# Emergence of Tetracycline ResistantVibrio cholerae O1 Biotype El Tor Serotype Ogawa with ClassicalctxB Gene from a Cholera Outbreak in Odisha, Eastern India

Authors: M. Jain; P. Kumar; A. K. Goel
Journal: Journal of Pathogens
 (2016)

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## Abstract

In September 2010, a cholera outbreak was reported from Odisha, Eastern India.V. cholerae isolated from the clinical samples were biochemically and serologically confirmed as serogroup O1, biotype El Tor, and serotype Ogawa. Multiplex PCR screening revealed the presence of various genes, namely,ompW,ctxB,zot,rfbO1,tcp,ace,hlyA,ompU,rtx, andtoxR, in all of the isolates. The isolates were resistant to co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). Minimum inhibitory concentration of tetracycline decreased in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), suggesting the involvement of efflux pumps. PCR analysis confirmed the presence of class I integrons as well as SXT elements harbouring antibiotic resistance genes in all isolates. Sequencing revealed the presence ofctxB gene of classical biotype in all the isolates. The isolates harboured an RS1-CTX prophage array with El Tor typerstRand classicalctxBon the large chromosome. The study indicated that theV. cholerae El Tor variants are evolving in the area with better antibiotic resistance and virulence potential.


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## Body

## 1. Introduction

Cholera is still a major health problem of global importance as evidenced by the recent outbreaks in Haiti and Zimbabwe. In 2013, a total of 47 countries from all continents reported 129 064 cases of cholera including 2102 deaths with a case fatality rate of 1.63% [1]. ToxigenicVibrio cholerae,the causative agent of cholera, has exhibited several changing patterns in its biotype as well as in drug resistance for its better survival and infection [2, 3]. Classical biotypeV. cholerae strains after causing earlier six cholera pandemics were replaced by El Tor biotype strains in the seventh pandemic. The variant ofV. cholerae El Tor strain possessing cholera toxin of classical biotype appeared after the emergence of O139 strain and has now become prevalent worldwide [4]. Antimicrobial susceptibility pattern has continuously changed forV. cholerae[2]. Tetracycline is an important broad spectrum antibiotic used for the prophylaxis and treatment of variety of bacterial infections including listed biothreat agentsBacillus anthracis,Francisella tularensis, andYersinia pestis[5]. It has been a preferred choice for control of cholera as recommended by WHO [6]. Earlier, we reported a novel mutation inctxB gene of outbreak isolates in 2007 from Rayagada (19.09°N 83.27°E), Odisha, and later on the same allelic variation was observed in the Haitian outbreak isolates [7, 8]. These isolates were multidrug resistant but sensitive to tetracycline [8]. In September-October, 2010, another cholera outbreak was observed in the same area of Rayagada, Odisha. In this communication, we report the emergence of tetracycline resistantV. cholerae from this outbreak of Eastern India.


## 2. Materials and Methods

### 2.1. Isolation and Identification ofV. cholerae

A total of 27V. cholerae isolates were recovered from randomly selected patients during the cholera outbreak in the month of September, 2010, from Rayagada district of Odisha (19.09°N 83.27°E). Samples were collected using sterile rectal swabs from the patients and processed as described earlier [9].V. cholerae isolates were identified and confirmed by using standard biochemical methods. Serological identification of the isolates was done by slide agglutination using commercially available polyvalent antiserum againstV. cholerae O1 (Difco, USA). Voges Proskauer (VP) test, sheep erythrocyte haemolysis, and polymyxin B susceptibility tests were performed for determination of biotype of isolates. Other bacterial reference strains used in the study wereV. cholerae O1 El Tor (ATCC 14033),V. cholerae O1 classical (ATCC 11623), andV. cholerae O139 (ATCC 51394).


### 2.2. PCR Assays for Virulence and Species-Specific Genes

Genomic DNA was extracted from each of the isolates using genomic DNA purification kit (MBI Fermentas, Vilnius, Lithuania) and screened for the presence of diverse gene traits by two sets of multiplex PCR (mPCR) as described elsewhere [10]. The first mPCR detected the presence of genes encoding the outer-membrane protein (ompW), cholera toxin (ctxB), zonula occludens toxin (zot), O1 somatic antigen (rfbO1), and toxin coregulated pilus (tcp). The second mPCR amplified other virulence and toxigenic genes encoding the accessory cholera enterotoxin (ace), haemolysin (hlyA), outer-membrane protein (ompU), repeat in toxin protein (rtx), and toxin regulator (toxR).


### 2.3. Antimicrobial Drug-Susceptibility Testing

The antimicrobial susceptibility of theV. cholerae isolates was determined by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar [11], and minimum inhibitory concentration (MIC) was calculated by broth dilution method according to the guidelines of Clinical Laboratory Standards Institute [12]. The antibiotic discs and antibiotic powder were procured from Oxoid limited, UK, and Sigma Aldrich, USA, respectively. The antibiotic discs used were ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), co-trimoxazole (25 μg), erythromycin (10 μg), nalidixic acid (30 μg), polymyxin B (50 μg), rifampicin (5 μg), spectinomycin (100 μg), streptomycin (10 μg), sulphamethoxazole (100 μg), tetracycline (30 μg), trimethoprim (5 μg), and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine, O/129 (150 μg). The MIC for selected antibiotics was calculated in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP) at subinhibitory concentration. Reference strains ofEscherichia coli ATCC 25922,Staphylococcus aureus ATCC 25923, andPseudomonas aeruginosa ATCC 27853 were used in each test for internal quality control.


### 2.4. Mobile Genetic Elements and Their Gene Cassettes

The presence of class 1 integron and SXT constins was detected by PCR as described by Hochhut et al. [13]. The presence of gene cassettes foraadA2(encoding resistance for streptomycin and spectinomycin),strA(encoding resistance for streptomycin),blaP1 (encoding resistance for β-lactams),floR (encoding resistance for chloramphenicol), anddfrA1 (encoding resistance for trimethoprim) was determined by PCR as described earlier [14, 15].


### 2.5. Sequencing of thectxB Gene

ThectxB gene was amplified from the isolates using primers described previously [16]. Sequencing was carried out using the same PCR primers on a 96-capillary model 3730xl system using the Big Dye Terminator kit from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The sequences were edited with SeqED program (Applied Biosystems) and were aligned with sequences ofctxB gene of reference strains ofV. cholerae O1 El Tor, classical, and O139. The deduced amino acid sequences of thectxB gene from all the strains were aligned using CLUSTAL W.


### 2.6. Analysis of RS1 and CTX Prophage Array

Arrangement of RS1 and CTX phage was determined by PCR. Presence of the RS1 element was confirmed by PCR using therstCspecific primers as described elsewhere [17]. The primers for r
s
t
R
E
l
T
o
r, r
s
t
R
C
l
a
s
s
i
c
a
l, and r
s
t
R
C
a
l
c
u
t
t
a were used forrstRtyping of RS element and for differentiation of classical and El Tor biotype ofV. cholerae [18]. The location of an RS1-CTX prophage array on the large chromosome, insertion of the CTX prophage on the small chromosome, and tandem repeats of RS1 and CTX prophage on chromosome were determined by PCR as described earlier [17].


## 2.1. Isolation and Identification ofV. cholerae

A total of 27V. cholerae isolates were recovered from randomly selected patients during the cholera outbreak in the month of September, 2010, from Rayagada district of Odisha (19.09°N 83.27°E). Samples were collected using sterile rectal swabs from the patients and processed as described earlier [9].V. cholerae isolates were identified and confirmed by using standard biochemical methods. Serological identification of the isolates was done by slide agglutination using commercially available polyvalent antiserum againstV. cholerae O1 (Difco, USA). Voges Proskauer (VP) test, sheep erythrocyte haemolysis, and polymyxin B susceptibility tests were performed for determination of biotype of isolates. Other bacterial reference strains used in the study wereV. cholerae O1 El Tor (ATCC 14033),V. cholerae O1 classical (ATCC 11623), andV. cholerae O139 (ATCC 51394).


## 2.2. PCR Assays for Virulence and Species-Specific Genes

Genomic DNA was extracted from each of the isolates using genomic DNA purification kit (MBI Fermentas, Vilnius, Lithuania) and screened for the presence of diverse gene traits by two sets of multiplex PCR (mPCR) as described elsewhere [10]. The first mPCR detected the presence of genes encoding the outer-membrane protein (ompW), cholera toxin (ctxB), zonula occludens toxin (zot), O1 somatic antigen (rfbO1), and toxin coregulated pilus (tcp). The second mPCR amplified other virulence and toxigenic genes encoding the accessory cholera enterotoxin (ace), haemolysin (hlyA), outer-membrane protein (ompU), repeat in toxin protein (rtx), and toxin regulator (toxR).


## 2.3. Antimicrobial Drug-Susceptibility Testing

The antimicrobial susceptibility of theV. cholerae isolates was determined by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar [11], and minimum inhibitory concentration (MIC) was calculated by broth dilution method according to the guidelines of Clinical Laboratory Standards Institute [12]. The antibiotic discs and antibiotic powder were procured from Oxoid limited, UK, and Sigma Aldrich, USA, respectively. The antibiotic discs used were ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), co-trimoxazole (25 μg), erythromycin (10 μg), nalidixic acid (30 μg), polymyxin B (50 μg), rifampicin (5 μg), spectinomycin (100 μg), streptomycin (10 μg), sulphamethoxazole (100 μg), tetracycline (30 μg), trimethoprim (5 μg), and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine, O/129 (150 μg). The MIC for selected antibiotics was calculated in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP) at subinhibitory concentration. Reference strains ofEscherichia coli ATCC 25922,Staphylococcus aureus ATCC 25923, andPseudomonas aeruginosa ATCC 27853 were used in each test for internal quality control.


## 2.4. Mobile Genetic Elements and Their Gene Cassettes

The presence of class 1 integron and SXT constins was detected by PCR as described by Hochhut et al. [13]. The presence of gene cassettes foraadA2(encoding resistance for streptomycin and spectinomycin),strA(encoding resistance for streptomycin),blaP1 (encoding resistance for β-lactams),floR (encoding resistance for chloramphenicol), anddfrA1 (encoding resistance for trimethoprim) was determined by PCR as described earlier [14, 15].


## 2.5. Sequencing of thectxB Gene

ThectxB gene was amplified from the isolates using primers described previously [16]. Sequencing was carried out using the same PCR primers on a 96-capillary model 3730xl system using the Big Dye Terminator kit from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The sequences were edited with SeqED program (Applied Biosystems) and were aligned with sequences ofctxB gene of reference strains ofV. cholerae O1 El Tor, classical, and O139. The deduced amino acid sequences of thectxB gene from all the strains were aligned using CLUSTAL W.


## 2.6. Analysis of RS1 and CTX Prophage Array

Arrangement of RS1 and CTX phage was determined by PCR. Presence of the RS1 element was confirmed by PCR using therstCspecific primers as described elsewhere [17]. The primers for r
s
t
R
E
l
T
o
r, r
s
t
R
C
l
a
s
s
i
c
a
l, and r
s
t
R
C
a
l
c
u
t
t
a were used forrstRtyping of RS element and for differentiation of classical and El Tor biotype ofV. cholerae [18]. The location of an RS1-CTX prophage array on the large chromosome, insertion of the CTX prophage on the small chromosome, and tandem repeats of RS1 and CTX prophage on chromosome were determined by PCR as described earlier [17].


## 3. Results

### 3.1. Identification of Outbreak Strains

A total of 27 clinical strains ofV. cholerae were isolated from the affected patients during cholera outbreak in Odisha, Eastern India. On the basis of biochemical tests, all the isolates were found to belong to El Tor biotype as the isolates were VP test positive, hemolytic on sheep blood agar, and resistant to polymyxin B.


### 3.2. Gene Traits

Two sets of multiplex PCR revealed that all the outbreak isolates were invariably positive for various virulent and species-specific genes, namely,ace(309 bp), ctxAB(536 bp), hlyA(480 bp),ompU(655 bp), ompW(304 bp), rfbO1(638 bp), rtxC(265 bp), tcpA(805 bp), toxR(779 bp), andzot(947 bp) genes (Table 1).Table 1
Comparison ofV. choleraestrains isolated during two cholera outbreaks in 2007 and 2010 from Rayagada, Odisha.
Year of isolation
V. cholerae strain
Gene traits
Antibiogram
Class 1 integrons
SXT
Antibiotic resistant gene
Reference
2007
O1 El Tor Ogawa
ompW
+, ctxB
+, zot
+, rfbO1
+, tcp
+, ace
+, hlyA
+, ompU
+, rtx
+, and toxR
+
CotR, NalR, PolR, SpeR, StrS, SulR, TriR, AmpR, TetS, ChlS, EryR. CipS, and RifS
+
+
aadA2
+, blaP1
−, dfrA1
+, floR
−, and strA
+
[10, 19]
2010
O1 El Tor Ogawa
ompW
+, ctxB
+, zot
+, rfbO1
+, tcp
+, ace
+, hlyA
+, ompU
+, rtx
+, and toxR
+
CotR, NalR, PolR, SpeR, StrR, SulR, TetR, TriR, O/129R, AmpS, ChlS, EryS. CipS, and RifS
+
+
aadA2
+, blaP1
−, dfrA1
+,floR
−, and strA
+
This study
Cot: co-trimoxazole, Nal: nalidixic acid, Pol: polymyxin B, Spe: spectinomycin, Str: streptomycin, Sul: sulfamethoxazole, Tet: tetracycline, Tri: trimethoprim, Amp: ampicillin, and Ery: erythromycin.


### 3.3. Antibiotic Drug Resistance Pattern

The antimicrobial disc diffusion test revealed that all the isolates were resistant to co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). However, the isolates were sensitive to ampicillin, chloramphenicol, erythromycin, ciprofloxacin, and rifampicin. The MIC for selected antibiotics is given in Table2. The presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), an inhibitor of major facilitator superfamily (MFS) proteins in broth at subinhibitory concentration (0.5 μg/mL), reduced MIC for tetracycline to eightfold (16 to 2 μg/mL) and the isolates became susceptible. CCCP also reduced the MIC for trimethoprim from 1024 μg/mL to 256 μg/mL, whereas MIC for nalidixic acid, streptomycin, sulfamethoxazole, and ampicillin remained unaffected (Table 2).Table 2
Effect of CCCP on minimum inhibitory concentration (MIC) of various antibiotics onV. cholerae isolates.
Isolate
Tet
Nal
Str
Sul
Tri
Amp
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
VCR4
16
32
≥1024
≥4096
≥1024
4
VCR4 + CCCP
2
32
≥1024
≥4096
256
4


### 3.4. Prevalence of Multidrug Resistant Elements

PCR analysis revealed the presence of class I integron as well as SXT element harbouring antibiotic resistance traits against multiple drugs in all the isolates. The isolates were positive foraadA2(streptomycin and spectinomycin) anddfrA1(trimethoprim) gene cassettes but negative forblaP1(β-lactam antibiotics) cassettes [14]. The strains were also positive forstrA carried on SXT constin that confers resistance for streptomycin. However, all the isolates were negative forfloR(chloramphenicol) genes.


### 3.5. Sequence Analysis ofctxB

The deduced amino acid sequence analysis of CT-B from the representative isolates varied from CT-B of reference El Tor strain N16961 and O139 strain at position 39 (histidine in place of tyrosine) and position 68 (threonine in place of isoleucine) but was similar to CT-B subunit of reference classical strains 569B (Figure1). However, no mutation was observed at position 20, which was found in theV. cholerae isolates from the previous outbreak in 2007 (Figure 1).Figure 1
Amino acid sequence alignment of CTB subunit ofV. cholerae O1 El Tor isolates from Odisha outbreak with preexisting reference El Tor and classical strains. Identical amino acid residues are indicated by dots. Amino acid sequences ofV. cholerae CTB used in alignment were taken from GenBank.


### 3.6. Phage Array

The cholera toxin genectxAB lies on the single stranded DNA of CTX phage which is a filamentous phage [20]. There is specific site for integration of CTX phage DNA into theV. cholerae chromosome. Toxigenic strains contain a genetic element RS1 which is related to CTX phage. RS1 element is found adjacent (5′ or 3′) to CTX prophage. Several types of CTX phage/RS1 arrays have been found in the epidemicV. cholerae strains [21]. In this study,rstC specific primer setrstCF/rstCRamplified the DNA, indicating the presence of the RS1 element in the chromosome. A PCR product was amplified withrstCF/cepR showing the existence of RS1-CTX prophage array. In contrast,ctxBF andrstCR primer pair did not give any amplification, indicating the absence of CTX prophage-RS1 array. No DNA fragment was amplified using the primer pairs ofctxBF/cepR andrstCF4/rstCR4, indicating the absence of tandem repeats of CTX prophage and RS1 element on the genome, respectively. Location of RS1-CTX prophage array on the large chromosome was confirmed with the PCR amplified products usingCh1F/rstARandctxBF/Ch1Rprimers. The isolates yielded PCR products usingCh2F/Ch2Rprimers indicating the absence of the CTX prophage or RS1 element on small chromosome. The RS1-CTX prophage array has been shown in Figure 2.Figure 2
Genetic structures and CTX prophage array ofV. cholerae strains. Block arrows indicate the transcription direction of each gene. Black triangles on the genome indicate the repeat sequence flanking the integrated phage DNA.V. choleraeisolates in this study contain a single RS1 element and a single classical CTX prophage after the TLC element on chromosome I.


## 3.1. Identification of Outbreak Strains

A total of 27 clinical strains ofV. cholerae were isolated from the affected patients during cholera outbreak in Odisha, Eastern India. On the basis of biochemical tests, all the isolates were found to belong to El Tor biotype as the isolates were VP test positive, hemolytic on sheep blood agar, and resistant to polymyxin B.


## 3.2. Gene Traits

Two sets of multiplex PCR revealed that all the outbreak isolates were invariably positive for various virulent and species-specific genes, namely,ace(309 bp), ctxAB(536 bp), hlyA(480 bp),ompU(655 bp), ompW(304 bp), rfbO1(638 bp), rtxC(265 bp), tcpA(805 bp), toxR(779 bp), andzot(947 bp) genes (Table 1).Table 1
Comparison ofV. choleraestrains isolated during two cholera outbreaks in 2007 and 2010 from Rayagada, Odisha.
Year of isolation
V. cholerae strain
Gene traits
Antibiogram
Class 1 integrons
SXT
Antibiotic resistant gene
Reference
2007
O1 El Tor Ogawa
ompW
+, ctxB
+, zot
+, rfbO1
+, tcp
+, ace
+, hlyA
+, ompU
+, rtx
+, and toxR
+
CotR, NalR, PolR, SpeR, StrS, SulR, TriR, AmpR, TetS, ChlS, EryR. CipS, and RifS
+
+
aadA2
+, blaP1
−, dfrA1
+, floR
−, and strA
+
[10, 19]
2010
O1 El Tor Ogawa
ompW
+, ctxB
+, zot
+, rfbO1
+, tcp
+, ace
+, hlyA
+, ompU
+, rtx
+, and toxR
+
CotR, NalR, PolR, SpeR, StrR, SulR, TetR, TriR, O/129R, AmpS, ChlS, EryS. CipS, and RifS
+
+
aadA2
+, blaP1
−, dfrA1
+,floR
−, and strA
+
This study
Cot: co-trimoxazole, Nal: nalidixic acid, Pol: polymyxin B, Spe: spectinomycin, Str: streptomycin, Sul: sulfamethoxazole, Tet: tetracycline, Tri: trimethoprim, Amp: ampicillin, and Ery: erythromycin.


## 3.3. Antibiotic Drug Resistance Pattern

The antimicrobial disc diffusion test revealed that all the isolates were resistant to co-trimoxazole, nalidixic acid, polymyxin B, spectinomycin, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). However, the isolates were sensitive to ampicillin, chloramphenicol, erythromycin, ciprofloxacin, and rifampicin. The MIC for selected antibiotics is given in Table2. The presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), an inhibitor of major facilitator superfamily (MFS) proteins in broth at subinhibitory concentration (0.5 μg/mL), reduced MIC for tetracycline to eightfold (16 to 2 μg/mL) and the isolates became susceptible. CCCP also reduced the MIC for trimethoprim from 1024 μg/mL to 256 μg/mL, whereas MIC for nalidixic acid, streptomycin, sulfamethoxazole, and ampicillin remained unaffected (Table 2).Table 2
Effect of CCCP on minimum inhibitory concentration (MIC) of various antibiotics onV. cholerae isolates.
Isolate
Tet
Nal
Str
Sul
Tri
Amp
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
VCR4
16
32
≥1024
≥4096
≥1024
4
VCR4 + CCCP
2
32
≥1024
≥4096
256
4


## 3.4. Prevalence of Multidrug Resistant Elements

PCR analysis revealed the presence of class I integron as well as SXT element harbouring antibiotic resistance traits against multiple drugs in all the isolates. The isolates were positive foraadA2(streptomycin and spectinomycin) anddfrA1(trimethoprim) gene cassettes but negative forblaP1(β-lactam antibiotics) cassettes [14]. The strains were also positive forstrA carried on SXT constin that confers resistance for streptomycin. However, all the isolates were negative forfloR(chloramphenicol) genes.


## 3.5. Sequence Analysis ofctxB

The deduced amino acid sequence analysis of CT-B from the representative isolates varied from CT-B of reference El Tor strain N16961 and O139 strain at position 39 (histidine in place of tyrosine) and position 68 (threonine in place of isoleucine) but was similar to CT-B subunit of reference classical strains 569B (Figure1). However, no mutation was observed at position 20, which was found in theV. cholerae isolates from the previous outbreak in 2007 (Figure 1).Figure 1
Amino acid sequence alignment of CTB subunit ofV. cholerae O1 El Tor isolates from Odisha outbreak with preexisting reference El Tor and classical strains. Identical amino acid residues are indicated by dots. Amino acid sequences ofV. cholerae CTB used in alignment were taken from GenBank.


## 3.6. Phage Array

The cholera toxin genectxAB lies on the single stranded DNA of CTX phage which is a filamentous phage [20]. There is specific site for integration of CTX phage DNA into theV. cholerae chromosome. Toxigenic strains contain a genetic element RS1 which is related to CTX phage. RS1 element is found adjacent (5′ or 3′) to CTX prophage. Several types of CTX phage/RS1 arrays have been found in the epidemicV. cholerae strains [21]. In this study,rstC specific primer setrstCF/rstCRamplified the DNA, indicating the presence of the RS1 element in the chromosome. A PCR product was amplified withrstCF/cepR showing the existence of RS1-CTX prophage array. In contrast,ctxBF andrstCR primer pair did not give any amplification, indicating the absence of CTX prophage-RS1 array. No DNA fragment was amplified using the primer pairs ofctxBF/cepR andrstCF4/rstCR4, indicating the absence of tandem repeats of CTX prophage and RS1 element on the genome, respectively. Location of RS1-CTX prophage array on the large chromosome was confirmed with the PCR amplified products usingCh1F/rstARandctxBF/Ch1Rprimers. The isolates yielded PCR products usingCh2F/Ch2Rprimers indicating the absence of the CTX prophage or RS1 element on small chromosome. The RS1-CTX prophage array has been shown in Figure 2.Figure 2
Genetic structures and CTX prophage array ofV. cholerae strains. Block arrows indicate the transcription direction of each gene. Black triangles on the genome indicate the repeat sequence flanking the integrated phage DNA.V. choleraeisolates in this study contain a single RS1 element and a single classical CTX prophage after the TLC element on chromosome I.


## 4.  Discussion

V. choleraeO1 El Tor strains are supposed to have better adaptability in the environment and colonize more efficiently in the intestinal lumen as compared to the classical one [22]. However, cholera toxin of classical type causes greater fluid accumulation in the intestinal lumen as compared to toxin of El Tor type [23]. Therefore, hybrid biotype property ofV. cholerae enhances the infection potential and adaptability in the environment showing their epidemiological successfulness. Outbreaks of such variant type strains are associated with more fluid loss and increased case fatality rate [24]. It has now become a global threat as most of the outbreak strains are associated with variant El Tor strains in India and other countries [3, 7, 16].All isolates from this outbreak were identified asV. cholerae O1 biotype El Tor serotype Ogawa. The isolates harboured various toxigenic and pathogenic genes (Table 1). The presence ofompW, ctxB, andrfbO1 genes confirmed theV. cholerae species, its toxigenicity, and group 1 antigen of the “O” side chain of the LPS (serogroup), respectively. Presence ofrtxA orrtxC genes determines the biotypes ofV. choleraeO1 [25, 26]. All isolates in this study belonged to the El Tor biotype on the basis of the repeat in toxin gene (rtxC).Sequencing ofctxB gene revealed that allV. cholerae O1 El Tor isolates in this study hadctxBsequence of the classical biotype. Classical biotype strains were replaced by the El Tor biotype in the seventh and current pandemic of cholera. Both biotypes ofV. choleraeO1 are closely related in their O-antigen biosynthetic genes. However, the genomic structure of the CTXΦ, in which the cholera toxin genes are contained, differs between the classical and El Tor biotypes [20, 27]. On the basis of genetic structures of the CTX prophage and RS1 element on each chromosome, atypical El tor strains have been classified into two different groups [17, 28]. Group 1 strains harbor a tandem repeat of the classical CTX prophage on the small chromosome and Group II strains contain the RS1 and CTX prophage with El Tor typerstR and classicalctxB on the large chromosome [28, 29]. All the strains used in this study belonged to Group II and contained the RS1 and CTX prophage with El Tor typerstRand classicalctxBon the large chromosome.Odisha is situated at the coast of Bay of Bengal and in general outbreaks in the state are linked to flooding and cyclones [10, 30]. The variant El Tor strains are circulating in Odisha since 2000 and appeared in Rayagada district in 2002 but the first outbreak due to this strain was recorded in 2007 [10, 31]. To the best of our knowledge, this was the first time when tetracycline resistant clone of variant El Tor strain caused an outbreak in Odisha. Recently, tetracycline resistantV. cholerae strains have been reported from several other parts of India [32, 33]. Tetracycline has been a drug of choice for control of severe cases of cholera [6]. Therefore, resistance for tetracycline further curtails the list of cost effective first-line drugs for control of cholera.All theVibrio choleraeisolates in this study harboured class 1 integron and SXT constins. In this region,qacEΔ1 andsul1 genes provide resistance to ammonium compounds and sulphonamides, respectively. All the isolates were positive foraadA2 anddfrA1 genes, which confer resistance to streptomycin and trimethoprim, respectively. However, all the isolates were PCR negative forblaP1involved in resistance for β-lactam antibiotics (Table 1). The SXT constin is a conjugative, self-transmissible, and integrative element that provides resistance to sulphamethoxazole, trimethoprim, chloramphenicol, and streptomycin. In this study, all isolates were susceptible to chloramphenicol and PCR negative forfloRgene. In addition, the gene for streptomycin resistance (strA) was invariably present in all the isolates.Integrons provide natural genetic engineering platforms that incorporate gene cassettes and convert them to functional genes by ensuring correct expression in bacteria. The present study showed that nowV. choleraeoutbreak strains carry the mobile antibiotic genetic elements and the horizontal transfer of these genes is the major cause of emergence of multidrug resistance strains. The commonly prescribed antibiotics in India for the treatment of diarrhea are fluoroquinolones (DNA damaging drugs) that induce horizontal transfer of SXT elements leading to inter- and intraspecies spread of multidrug resistance [34]. Therefore, use of such drugs should be closely monitored as most of the recent outbreak strains carry these elements [16, 19, 35].MIC of tetracycline decreased eightfold in the presence of CCCP, indicating the role of efflux pumps in tetracycline resistance [36]. However, the MIC for nalidixic acid (32 μg/mL) remained unaffected in the presence of CCCP indicating the involvement of some other mechanisms also for antibiotic resistance. Tetracycline resistance in Gram negative bacteria occurs due to the acquisition of genes encoding efflux pump and ribosomal protection proteins. Most of the efflux pumps belong to major facilitator superfamily (MFS), which export tetracycline from the cell and exchange a proton against the concentration gradient [37]. The isolates from this outbreak were PCR negative for the reported ribosomal protection protein Tet(M) in Vibrios (data not shown). Efflux mediated tetracycline resistance (tet genes) in Vibrios is mostly characterized in non-V. cholerae species [37]. Therefore, the detailed investigation of the mechanism is needed. In recent years, rise in antimicrobial resistance in clinical strains is a serious concern as it hampers outbreak control policies [10, 19, 35]. Thus, antimicrobial resistance can increase the outbreak size, duration, and case fatality rates. Continued monitoring of antimicrobial susceptibility as well as strain-tracking is important in adapting policies for cholera control at national and global levels.



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*Source: 1695410-2016-01-03.xml*