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Inland cholera in freshwater environs of north India

Neelam Taneja ⇑, Arti Mishra, Nitya Batra, Parakriti Gupta, Jaspreet Mahindroo, Balvinder Mohan
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

a r t i c l e i n f o a b s t r a c t

Article history: In the freshwater environment of north India, cholera appears seasonally in form of clusters as well as
Available online xxxx sporadically, accounting for a significant piece of the puzzle of cholera epidemiology. We describe a
number of cholera outbreaks with an average attack rate of 96.5/1000 but an overall low case fatality
Keywords: (0.17). Clinical cholera cases coincided with high rainfall and elevated temperatures, whereas isolation
Cholera outbreak of V. cholerae non-O1 non-O139 from water was dependent on temperature (p < 0.05) but was indepen-
Freshwater dent of rainfall and pH (p > 0.05). However, isolation from plankton samples correlated with increased
Sporadic cholera
temperature and pH (p < 0.05). A lag period of almost a month was observed between rising temperature
Vibrio cholerae
and increased isolation of V. cholerae from the environment, which in succession was followed by an
appearance of cholera cases in the community a month later. Our results suggested that the aquatic envi-
ronment can harbor highly divergent V. cholerae strains and serve as a reservoir for multiple V. cholera
virulence-associated genes that may be exchanged via mobile genetic elements. In agreement with
PFGE, AFLP data also proved that the V. cholerae O1 population was not clonal but was closely related.
Our investigation did not support the concept that seasonal cholera outbreaks occur by movement of a
single clonal strain across the region, as the clinical isolates from the same years were clearly different,
implying that continuous evolution of V. cholerae O1 strains occurs in the cholera endemic area.
Interestingly, the viable but non-culturable (VBNC) V. cholerae O1 cells were demonstrated in 2.21% sam-
ples from natural water bodies in addition to 40.69% samples from cholera-affected areas respectively.
This suggests that aquatic environs do harbor the pathogenic O1 strain, though the isolation of culturable
V. cholerae O1 is a rare event in the presence of relatively abundant non-O1 non-O139 isolates.
Ó 2019 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creative-
commons.org/licenses/by/4.0/).

1. Introduction have led to an expansion of slums and underdeveloped areas in

the adjacent regions of the city without proper drinking water sup-
Cholera is an ancient acute dehydrating illness which is both ply and sewage systems, resulting in certain endemic pockets from
easy to prevent and to treat, yet remains a deadly and ever- where the cholera cases are reported annually. In this region, cho-
present scourge, especially in low and middle income countries. lera is endemic and seasonal, occurring during hot and humid
One-third of India’s population lives under the threat of cholera months peaking with monsoons. This region does not exhibit the
with 411,700,175 persons being at risk [1]. Of 641,78 districts in bi-annual cycle (pre- and post-monsoon) of cholera due to distinct
India were designated as hotspots, which include Chandigarh and climatic factors and experiences a peak only during the monsoon
adjoining region in Punjab and Haryana [2]. Chandigarh was orig- months. The freshwater bodies in North India have zero salinity
inally built to house a population of a hundred thousand. However, [3]. Two epidemiological pictures are seen in seasonal cholera:
now the burgeoning population of the city has crossed one million. clusters/outbreaks of cholera affecting a defined geographical area
The city has an area of 114 square kilometers, of which 79.4 km is mostly due to accidental contamination of drinking water supplies
urban, 34.6 km is semi-urban, and 18 villages that surround the with sewage. Such clusters are well defined and may have satellite
city. Treated and chlorinated underground water is supplied cases appearing in nearby areas. The second scenario is sporadic
through pipes in most of the areas. However, water from tube wells cases not related to these clusters. These cases do not flare into
and hand pumps are socially acceptable for drinking. Some vil- outbreaks, have no conclusive evidence of contamination of drink-
lagers and pilgrims use water from ponds and wells because of reli- ing water supplies with sewage, and there is no history of travel or
gious reasons. The rapid growth of population and urbanization visit to an area having an outbreak. They mostly occur due to inter-
mittent contamination of water that may be linked to well water,
⇑ Corresponding author. tube well water or hand pump water or due to consuming
E-mail address: drneelampgi@yahoo.com (N. Taneja). untreated water. The formative work by Colwell et al. [4] and

https://doi.org/10.1016/j.vaccine.2019.06.038
0264-410X/Ó 2019 Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: N. Taneja, A. Mishra, N. Batra et al., Inland cholera in freshwater environs of north India, Vaccine, https://doi.org/10.1016/j.
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several investigators in the recent past has traced the potential occurred in July 1994 [7]. After that, V. cholerae O139 has not been
environmental and ecological niche where vibrios flourish and per- isolated and the majority of cases have been due to V. cholerae O1
sist. Several biotic and abiotic factors, viz. salinity, temperature, Ogawa. Since 2002, the region has witnessed many outbreaks
rainfall, and plankton are known to affect the ecology of V. cholerae, occurring annually. Phenotypic characterization showed that
and influence the transmission of cholera [5,6]. The ecology of the these outbreaks were due to V. cholerae O1 Ogawa except those
organism in the freshwater aquatic environs is poorly understood. in 2004, which were due to the Inaba serotype which emerged
In this review, we describe the cholera epidemiology in this region in this region and almost replaced Ogawa for a short period of
and try to explain the seasonality of cholera. Most of the work was time [8].
carried out at the Postgraduate Institute of Medical Education and The attack rate ranged from 8.6 to 280/1000 population with an
Research (PGIMER), a 2200-bed tertiary care referral center in average of 96.5/1000. A shift in the age distribution was noted. Till
Chandigarh, catering to a large population in neighboring states 2006, on an average, children below the age of 14 years accounted
of Punjab, Haryana, Himachal Pradesh, Jammu and Kashmir, Uttar- for more than 54% of cases. From 2006 onwards, on an average only
akhand and parts of Rajasthan. It provides surveillance and referral 35.8% of those affected were children. However, in the outbreaks of
services investigating and managing outbreaks of cholera, food Panchkula in 2009 and Ambala in 2011, 58% and 86% of persons
poisoning, and other food and water-borne infections which are affected were children. The male and female population appeared
very common in this geographical area. In a study conducted in to be equally affected, with a low case fatality (0.17), in comparison
collaboration with WHO, we found that every year 1400–31,000 to Kolkata (3%) [12] and Odisha (8.6%) [13]. The low case fatality
cases of suspected food- and water-borne infections were being reflects good health infrastructure and prompt investigation and
reported at the district public health labs (DPHLs) across Punjab, management of cholera cases. All the outbreaks were managed
Haryana and Uttarakhand [Unpublished data, WHO-PGI, APW by rapid response public health teams which supplied drinking
project]. water from tankers, repaired the leaks in drinking water pipes, dis-
tributed ORS packets and provided intravenous hydration to
patients with moderate to severe dehydration. In many areas,
2. Outbreaks of cholera
hyper-chlorination of contaminated water supplies was tried but
was not found to be effective. Prophylactic antibiotics (doxycy-
Figs. 1a, 1b, 1c depicts the outbreak affected areas and year wise
cline/ciprofloxacin) were given to only household contacts of cho-
number of cases. An interactive map showing location of cholera
lera cases, especially children under the age of five years where
outbreaks with number of cases and deaths in and around Chandi-
mortality is expected to be high.
garh is available as a supplementary.kml file. The attack rates, age/
The outbreaks with a similar scenario quite commonly occur in
sex distribution are shown in Fig. 2a whereas Fig. 2b shows the
many parts of India. During 1997–2006, 68 outbreaks were
number of cholera cases reported, total stool samples tested and
reported in India with a total of 37,783 cases and 84 deaths [14].
culture confirmed cases from 2002 to 16. All outbreaks except of
Almost 90% of the cholera cases were reported from the states of
2012 were investigated by PGIMER. Published data is referred to
Odisha, West Bengal, Assam, and Chhattisgarh [14]. According to
[7–11]. Rest of the data is from the records maintained by PGIMER.
a descriptive analysis of published outbreaks, 59 outbreaks of cho-
Most of the outbreaks occurred due to contamination of piped
lera were reported in India from 2003 to 2012 [15]. However, a
drinking water supplies with sewage. In overcrowded rehabilita-
majority of cholera outbreaks go unreported in our country due
tion colonies, because of the low pressure in water supplies people
to limited laboratory diagnostic capacities and reluctance of health
have installed booster pumps on the direct supply line that leads to
authorities to admit and report cholera cases in their region for fear
sucking of sewage from alongside running sewage pipes. In one of
the areas, which are very prone to cholera outbreaks, the rusted of administrative and societal repercussions [14]. Some recent out-
water supply, and sewage pipelines run through a nullah (drain), breaks similar to the ones we have described have occurred in
which receives rainwater mixed with sewage. Rains increase the Medipally village and Medak district in Andhra Pradesh, Bagalkot
level of surface water and further enhance the chances of mixing in Karnataka and rural North Karnataka [16–19].
of stagnant water through broken pipelines.
The number of cases in each cluster varied from a few cases to 3. Sporadic cholera
>1000 cases. Cholera was quiescent before 2002 in Chandigarh,
with few cases (four confirmed cases during 2000–01). A limited Overall PGIMER investigated 1117 cases of suspected cholera
outbreak due to V. cholerae O139 (36 cases, unpublished data) from 2000 to 15, out of which 17% were confirmed by culture

Fig. 1a. Study area in northern part of India.

Please cite this article as: N. Taneja, A. Mishra, N. Batra et al., Inland cholera in freshwater environs of north India, Vaccine, https://doi.org/10.1016/j.
vaccine.2019.06.038
 N. Taneja et al. / Vaccine xxx (xxxx) xxx 3

Fig. 1b. Cholera outbreaks reported in Chandigarh and nearby areas in states of Punjab and Haryana from 2002 to 2008.

Fig. 1c. Cholera outbreaks reported in Chandigarh and nearby areas in states of Punjab and Haryana from 2009 to 2016.

(Table 1). In all cases, 59% were children, out of which 13% were ranged 5% to 95% with an average of 31% (457/1457). In areas with
below the age of two years, and 33% were below the age of five gross contamination of water, other etiological agents were also
years. We have observed a decline in sporadic cases similar to isolated. During 2011, we collected a total of 336 stool samples
results from the IDH-based surveillance of cholera in Delhi where by active surveillance during cholera season at the primary health
prevalence of cholera positive cases are decreasing over the years care level from cases of moderate to severe acute diarrhea (Table 2).
from 48% in 2003 to 26% in 2012 [20]. These were submitted from the following district public health
labs: Moga (N = 58), Ambala (N = 38), Sangrur (N = 19), Panchkula
(N = 72), Chandigarh (N = 118) and Pauri (N = 32). These samples
4. Not all is cholera were collected using PGI-WHO referral system established by us.
In 8% of cases, multiple bacterial pathogens are also found includ-
Though we collected a relatively large number of stool speci- ing concomitant Enterotoxigenic E. coli (ETEC) and Enteroaggregative
mens (7603) during the outbreaks, recovery of cholera bacteria E. coli (EAEC) mainly.

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5. Ecological and environmental factors responsible for

seasonal cholera

We conducted an environmental and ecological surveillance in

our region to understand the seasonality of cholera. The influence
of environmental parameters, including abiotic factors (tempera-
ture, salinity, pH, rainfall) and biotic factors (phytoplanktons and
zooplanktons) on the prevalence and isolation of V. cholerae was
measured [2]. The northern part of India has a dense network of
many major rivers and several freshwater lakes. This region
receives heavy rainfall during the monsoon months (July–October)
and exhibits high temperatures >30 °C during the summer and
rainy season (April–October). The winter months (November–
March) exhibit temperature below 20 °C and little rainfall. The
clinical cholera cases generally exhibit an annual peak from May
to October, coinciding with high rainfall and elevated temperatures
(Fig. 3).
Since isolation of serogroups O1 and O139 from the environ-
ment samples using conventional culture methods is a rare event
Fig. 2a. The cholera attack rate observed in males and children from 2002 to 16. [21], we found that in this case as well, the pathogenic isolates
were cultured from the water and sewage during the epidemic per-
iod, so we correlated the isolation of V. cholerae non-O1 non-O139
with other environmental and ecological factors to understand
cholera dynamics. All the abiotic and biotic factors vary in this
region with season, except salinity which was almost constant
throughout the year. The isolation of V. cholerae non-O1 non-
O139 varied in different seasons, with a peak during summers
(69%) and monsoons (46.5%) and was minimal in winters (15.5%,
p < 0.05) (Fig. 4), although isolation either from water and plankton
samples was comparable. The phytoplanktonic growth was
assessed by measuring chlorophyll a levels [3]. The isolation of
V. cholerae non-O1 non-O139 from water was dependent on tem-
perature (p < 0.05) but was independent of rainfall, chlorophyll a
and pH (p > 0.05). However, the isolation from the plankton sam-
ples correlated with increased temperature and pH (p < 0.05).
The plankton population in the freshwater aquatic ecosystem
was composed mainly of phytoplanktons and zooplanktons. It is
understandable as the temperature, along with nutrients and sun-
light, is known to affect the growth of these phytoplanktons; which
in succession modify the dissolved O2 and CO2 content of water
and, therefore, alter the pH of water [22]. The pH of water in fresh-
Fig. 2b. The number of cholera cases reported, total stool samples tested and water lakes significantly increased with plankton blooms from
culture confirmed cases from 2002 to 16.
5.25 to –7.0. These algae sometimes result in bright green blooms

Table 1
Presentation of sporadic cholera cases at PGIMER, Chandigarh.

Year Suspected Confirmed Age 0– Age >2 to Age >5 to Age >14 to Age Sex distribution Male:
cases cases 2 years 5 years 14 years 40 years >40 years Female
2000 27 3 3 0 0 0 0 3:0
2001 35 2 0 0 2 0 0 2:0
2002 69 8 2 3 1 2 0 4:4
2003 87 13 3 1 3 6 0 5:8
2004 133 14 2 0 1 11 0 9:5
2005 59 2 0 0 0 2 0 2:0
2006 71 8 0 0 1 5 2 6:2
2007 113 19 2 2 7 5 3 13:6
2008 87 36 4 9 13 5 4 20:16
2009 87 22 2 3 6 7 4 14:8
2010 44 5 0 0 1 1 3 3:2
2011 40 7 0 1 1 2 3 4:3
2012 125 28 3 13 5 1 6 16:12
2013 56 11 3 1 0 2 5 9:2
2014 50 11 1 6 2 1 1 10:1
2015 34 2 0 0 1 1 0 0:2
Total 1117 191 (17.1%) 25 (13%) 39 (20.4%) 44 (25.6%) 51 (25.6%) 31 (16.2%) 120:71

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Table 2
Result of stool samples collected during 2011 surveillance from District public health labs (DPHLS) through PGI-WHO network.

Isolated pathogens Region wise stool samples tested Total

Ambala Sangrur Moga Panchkula Uttarakhand Chandigarh
EAEC 15 5 21 30 24 18 113 (33.6%)
EPEC 0 4 9 8 1 21 43 (12.7%)
ETEC 1 1 3 5 1 5 16 (4.7%)
V. cholerae 4 0 4 13 1 1 23 (6.8%)
Aeromonas spp. 0 1 3 1 5 0 10 (2.8%)
Salmonella 0 0 1 1 1 0 3 (0.9%)
Shigella flexneri 1 0 0 2 0 4 7 (2.1%)
Bacillus cereus 2 0 0 1 0 0 3 (0.9%)
38 19 57 72 32 118 336

Fig. 3. Mapping of cholera cases with respect to temperature, rainfall and maximum relative humidity from 2009 to 16.

Fig. 4. Relationship between temperature, percentage isolation of V. cholerae non-O1 non-O139 from the environment, rainfall, appearance of V. cholerae O1 in environment
and cholera cases. The bidirectional arrows () represent the lag observed between rise in temperature, percentage isolation of V. cholerae non-O1 non-O139, rainfall and
appearance of cholera cases in the community. The figure has been adapted from Mishra et al., 2012 [12].

and further enhance the zooplankton population, which in succes- with copepods [22,24]. We also observed significant contribution
sion result in an increase in V. cholerae population. The plankton of these variables on the isolation of V. cholerae from the plankton
blooms are known to increase the pH up to 8.5 which influence samples. Fig. 4 explains the relationship between various environ-
the attachment of V. cholerae to copepods [23]. Temperature and mental parameters and appearance of cholera cases in our region.
salinity are also known to increase the association of V. cholerae With the onset of the rainfall, chances of a breach in sewage and

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contamination of drinking water supplies increases, and as gene distribution data from our region was similar to reports from
expected with a lag period of around a month from the onset of other studies from India and other parts of the world [21,36–42]. A
the rainy season the peak of cholera cases were seen. It was during study conducted in Bangladesh stated that, though V. cholerae iso-
this peak that V. cholerae O1 could be isolated from sewage and lates obtained by conventional culture methods were negative for
drinking water samples. the major virulence genes tcpA and ctxA, enrichment in rabbit
intestines resulted in the isolation of colonization-competent
6. Virulence factors and genetic diversity of environmental strains, and almost 56% of them carried genes encoding tcpA alone
V. cholerae vis-à-vis clinical isolates or both tcpA and ctxA; confirming the environmental reservoir of
pathogenic strains [21].
V. cholerae is known to be autochthonous to marine and estuar- We also studied the molecular epidemiology of clinical isolates
ine ecosystems, which act as a source and reservoir for human causing outbreaks as well as sporadic infections and genetic rela-
infections [25]. Based on variations in cell-surface lipopolysaccha- tionships between environmental V. cholerae and clinical isolates
rides, almost 200 serogroups (O1-O200) are known for V. cholerae, using molecular fingerprinting methods including – Pulse Field
out of which only serogroups O1 and O139 are pathogenic, and are Gel Electrophoresis (PFGE), ribotyping [46] and amplified frag-
associated with cholera [26–30]. The other serogroups are collec- ment length polymorphism (AFLP) [47]. It was established that
tively called as non-O1 non-O139 V. cholerae, which are seldom multiple pulsotypes were responsible for each outbreak and differ-
associated with diarrheal diseases less severe than cholera [31]. ent pulsotypes caused sporadic outbreak of cholera in the same
The virulence of V. cholerae is multi-factorial and is dependent on region. Ribotyping of the strains revealed the involvement of more
the synergistic action of a set of virulence genes. The virulence than one ribotypes during 2002 (RIII, RIV, UN1), 2003 (RIII, RIV),
genes include the CTXU element [32], and vibrio pathogenicity 2007 (RIV, UN1) and 2008 (RIV, UN2), while uniform ribotype pat-
island (VPI) [33], which encode for the main cholera toxin (CT) tern was observed during 2004 and 2006 (RIV). In our region, the
and a major colonization factor, toxin-coregulated pilus (TCP), ribotype RIII disappeared after 2003 and RIV became dominant
respectively. V. cholerae has an extensive horizontal gene acquisi- 2004 onwards [46]. The RIII is known to emerge in 1993, after
tion capability [34]. Therefore, the conversion of non-toxigenic the spread of O139 serogroup [48], while RIV appeared during
strains into pathogenic strains is always possible by the horizontal 2004–05 [49], however, in this region, RIV started existing from
gene transfer of CTXU and VPI elements. It is believed that the 2002 onwards.
transduction process in the environment can result in the conver- AFLP analysis helped in further exploring and understanding
sion of non-toxigenic environmental strains into toxigenic strains the relationship between environmental non-O1non-O139 iso-
[35]. The distribution of virulence genes in environmental strains lates, which are autochthonous to the aquatic ecosystem, and clin-
of V. cholerae have been shown in several published reports, further ical O1 pathogenic isolates. The AFLP was performed with six
supporting the possibility of an environmental origin of pathogenic primer combinations on 52 V. cholerae isolates, collected over a
V. cholerae [3,21,36–42, Table 3]. narrow time frame. We did a random comparison of almost 2.0%
The Vibrio cholerae was found abundantly in natural freshwater of the genome with an excellent resolving power of 0.99 [47]. All
bodies with isolation rate of 59.5% (N = 240) from water and plank- the V. cholerae O1 isolates irrespective of their origin, both from
ton samples; however, all of the isolates were confirmed as non-O1 patients or the environment, harbored the major virulence factors
non-O139 by agglutination and by the absence of rfb gene encod- ctxA and tcpA and belonged to a single sub-clade. The environmen-
ing for somatic O-antigen biosynthesis (O1 and O139) [43]. V. cho- tal non-O1non-O139 isolates, which did not possess major viru-
lerae O1 was isolated from 11.1% (N = 117) of environmental lence genes, were heterogeneous in their distribution. In
samples including water and sewage samples collected from agreement with PFGE, AFLP data also proved that the V. cholerae
cholera-affected areas during the time of outbreaks, signifying O1 population was not clonal but was closely related. This interro-
the presence of O1 isolates in contaminated water in cholera- gation does not support the concept that seasonal cholera out-
affected areas. Interestingly, the viable but non-culturable (VBNC) breaks occur by movement of a single clonal strain across the
V. cholerae O1 cells were demonstrated in 2.21% samples from nat- region, because the clinical isolates from the same years were
ural water bodies in addition to 40.69% samples from cholera- clearly different, implying that continuous evolution of V. cholerae
affected areas respectively [44]. This suggests that aquatic environs O1 strains occurs in the cholera-endemic area. The clinical isolates
do harbor the pathogenic O1 strain, though the isolation of cultur- from patients and V. cholerae O1 from the aquatic environment
able V. cholerae O1 is a rare event in the presence of relatively belonged to a single cluster, demonstrating that the aquatic
abundant non-O1 non-O139 isolates. ecosystem does play a role in the spread of cholera. The non-
All V. cholerae O1 isolates, whether clinical or environmental, O1non-O139 strains were very heterogeneous in their patterns
possessed ctxA, ctxB (classical type), tcpA and rstR (ElTor type), con- and were genetically different from the O1 strains. The precise role
firming circulation of ElTor variants in our region. All non-O1non- of non-O1non-O139 strains in the dynamics of cholera outbreaks is
O139 isolates were free of CTXUphage, as both ctxA and rstR were very difficult to explain. The possibility of conversion ofnon-O1
absent. The majority of non-O1 non-O139 isolates were confirmed non-O139 environmental strains into pathogenic O1 strains seem
free of another genetic element, VPI; as tcpA and toxT were absent. to be remote in our region, as the non-O1non-O139 isolates were
The gene tcpA was present in one, and toxT was present in three nonpathogenic and diverse, and moreover, only one clinical isolate
non-O1 non-O139 isolates only. Such tcpA+ strains are considered clustered with the environmental non-O1non-O139 strains [47]. In
to be selected in human intestines and become pathogenic upon a recent study from Vietnam, the phylogenetic analysis based on
transduction with CTXU lysogenic phage [28]. Further amplifica- whole-genome sequencing of four environmental V. cholerae O1
tion of ctxA and tcpA-positive V. cholerae is known to occur inside (ctxA- negative) isolates revealed a close genetic relationship with
the host, contributing to the beginning of an epidemic cycle which pathogenic strains [37]. This establishes that the aquatic environ-
spreads rapidly through environmental waters that are otherwise ment harbors genetically divergent V. cholerae strains which carry
free of pathogenic isolates [45]. Among the other accessory, repertoire of virulence-associated genes and these genetic ele-
genes studied – toxR, rtxA, rtxC and hylA – showed an equal distri- ments can be exchanged via mobile genetic elements. However,
bution in environmental and clinical isolates, depicting the envi- it is highly challenging to score the conversion of a non-toxigenic
ronmental repertoire of accessory virulence genes. This virulence strain to a pathogenic strain of epidemic potential.

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Table 3
Distribution of virulence genes reported among the V. cholerae isolated from environment.

Country & Year Serogroup Virulence Genes

ctxA ace zot tcpA tcpI toxR toxT rstR rtxA rtxC hlyA mshA vasA vasK vasH chxA TTSS
Vietnam 2017 O1 (n = 4) – NT NT NT +* + NT NT NT NT NT NT NT NT NT NT NT
[37] (100.0)
Thailand 2017 O1 (n = 4) – – – – NT + NT NT + (100.0) NT + (100.0) + NT NT NT 1 1
[38] (100.0) (75.0) (25.0) (25.0)
O139 (n = 4) – – – – NT + NT NT + (100.0) NT + (100.0) NT NT NT NT (0) (0)
(100.0)
Non-O1 non-O139 – – – – NT + NT NT + (100.0) NT + (100.0) + NT NT NT (0)
(n = 16) (100.0) (18.8) (18.8)
Kenya 2014 [39] V. cholerae (n = 50) + NT + + (12.0) + (8.0) + NT NT NT NT + [ElTor] NT NT NT NT NT NT
(12.0) (22.0) (24.0) (10.0)
+ [Classical]

N. Taneja et al. / Vaccine xxx (xxxx) xxx

(26.0)
Brazil 2014 [40] V. cholerae (n = 5) – NT – – NT NT NT NT NT NT NT NT NT NT NT NT NT
Bangladesh 2013 O1 (n = 50) + NT + NT + + NT NT + NT + NT + + + NT NT
[41] O139 (n = 31) + NT + + + + NT NT + NT + NT + + + NT NT
Non-O1 non-O139 + (2.0) NT + (2.0) + (0.2) + (2.0) + NT NT + (82.0– NT + (82.0) NT + + + NT NT
(n = 601) (90.0) 99.0) (28.0) (28.0) (28.0)
India 2011 [3] O1 (n = 13) + NT NT + [ElTor] NT + + + + (100.0) + + (100.0) + NT NT NT NT NT
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
Non-O1 non-O139 – – – + [ElTor] – + + (2.2) – + (94.7) + + (99.2) + NT NT NT NT NT
(n = 134) (0.7) (100.0) (97.7) (100.0)
Bangladesh 2004 O1 ElTor (n = 9) + NT NT + (66.6) NT NT NT + + (100.0) + + (100.0) + NT NT NT NT NT
[21] (44.4) (44.4) (100.0) (100%)
O139 (n = 2) + NT NT + (100.0) NT NT NT + + (100.0) + + (100.0) + NT NT NT NT NT
(100.0) (100.0) (100.0) (100.0)
Non-O1 non-O139 + (1.7) NT NT + (13.0) NT NT NT + (1.7) + (99.4) + + (98.2) + NT NT NT NT NT
(n = 169) (97.0) (100.0)
India 2001 [42] O1ElTor (n = 3) + + + + (100.0) + + NT NT NT NT + (100.0) NT NT NT NT NT NT
(100.0) (100.0) (100.0) (100.0) (100.0)
O139 (n = 4) + + + + (100.0) + + NT NT NT NT + (100.0) NT NT NT NT NT NT
(100.0) (100.0) (100.0) (100.0) (100.0)
Non-O1 non-O139 – – – – – + NT NT NT NT + (80.0) NT NT NT NT NT NT
(n = 5) (100.0)
India 2000 [36] Non-O1 non-O139 + NT NT + (79.1) NT + + NT NT NT NT NT NT NT NT NT NT
(n = 24) (20.8) (100%) (33.3)

Negative, + Positive, NT – Not tested.

*
new genotype cluster_11

7
8 N. Taneja et al. / Vaccine xxx (xxxx) xxx

7. Persistence of V. Cholerae as viable but nonculturable forms cells; however, the resuscitation requirements change with time.
in freshwater environs In the initial phase, the non-culturable cells could be recovered
by addition of sodium pyruvate, catalase, and cells extracts or tem-
V. cholerae is known to survive and persist in the adverse perature upshift but with extended non-conducive conditions, the
growth conditions by entering a dormant, viable but non- VBNCs cannot be resuscitated [68–70]. The VBNC state is a tran-
culturable (VBNC) state [50]. The VBNC bacteria are reduced in sient state and the heterogeneity in the bacterial population con-
size, alive, and metabolically active, but fail to grow on conven- taining dead, injured, viable cells make resuscitation process
tional bacteriological media on which they typically form colonies difficult to be measured accurately. However, the resuscitated pop-
[51,52]. The factors known to induce this state include – reduced ulation could be differentiated from regrowth based on the expres-
temperatures, nutrient starvation, elevated salinity, oxygen con- sion of vital genes [60,68]. In addition, the presence of viable cells
centration, and alkaline pH [53–55]. The bacterial cells in VBNC is a prerequisite for resuscitation. As a part of survival strategy, few
state become coccoid, exhibit reductive division and a decrease viable cells called scouts are retained by the dormant population.
in cell volume [56,57]. V. cholerae O1 VBNC cells retain its When conditions are conducive for growth, these scouts signal
pathogenicity as demonstrated – in rabbit ileal loops [58], gut col- the dormant cells to wake up by producing growth-promoting fac-
onization assay with iron-dextran-treated mice [59]– and continue tors, but if unfavorable conditions persist, the scouts simply die
to express virulence determinants including ctxAB, tcpA, rtxA and [71]. This is why an alternative term, ‘metabolically active but
hylA [60]. VBNCs resuscitate back into normal actively propagating non-culturable’ is applied to VBNC state as it remains uncertain,
forms on exposure to favorable growth conditions, such as– tem- whether it is a survival strategy or a consequence of deterioration
perature upshift [61], nutrients availability [62], passage through at a molecular level which protects partial features of viable cells
animal digestive tract [59,60,63] and ingestion by human volun- [72,73]. Nevertheless, there is tremendous support in the literature
teers [64]. Therefore, the role of VBNC state in the epidemiology for the existence of V. cholerae VBNCs and their potential role in the
of cholera is important also because of their potential to revert to cholera epidemiology. In our region, we could demonstrate VBNC
fully potent pathogenic form and presumably causing disease. V. cholerae O1 cells in 2.21% samples from natural water bodies
The viable but non-cultivable state represents an important in addition to 40.69% samples from cholera-affected areas respec-
environmental reservoir of pathogenic V. cholera O1. These have tively [43].
frequently been detected in the environmental water samples in
the cholera-endemic areas using direct fluorescent antibody- Declaration of Competing Interest
direct viable counting (DFA-DVC), where the bacterium couldn’t
be isolated using standard culture methods [62,65]. It was reported The authors declare that they have no known competing finan-
in V. vulnificus that in response to cold stress the cells become inca- cial interests or personal relationships that could have appeared
pable of detoxifying hydrogen peroxide, which is a lethal metabo- to influence the work reported in this paper.
lite, because of the absence of catalase activity or de novo synthesis
[66]. The variable gene expression in V. cholerae VBNCs in compar- Acknowledgements
ison to culturable cells have been reported, especially genes that
encode DNA polymerase II (polB), flagellar motor switch protein We acknowledge the help and support provided by local health
(fliG), iron (III) ABC transporter, and flagellin subunit protein C authorities in investigating and managing the outbreaks of cholera.
(flaC) were significantly up-regulated[59]. VBNC cells also express We also acknowledge the financial support of WHO India office,
genes involved in protein synthesis (tuf) and stress response (relA ICMR and Department of Science and Technology Chandigarh in
or rpoS) [67]. This continued expression of genes involved in cellu- carrying out most of this work. Technical support for PFGE and
lar processes and metabolism support that vital macromolecules reference strains were provided by NICED, Kolkata (kind courtesy
(RNA, DNA, proteins) are sufficiently available for VBNC cells to Dr. Ramamurthy and Dr. Nair). Dr. Ram Kumar of Institute of
resuscitate from dormant coccoid cells to active vibrioids. Himalayan Bioresource and Technology, Palampur, Himachal
Pradesh provided technical help for AFLP work.
All authors attest they meet the ICMJE criteria for authorship.
8. Induction and resuscitation of V. Cholerae O1 in VBNC state

We successfully demonstrated that V. cholerae O1 undergoes Appendix A. Supplementary material

both phenotypic and genotypic modulation to protect itself from
stress in fresh water [68]. Since the seminal work by Xu et al. in Supplementary data to this article can be found online at
1982 [50], different researchers have induced VBNCs in various https://doi.org/10.1016/j.vaccine.2019.06.038. These data include
microcosms, including autoclaved river water [58], alkaline seawa- Google maps of the most important areas described in this article.
ter [59,60,67,69] and river water [70]. The effect of abiotic factors
like different temperatures [67,70], nutrition starvation [59,68] References
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vaccine.2019.06.038
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