(1)2(38)
95412LH1111(7)1(11)1(4)3(9)1(12)1(1)2(38)
V52LH1111(7)1(11)1(4)S(17)S(23)1(1)S(35)
HE-46/others (n = 3)M7442(19)3(1)2(14)4(66)2(39)3(1)3(45)Others
HE-48 M19 5 S S(45)4(46)3(3)S(new)S(95)4(1)S(123)
CP-1035(8) M11 S S 3(26)5(5)S(2)5(50)3(30)S(18)3(45)
HE-45 M15 5 S 4(1)5(5)4(14)5(50)4(31)5(14)S(12)
HC-41B1M4553(26)6(5)5(new)5(50)4(31)5(14)3(45)
HC-43B1M4553(26)6(5)5(new)5(50)4(31)5(14)3(45)
HC-44C1M4553(26)6(5)5(new)5(50)4(31)5(14)3(45)
HC-46B1M4553(26)6(5)5(new)5(50)4(31)5(14)3(45)
116063M4553(26)6(5)4(14)6(54)5(28)5(14)3(45)
TM11079-80M4553(26)6(5)4(14)6(54)5(28)5(14)3(45)
AmazoniaM4553(26)6(5)4(14)6(54)5(28)5(14)3(45)
HC50A1/others (n = 16)M6665(S)7(36)2(14)7(40)3(30)5(14)4(4)
HE_16 LH12 S 76(23)8(34)6(26)S(78)S(36)6(10)5(29)
HE_09 M5 S 76(23)4(46)6(26)S(32)6(37)6(10)5(29)
87395 LH4M5 S S 6(23)8(34)6(26)S(57)6(37)6(10)S(25)
SX_4M8787(32)9(21)S(38)8(26)7(46)7(25)6(34)
VM573M8787(32)9(21)S(38)8(26)7(46)7(25)6(34)
CAIM602 M20 S S S(31)S(18)S(41)S(52)8(40)S(23)S(33)
VM223 M22 S S S(30)S(16)S(36)S(62)8(40)S(24)S(30)
MB-451 M21 S S S(31)S(22)S(40)S(24)S(41)S(28)S(17)
VM603 M23 S S S(35)S(19)S(37)S(25)S(45)S(26)S(32)
V51 M14 S S 8(25)S(5)7(22)9(16)9(21)8(8)S(11)
CP1110/others (n = 8)M3898(25)11(S)7(22)9(16)9(21)9(new)7(new)
MZO-3 LH9 S S S(4)S(23)S(28)S(13)10(25)4(1)S(9)
CT 5369-93 LH15 S S NA(NA)NA(NA)S(30)S(59)11(3)S(1)3(45)
EM-1676A LH6 S S 4(1)S(90)S(new)S(new)S(59)3(1)S(new)
MZO-2 LH8 S S 9(2)S(23)8(15)S(42)12(18)S(13)S(10)
1587 LH7 11S(NA)S(6)S(29)S(2)10(25)10(1)S(3)
TMA21 M18 S S S(9)S(25)3(3)S(41)S(6)10(1)8(5)
NHCC-008D M12 S S S(new)S(90)S(46)10(19)S(18)S(1)S(new)Others
HE-25 M10 9109(2)5(5)S(44)10(19)13(6)1(1)S(51)
AM19226 M9 9109(2)5(5)S(13)S(19)13(6)S(6)8(5)
623-39 M17 S S S(20)S(41)S(5)S(3)12(18)S(2)S(4)
CP1037(10) M16 S S 1(7)S(57)8(15)S(96)S(new)10(1)S(75)
BJG-01 LH11 S S S(13)S(30)2(14)S(44)11(3)3(1)S(39)
RC385 LH10 S S S(2)NA(NA)S(18)S(45)S(4)8(8)S(48)
PS15 LH13 S S S(new)S(5)S(14)S(130)S(66)S(new)S(new)
VL426 LH14 S S S(15)S(29)S(16)S(18)S(10)10(1)S(22)
12129(1) LH5 S S S(14)11(31)S(20)S(39)S(16)1(1)9(13)
VC35 M13 S S S(56)11(31)S(56)4(66)S(68)S(48)9(13)
RC341 M24 S S S(28)S(new)S(31)S(14)S(27)S(22)S(8)
RC586 M25 S S S(29)S(35)S(new)S(51)S(new)S(32)S(21)
Total
NA0011 (2)2 (3)0 (0)1 (1)0 (1)0 (3)0 (2)
variations40363527(24)30(24)30(27)36(34)30(27)27(18)35(32)

aSequence types in each gene. In italics and bold-face, singleton sequence type; NA, not available. Numbers in parentheses after the names of genes used for MLST indicate the allele number based on the profile definitions in the Vibrio cholerae MLST databases. bGroup assigned in Fig. 3.

Phylogenetic relationships among Vibrio cholerae and other Vibrio spp. and distribution of genes M and LH. A maximum likelihood tree showed phylogenetic relationships among 178 strains of V. cholerae, 6 V. mimicus, 1 V. metecus, and 1 V. parilis. Color coding is based on the presence of M: red; LH: blue; and M and LH: green. Bootstrap supports (%) are indicated at branching points. Branch lengths are proportional to sequence differences. Pathogenic O1 strains were classified into five phylogenic groups, A to E. Asterisk indicates a possible recombination event for the M gene through horizontal gene transfer. Sequence variations among the 186 vibrios as revealed by the M/LH sequence profiling and other 9 single locus sequence typing assays. aSequence types in each gene. In italics and bold-face, singleton sequence type; NA, not available. Numbers in parentheses after the names of genes used for MLST indicate the allele number based on the profile definitions in the Vibrio cholerae MLST databases. bGroup assigned in Fig. 3. We found a similar gene arrangement in the loci between MS6_A0926 and MS6_A0928 containing M and LH in the two strains N2_17 and 87395 (Fig. S2). Strain 87395 was phylogenetically closely related with HE-09, which exhibited M5 in common. In addition, the DNA sequence of LH was clearly different from those in V. cholerae, except for the four strains N56, N79, N80, and N83 of subclade LH4. Based on these observations, V. cholerae commonly carried a single copy of the M and/or LH genes in the specific locus. It is likely that V. cholerae originally carried the M gene; however, horizontal gene transfer led to the emergence of V. cholerae carrying LH in ancient times. In some subclades of V. cholerae, such as those in groups C and D and CP1037(10), gene replacement with M occurred in succession. As observed in strains 87395 and N2.17, an incomplete choice between the two alternatives of M and LH could occur, resulting in incidental possession of both M and LH in these strains. The alternative choice of M or LH might be of benefit to the survival of V. cholerae in different environmental conditions. Very recently, Das et al. (29) reported that the product of H in V. cholerae O1 classical biotype strain O395 was endowed with molecular chaperone, aminopeptidase, and robust methylglyoxalase activities. The functional roles of these genes in V. cholerae are now under investigation.

Genetic diversity and population structure of V. cholerae revealed by M/LH sequence profiling

The above-described data show that V. cholerae have maintained M or LH in MS6_A0927 on their genomes and indicate that the subclades corresponding well to clusters generated from a genome-wide phylogenetic analysis. Therefore, we considered the advantages and limitations of using our targeted gene sequencing for V. cholerae investigations. The M/LH sequence profiling exhibited the highest discrimination index (D = 0.63) as compared with those of the nine SLST analyses that targeted the two gene loci kbl and lysR and seven housekeeping genes adk, gyrB, mdh, metE, pntA, purM, and pyrC (Table 2). The kbl and lsyR genes encode 2-amino-3-ketobutyrate coenzyme A ligase and the LysR family of transcriptional regulators, respectively. M/LH sequence profiling differentiated between the four groups, i.e., A/B, C, D, and E, whereas the 9 other SLSTs failed to differentiate these groups. The conventional MLST analysis based on the seven loci was able to differentiate between groups A and B, but not between C and D. The ability to distinguish C from D was critical to trace the pathogen MS6 back to its likely origin in China or vice versa. Recently, sporadic cholera outbreaks caused by US Gulf-like V. cholerae O1 (non-7th pandemic clone) occurred in Zhejiang province, China[29]. MLST analysis separated 13 Zhejiang strains into 3 sequence types—ST75, ST169, and ST170—and of them, 10 strains with ST75 sequence type were identical to US Gulf coast strains, while M/LH sequence profiling showed that all the 13 strains belonged to group D (subclade M1), differentiating them from US Gulf coast strains (M2). The results of M/LH sequence profiling corresponded well with those of the clusters deduced by their whole genome sequence-based phylogenetic analysis, and consequently, it demonstrated higher epidemiological relevance than MLST did. Vibrios with M or LH were isolated from clinical and environmental sources. Subclades LH1, LH2, M4, and M6 were dominantly found in test strains (Fig. 2). Among them, LH2 mostly comprised the seventh cholera pandemic El Tor and toxigenic O139 strains, whereas the classical type of V. cholerae, the major player in the sixth cholera pandemic, belonged to subclade LH1. Interestingly, there have been no reports on the toxigenic and non-toxigenic O1 strains in group C and D that replaced LH with M causing epidemics and severe diarrhea (Fig. 3)[28-32]. Furthermore, all 20 clinical strains of non-O1/non-O139 V. cholerae obtained in the Haiti cholera epidemic belonged to two subclades M4 and M6 (Table S1), which corresponded to clusters HC-1 and −2 as indicated by the comparative genomic analysis of Hassan et al. (29). The three environmental strains from Haiti from 2010 were in subclade M7. In addition, eight strains of serogroup O75 CP1110-CP1117 from an oyster-borne cholera outbreak in Florida[33] belonged to subclade M3. In this study, 130 strains including V. cholerae O1 and O139 were separated into 13 subclades. Subclade LH2 contained current pandemic/epidemic clone and normally possessed the genes ctxAB/tcpA/toxR/VC2346/LH, which were targeted by our multiplex PCR. Most of the non-toxigenic V. cholerae of O1 serogroup, such as strains 12129(1) and TM11079-80, belonged to different subclades, and phylogenetic lineages from the toxigenic O1 strains and their O1 antigen phenotype probably arose from horizontal gene exchange in the evolution of V. cholerae[13]. The alterations in the cell surface antigens of V. cholerae can lead to new epidemics/pandemics, especially in populations without adequate immunity against the serogroup. V. cholerae O139 Bengal evolved from a V. cholerae O1 El Tor strain by exchange of genes encoding cell surface polysaccharides[34,35], and cholera caused by V. cholerae O139 Bengal has rapidly spread in southeast Asian countries following its initial isolation in Madras, India, in 1992. A high incidence of cholera has been observed in both adults and children in the areas where cholera is endemic[36,37]. In conclusion, our targeted gene sequencing of MS6_A0927 revealed divergent genetic traits among V. cholerae species.

Methods

Bacterial strains and genomes

This study analyzed 341 genomes consisting of i) O1/O139 (n = 128), non-O1/non-O139 (n = 48), and unknown serogroups (n = 2) of V. cholerae strains; V. mimicus (n = 6); V. metecus (n = 1); and V. parilis (n = 1) deposited in GenBank; and ii) serotyped strains of non-O1 V. cholerae (n = 153) and V. mimicus (n = 2). Detailed information on the genomes/strains used in this study is presented in Table S1 and Table 1.

Multiplex PCR method

In total, 186 genomes of Vibrio species (Table S1) were referenced for the in-house design of the multiplex PCR method and targeted gene sequencing. Primers for the six target genes toxR, ctxAB, luxR-hchA (LH), metY (M), VC2346[38,39], and tcpA were designed to target each consensus region. The multiplex PCR assay for detection of those genes was performed in a 25-µl reaction mixture containing 0.2 mM dNTPs, 1 × Ex Taq buffer, 2 mM MgCl2, each primer at 1 µM, 0.75 U of Ex Taq DNA polymerase (Takara Bio, Otsu, Japan), and 100 ng of genomic DNA extracted using the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany). Primer sequences are shown in Table S2. Thermal cycling conditions were as follows: 94 °C for 30 s and 30 cycles of 94 °C for 30 s, 59 °C for 1 min, and 72 °C for 1 min. Amplicons were separated by 2% agarose gel electrophoresis and bands were visualized under ultraviolet transillumination after staining of the gel with ethidium bromide (Fig. S3).

Targeted gene sequencing

PCR amplification of the locus MS6_A0927 was performed in a 50-µl reaction mixture containing 1 × Ex Taq buffer, 2 mM MgCl2, 0.2 mM dNTPs, each external primer at 1 µM (MS6_A0926F and MS6_A0928R) (Fig. 1), 1.25 U of Ex Taq DNA polymerase, and 100 ng of purified genomic DNA. Thermal cycling conditions were as follows: 95 °C for 4 min and 30 cycles of 95 °C for 30 s, 62 °C or 55 °C for 30 s, and 72 °C for 1.5 min. PCR products were purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) or the QIAquick Gel Extraction kit (Qiagen, Valencia, CA, USA). Samples positive for either M or LH were sequenced within these regions (Fig. 1). The PCR amplification for sequencing was performed in a 10-µl reaction mixture containing 1.5 µl of BigDye Terminator v.3.1 Ready Reaction Mix, 1.25 µl of 5 × BigDye Sequencing Buffer, each internal primer at 0.32 µM (lh_uni1–4 and m_uni1–4), and 40 ng of purified PCR product. The cycling condition was as follows: 96 °C for 1 min and 30 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. The reaction product was sequenced on the ABI 3130xl Genetic Analyzer platform (Applied Biosystems, Foster City, CA, USA). Sequence data determined in this study were submitted to DDBJ (DNA Data Bank of Japan, National Institute of Genetics, Mishima, Shizuoka, Japan) and published with the accession no. LC202659-LC202813. For M/LH sequence profiling, DNA sequences of the targeted locus were aligned to one of two references: the 1,296-bp region of the MS6 strain (MS6_A0927) or the 1,600-bp region of the classical O1 strain O395 (from VC395_A0912 to VC395_A0913). The subclades were numbered by sequential assignment to each nucleotide sequence variation. The sequence variations based on M and LH were compared with those from the nine SLST analyses. The sequence data of kbl (MS6_A0926) and lysR (MS6_A0928) and of the seven gene regions targeted by a multilocus sequence typing (MLST) procedure[21] were extracted from the 186 genomes. Regions of sequences of the primer sets in each locus of the MLST were used. DNA sequences of each target gene were aligned to each reference sequence from MS6 (kbl and lysR) or N16961 (adk, gyrB, mdh, metE, pntA, purM, and pyrC). Arabic numbers were sequentially assigned to each sequence variation in each target region as described in Table 2. In addition, the number of alleles for MLST were determined based on the profile definitions in the Vibrio cholerae MLST databases (https://pubmlst.org/vcholerae/). The discrimination index was determined by calculation of the Simpson index of diversity, D[40].

Phylogenetic tree

Dendrograms were constructed by the neighbor-joining method using MEGA v.6.0[41]. Relationships among strains were assessed by genome-wide phylogenetic analysis. Coding sequences present as a single copy in genomes were analyzed using the Pan-genomes Analysis Pipeline v.1.02[26,42].
  42 in total

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Authors:  S M Faruque; K M Ahmed; A R Abdul Alim; F Qadri; A K Siddique; M J Albert
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Authors:  H M Frénay; A E Bunschoten; L M Schouls; W J van Leeuwen; C M Vandenbroucke-Grauls; J Verhoef; F R Mooi
Journal:  Eur J Clin Microbiol Infect Dis       Date:  1996-01       Impact factor: 3.267

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Authors:  Daniela Ceccarelli; Matteo Spagnoletti; Piero Cappuccinelli; Vincent Burrus; Mauro M Colombo
Journal:  Lancet Infect Dis       Date:  2011-04       Impact factor: 25.071

4.  US Gulf-like toxigenic O1 Vibrio cholerae causing sporadic cholera outbreaks in China.

Authors:  Yun Luo; Sophie Octavia; Dazhi Jin; Julian Ye; Ziping Miao; Tao Jiang; Shichang Xia; Ruiting Lan
Journal:  J Infect       Date:  2016-02-23       Impact factor: 6.072

5.  Development and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio cholerae.

Authors:  K L F Cooper; C K Y Luey; M Bird; J Terajima; G B Nair; K M Kam; E Arakawa; A Safa; D T Cheung; C P Law; H Watanabe; K Kubota; B Swaminathan; E M Ribot
Journal:  Foodborne Pathog Dis       Date:  2006       Impact factor: 3.171

6.  Type III secretion is essential for the rapidly fatal diarrheal disease caused by non-O1, non-O139 Vibrio cholerae.

Authors:  Ok S Shin; Vincent C Tam; Masato Suzuki; Jennifer M Ritchie; Roderick T Bronson; Matthew K Waldor; John J Mekalanos
Journal:  MBio       Date:  2011-06-14       Impact factor: 7.867

7.  Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

Authors:  Tomoo Sawabe; Yoshitoshi Ogura; Yuta Matsumura; Gao Feng; Akm Rohul Amin; Sayaka Mino; Satoshi Nakagawa; Toko Sawabe; Ramesh Kumar; Yohei Fukui; Masataka Satomi; Ryoji Matsushima; Fabiano L Thompson; Bruno Gomez-Gil; Richard Christen; Fumito Maruyama; Ken Kurokawa; Tetsuya Hayashi
Journal:  Front Microbiol       Date:  2013-12-27       Impact factor: 5.640

Review 8.  Whole genome sequencing in clinical and public health microbiology.

Authors:  J C Kwong; N McCallum; V Sintchenko; B P Howden
Journal:  Pathology       Date:  2015-04       Impact factor: 5.306

9.  Vibrio cholerae O1 isolate with novel genetic background, Thailand-Myanmar.

Authors:  Kazuhisa Okada; Amonrattana Roobthaisong; Witaya Swaddiwudhipong; Shigeyuki Hamada; Siriporn Chantaroj
Journal:  Emerg Infect Dis       Date:  2013-06       Impact factor: 6.883

10.  Population structure and evolution of non-O1/non-O139 Vibrio cholerae by multilocus sequence typing.

Authors:  Sophie Octavia; Anna Salim; Jacob Kurniawan; Connie Lam; Queenie Leung; Sunjukta Ahsan; Peter R Reeves; G Balakrish Nair; Ruiting Lan
Journal:  PLoS One       Date:  2013-06-11       Impact factor: 3.240
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