Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages.

Abstract

The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.

Full text

Page 1

Seasonal epidemics of cholera inversely correlate
with the prevalence of environmental cholera phages
Shah M. Faruque*, Iftekhar Bin Naser*, M. Johirul Islam*, A. S. G. Faruque*, A. N. Ghosh†, G. Balakrish Nair*,
David A. Sack*, and John J. Mekalanos‡§
*Molecular Genetics Laboratory, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka-1212, Bangladesh;†National Institute of Cholera
and Enteric Diseases, Calcutta 700010, India; and‡Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue,
Boston, MA 02115
Contributed by John J. Mekalanos, December 7, 2004
The relationship among (i) the local incidence of cholera, (ii) the
prevalence in the aquatic environment of Vibrio cholerae, and (iii)
bacterial viruses that attack potentially virulent O1 and O139
serogroup strains of this organism (cholera phages) was studied in
Dhaka, Bangladesh. Over nearly a 3-year period, we found that
significantly more environmental water samples contained either
a phage or a phage-susceptible V. cholerae strain than both (P <
0.00001). The number of cholera patients varied seasonally during
this period and frequently coincided with the presence of patho-
genic V. cholerae strains in water samples that otherwise lacked
detectable cholera phages. Interepidemic periods were character-
ized by water samples containing cholera phages but no viable
bacteria. Our data support the conclusion that cholera phages can
influence cholera seasonality and may also play a role in emer-
gence of new V. cholerae pandemic serogroups or clones.
bacteriophage ? seasonality ? epidemiology ? emergence ? lysogeny
E
health problem in many developing countries of Asia, Africa,
and Latin America (1). Cholera epidemics occur with seasonal
regularity in the Ganges delta region of Bangladesh and India.
Epidemics usually occur twice during a year, with the highest
number of cases just after the monsoon during September to
December. A somewhat smaller peak of cholera cases also is
observed during the spring, between March and May. Although
V. cholerae is a human pathogen, these bacteria constitute part
of the normal aquatic flora in estuarine environments, and water
is clearly a vehicle for transmission of V. cholerae. Although the
seasonality of cholera in Bangladesh and elsewhere has been
temporally associated with numerous physical and biological
parameters (2), these associations do not directly cause epidem-
ics, nor do they end them. More than a century of public health
experience has shown that toxigenic O1 and O139 V. cholerae
cells cause cholera epidemics and that the elimination of these
cells from drinking water ends cholera epidemics. The param-
eters that directly modulate the level of viable cells belonging to
the pathogenic clones of V. cholerae O1 and O139 in the Ganges
delta aquatic environment remain unknown. Furthermore, the
fact that pathogenic strains of V. cholerae are clonally distinct
from environmental, nonpathogenic V. cholerae strains (1) un-
dermines proposed mechanisms of seasonality and pandemic
spread that are based on data from studies measuring the
abundance of all Vibrio species in the aquatic environment (2).
Bacterial viruses (phages) are known to play a critical role in
the evolution of pathogenic bacterial species, and V. cholerae in
particular. For example, cholera toxin genes are transferred to
nontoxigenic strains by means of a lysogenic filamentous phage,
CTX? (3). Here we show that the presence of bacterial viruses
acting on V. cholerae O1 or O139 (cholera phages or vibrio-
phages) inversely correlates with the occurrence of viable V.
cholerae in the aquatic environment and the number of locally
reported cholera cases. We also demonstrate that some envi-
pidemics of cholera caused by toxigenic Vibrio cholerae
belonging to the O1 or O139 serogroups are a major public
ronmental V. cholerae strains of both epidemic and nonepidemic
serogroups carry lysogenic phages and produce phage particles
that kill epidemic strains. We identified at least one common O1
phage that is able to use several V. cholerae non-O1?non-O139
strains as alternative hosts. Such alternative hosts and lysogenic
environmentalV.choleraestrainsmaypotentiallyactascofactors
in promoting cholera phage ‘‘blooms’’ within aquatic environ-
ments and thus negatively influence transmission of phage-
sensitive, pathogenic V. cholerae strains by aquatic vehicles.
Materials and Methods
Detection and Isolation of Phages. Environmental water samples
were obtained in sterile containers, and initial processing of the
samples to detect vibriophages and to culture for V. cholerae was
done within 3 h of collection. For detection of phages, logarith-
mic-phase cells (500 ?l) of each of 10 host bacterial strains in
nutrient broth (Difco) were mixed with 3.5-ml aliquots of soft
agar (nutrient broth containing 0.8% Bactoagar, Difco), and the
mixtures were overlaid on nutrient agar plates. The 10 strains
used for screening vibriophages were G-3669 (El Tor), G-7555
(El Tor), P-27457 (El Tor), AM-33363 (El Tor), AP-13550 (El
Tor), AT-2352515 (El Tor), AL-30457 (El Tor), AI-885 (O139),
AI 1852 (O139), and AL-11089 (O139). Aliquots of water
(10–50 ?l), which were prefiltered through 0.22-?m-pore filters
(Millipore) to make them bacteria-free, were inoculated on the
plates, and the plates were incubated for 16 h at 37°C. Aliquots
of water samples mixed with serial dilutions of a control phage
strain (10–104particles per ml) were used as positive controls in
all phage assays. A sample was scored positive for phages when
aplaquewasobservedonthebacteriallawnintheplates.Plaques
were counted to estimate the concentration of phage particles in
the water. Phages from representative plaques were further
purified and used for the production of high-titer stocks. High-
titer phage stocks were used to prepare phage nucleic acids and
to test for host range, morphology by electron microscopy, and
DNA restriction patterns and hybridization (see Supporting
Materials and Methods, which is published as supporting infor-
mation on the PNAS web site).
Culture of Environmental Samples. Water samples were analyzed
for the presence of V. cholerae by methods described in ref. 4.
Briefly, an aliquot (35 ml) of each water sample was centrifuged
at 4,500 ? g, and the pellet was resuspended in 3.5 ml of 10 mM
PBS (pH 7.4). The suspension was vortexed to dislodge any
bacteria adhering to solid particles and then centrifuged at low
speed (1,000 ? g) to precipitate particulate matter. One milliliter
of this suspension was added to 10 ml of alkaline peptone water
(APW) [peptone 1% (wt/vol)?NaCl 1% (wt/vol), pH 8.5] con-
tained in 20-ml screw-cap glass tubes and incubated at 37°C with
shaking for 6–8 h. Dilutions of this APW culture were streaked
on taurocholate tellurite gelatin agar plates (5). Suspected Vibrio
§To whom correspondence should be addressed. E-mail: jmekalanos@hms.harvard.edu.
© 2005 by The National Academy of Sciences of the USA
1702–1707 ?
PNAS ?
February 1, 2005 ?
vol. 102 ?
no. 5 www.pnas.org?cgi?doi?10.1073?pnas.0408992102

Page 2

colonies were picked and subjected to biochemical and serolog-
ical tests to identify V. cholerae belonging to the O1 and O139
serogroups.
Data Analysis. Frequency of isolation of vibriophages and V.
cholerae from different water samples was summarized into
tables for group comparisons. Statistical analyses were per-
formed by using EPI INFO 6.0 (USD, Stone Mountain, GA). The
significance of difference in proportions was evaluated by the ?2
test.
Results
Influence of Environmental Vibriophages on Cholera. To study the
prevalence of vibriophages and V. cholerae in the environment,
we systematically analyzed water samples collected from two
major rivers and a lake in Dhaka, the capital of Bangladesh,
where cholera epidemics occur every year. Sampling was done
monthly during a period between January 2001 and November
2003. A panel of 10 different V. cholerae clinical isolates from
recent and previous epidemic outbreaks of cholera was used as
potential indicator strains to detect the presence of vibriophages
in water. All water samples also were analyzed for the presence
of V. cholerae by standard enrichment culture techniques and for
the presence of O1 and O139 serogroup strains by serological
methods.
We found that the majority of water samples showed an
inverse correlation between the presence of vibriophages capa-
ble of lysing a given serogroup of V. cholerae and the presence
of a strain of that same serogroup (Table 1). Of a total of 221
water samples analyzed from January 2001 to November 2003,
114 samples (51.5%) contained either a vibriophage or an
epidemic V. cholerae, whereas only 15 samples (7.2%) contained
both. Of these 15 samples, 10 samples contained V. cholerae that
were resistant to the phage present in the same sample. Overall,
the number of water samples containing both a vibriophage and
an epidemic V. cholerae strain susceptible to coisolated phages
was significantly less than that predicted by chance alone based
on the frequency of samples that contained either vibriophages
or phage-susceptible V. cholerae (P ? 0.00001). Thus, if a phage
lytic to epidemic V. cholerae O1 was present in a water sample,
the water was free of phage-susceptible V. cholerae O1 strains
(Table 3, which is published as supporting information on the
PNAS web site). Conversely, a majority of those water samples
that contained either V. cholerae O1 or O139 did not contain a
detectable phage that lysed the corresponding serogroup strain.
Many samples were obtained that carried an O1 strain but only
O139-specific phages (e.g., Gulshan Lake in July 2001, August
2002, January 2003, and March 2003 and Buriganga River in
October 2001 and October 2003). Similarly, other samples
contained an O139 strain but only O1-specific phages (e.g.,
Gulshan Lake in May 2002). If a V. cholerae strain of a specific
serogroup was present in a water sample together with a phage
thatrecognizedthesameserogroup,inmostofthesecases(73%)
the actual strain present was resistant to the coincidentally
isolated phage. To address the possibility that V. cholerae present
in the water sample were simply killed during processing of water
samples by phages present in samples, we performed control
experiments on ‘‘spiked’’ water samples containing known con-
centrations of V. cholerae O1 and O1 phages (Table 4, which is
published as supporting information on the PNAS web site).
These experiments supported the conclusions noted above in
that spiking water samples with phages only modestly reduced
the titers of viable V. cholerae and then only when phages were
added at levels ?100 plaque-forming units?ml.
Our finding of an inverse association between the presence of
virulent phages and phage-susceptible V. cholerae O1 or O139 in
environmental water led us to examine whether incidence of
cholera due to these two serogroups could also be correlated
with these findings. We investigated this possibility by using a
diarrhea surveillance program of the International Centre for
Diarrhoeal Disease Research in Dhaka, Bangladesh (ICDDR,
B) (6). Diarrhea patients in and around Dhaka city are usually
treated in the Dhaka diarrhea hospital of ICDDR, B. In the
Dhaka hospital, stool samples from 2% of all diarrhea patients
presenting for treatment are analyzed for the presence of all
known diarrheal pathogens, including V. cholerae. Monthly
numbers of cholera patients extrapolated from this 2% surveil-
lance system were compared with the monthly phage isolation
data during the same period. As shown in Fig. 1, cholera
epidemics due to either the O1 or O139 serogroup strains
generally coincided with a marked reduction in the prevalence
of vibriophages specific for the serogroup dominating a partic-
ular epidemic wave. For example, the early upward slope of
epidemic curves most often corresponded to periods of low
vibriophage isolation (e.g., see the O139-dominated epidemic
occurring from February to April of 2002 and the corresponding
bars for isolation of O139-specific bacteriophages in Fig. 1). This
inverse relationship between the prevalence of a serogroup-
specific vibriophage and the incidence of cholera due to that
serogroup of V. cholerae was seen on both sides of the epidemic
wave. Temporal blooms of serogroup-specific phages usually
coincided with the downward slope of epidemic curves domi-
nated by the corresponding serogroup of V. cholerae (e.g., see
May–September 2001, January–April 2003, and July–August
Table 1. Presence of specific vibriophages and susceptible V. cholerae strains belonging to the O1 and O139 serogroups in two major
rivers and a lake in Dhaka, Bangladesh, between January 2001 and November 2003
Source of water
No. of water
samples
analyzed*
(A)
No. of water samples (%)
P value†
Phage-positive and
V. cholerae-negative
(B)
Phage-negative and
V. cholerae-positive
(C)
Samples positive for
either phage or
V. cholerae
(B ? C)
Both phage- and susceptible
V. cholerae-positive
(D)
Gulshan Lake
Buriganga River
Turag River
Total samples
63
62
96
221
26 (41.2)
30 (48.3)
31 (32.2)
87 (39.3)
19 (30.1)
12 (19.3)
18 (18.7)
49 (22.1)
45 (71.4)
42 (67.7)
49 (51.0)
136 (61.5)
1 (1.5)
2 (3.2)
2 (2.0)
5 (2.2)
0.0001
0.004
0.02
?0.00001
*Samples were collected from two different points along each of the Gulshan Lake and Buriganga River and from three different points along the Turag River
inDhaka,Bangladesh.Sampleswerescoredasphage-positiveiftheycontainedphagesthatplaquedonO1orO139V.choleraeindicatorstrains.Sampleswere
scoredasV.cholerae-positiveiftheycontainedO1orO139strains.FifteensamplescontainedbothphagesandV.cholerae,but10ofthesestrainswereresistant
to the coisolated phage and, accordingly, were eliminated from column D in the analysis.
†Significance of D being lower numerically than expected by chance alone given A number of samples scored as B, C, or D. Statistical analyses were performed
by using EPI INFO 6.0. The significance of difference in proportions was evaluated by the ?2test.
Faruque et al.
PNAS ?
February 1, 2005 ?
vol. 102 ?
no. 5 ?
1703
MICROBIOLOGY

Page 3

2003 in Fig. 1). Additionally, on one occasion these dynamic
changes in serogroup-specific cholera incidence occurred out of
temporal register for O1 and O139. From January to March of
2002, when the incidence of O1 cases was falling, the incidence
of O139 cases was dramatically increasing (Fig. 1). Thus, envi-
ronmental factors (2) that might be conducive or inhibitory to
cholera epidemics (temperature, salinity, pH, UV light, aquatic
carbon, copepod, or plankton levels) either occasionally affect
the two serogroups of cholera differently, or, alternatively, some
other serogroup-specific parameter modulates temporal inci-
dence of the disease. If the presence of phages in the aquatic
environment has an adverse effect on cholera transmission, then
serogroup-specific phage predation provides a feasible param-
eter that might modulate the occasional temporal discordance
between epidemics caused by the two serogroups O1 and O139.
Diversity of Phages and Their V. cholerae Hosts. We characterized
vibriophages isolated from the environmental surveillance for
their genetic and phenotypic diversity (Table 2) and observed
their morphology by electron microscopy (Fig. 2 and Table 5,
which is published as supporting information on the PNAS web
site). During this period, we observed only six phage types, which
were designated JSF1, JSF2, JSF3, JSF4, JSF5, and JSF6 based
on the restriction patterns of their genomes. Whereas JSF1,
JSF4, and JSF5 genomes cross-hybridized, those of JSF2, JSF3,
and JSF6 were unique and did not cross-hybridize with any of the
other phages. Thus, the phages represented four genetically
unrelated groups. Phage JSF3 was specific for V. cholerae O139
strains, whereas all other phages were specific for V. cholerae O1
strains. However, JSF6 could use, in addition to O1 strains, a
number of V. cholerae non-O1?non-O139 strains as alternative
hosts; JSF6 phages produced from these non-O1?non-O139 host
strains were virulent against V. cholerae O1 strains (Fig. 3).
Production of Phages by Lysogenic V. cholerae. All phages except
JSF2 produced clear plaques, suggesting that these phages were
Fig. 1.
the International Centre for Diarrhoeal Disease Research hospital in Dhaka from 2001 to 2003. Number of cholera cases is extrapolated from a 2% surveillance
sample of all patients presenting for treatment. pfu, plaque-forming units.
Mean concentration of lytic vibriophages in the aquatic environment of Dhaka, Bangladesh, and the estimated number of cholera cases reporting to
Table 2. Characteristics of vibriophages isolated from surface water samples in Bangladesh between January
2001 and November 2003
Phage
designationHost specificity Plaque type
Isolation of
lysogens*
Phage production
by lysogens*Crossimmunity
DNA
homology
JSF1
JSF2
JSF3
JSF4
JSF5
JSF6
V. cholerae O1
V. cholerae O1
V. cholerae O139
V. cholerae O1
V. cholerae O1
V. cholerae O1 and
some non-O1
Clear
Turbid
Clear
Clear
Clear
Clear
?
?
?†
?†
?
?
?
?
?†
?†
?
?
JSF4, JSF5
None
None
JSF1, JSF5
JSF1, JSF4
None
JSF4, JSF5
None
None
JSF1, JSF5
JSF1, JSF4
None
*Minus sign indicates that the phage designated was negative for the property being scored; plus sign indicates that the phage was
positive for the property being scored.
†JSF3 and JSF4 phages appear to be primarily lytic but can occasionally persistently infect a host strain. Such persistently infected cells
shed virus and display viral immunity but do not show typical chromosomal junction fragments consistent with stable integration of
the viral genome into the bacterial chromosome.
1704 ?
www.pnas.org?cgi?doi?10.1073?pnas.0408992102 Faruque et al.

Page 4

lytic and highly virulent for V. cholerae. JSF2 was a temperate
phage related to the previously described ? group of phages (7,
8),asrevealedbyelectronmicroscopy(hexagonalisometrichead
of 52.3 ? 1.7 nm and tail of 89.7 ? 2.2 nm ? 15.9 ? 1.2 nm, with
cross-striations), and produced characteristic turbid plaques on
susceptible V. cholerae O1 strains. Temperate vibriophages
exemplified by the ? group of vibriophages can grow lytically
(killing the host cell and producing numerous progeny phage
particles) or form lysogenic V. cholerae cells (9, 10). Lysogenic
cells are immune to ? phages but carry dormant prophages that
can be induced to replicate lytically through signals that typically
cause DNA damage (e.g., UV irradiation). Furthermore, one
highly conserved gene (glo) encoded by ? vibriophages has been
shown to provide resistance to superinfection by other ? phages
through the process of surface exclusion (11). Thus, we reasoned
thatlysogenicbacterialcellsmightonoccasionshedviablephage
particles to which they as a population were clearly immune.
These phages in turn could amplify quickly if the aquatic
environment contained sufficient susceptible V. cholerae cells,
thus interrupting an ongoing cholera epidemic by reducing the
concentration of viable V. cholerae cells in water and their
transmission to new victims.
To evaluate this hypothesis, we analyzed all V. cholerae strains
isolated from the environment during the study period for the
presence of lysogenic phages by DNA hybridization using probes
derived from the genome of each of the six phage types isolated.
Several strains belonging to the O1, O139, or non-O1?non-O139
serogroups were found to carry lysogenic phages related to JSF2,
JSF3, or JSF4. Probing of Southern blots with labeled phage
DNA revealed patterns of hybridizing fragments derived from
these strains that matched those derived from the native phage
genomes (see Fig. 5, which is published as supporting informa-
tion on the PNAS web site). Probe-positive V. cholerae strains
were further tested for phage production in their culture super-
natant. These strains spontaneously produced significant titers
of the respective phages (between 102and 104particles per ml)
when freshly isolated. Furthermore, when treated with the DNA
damaging agent mitomycin C, most strains produced even higher
levels of phage particles. The phages produced by these lysogens
were active against V. cholerae O1 or O139 strains. However,
Fig. 2.Electron micrograph of vibriophages isolated from environmental waters in Bangladesh. (A–F) Morphology of phages JSF1–JSF6. (Scale bar: 50 nm.)
Fig.3.
platedonaV.choleraeO1clinicalstrainisolatedfromtherecentepidemic(A),JSF2phageisolatedfromwater(B)andfromalysogenicnon-O1?non-O139strain
(G) plated on an epidemic V. cholerae O1 strain, JSF3 phage plated on a V. cholerae O139 clinical strain (C), JSF6 phage plated on a V. cholerae O1 strain (F) and
an environmental non-O1?non-O139 strain (H), JSF6 phage produced from an environmental non-O1?non-O139 strain and plated on a clinical V. cholerae O1
strain (E), and JSF4 phage plated on a V. cholerae O1 strain (D).
PlaquemorphologyofdifferentvibriophagesonlawnsofsusceptibleV.choleraestrains.Descriptionofphagesandhoststrainsareasfollows:JSF1phage
Faruque et al.
PNAS ?
February 1, 2005 ?
vol. 102 ?
no. 5 ?
1705
MICROBIOLOGY

Page 5

consistent with their lysogenic nature, the environmental strains
of V. cholerae that produced phages after induction were resis-
tant to superinfection by the phage they elaborated.
Territorialism and V. cholerae Evolution. Together, these data sug-
gest a model for how environmental phages might influence the
occurrence of epidemics, cholera seasonality, and changes in
serogroup prevalence (Fig. 4). The inverse correlation between
the environmental concentration of vibriophages and the pres-
ence of susceptible V. cholerae strains in water suggests that
epidemics would most likely begin in periods of low phage
concentration (after floods and the monsoon season). Predict-
ably, this inverse correlation would not hold for V. cholerae
strains that were either phage-resistant (due to serogroup type)
or lysogenic for prevalent phages and thus immune to lysis by
these phages. However, such lysogenic cells might, after expo-
sure to DNA damaging agents such as sunlight (12), serve as a
source of phages that could attack nonlysogenic cells. If both
lysogenic strains and nonlysogenic strains competed for a similar
environmental niche, this competition would lead to a selective
pressure for strains to become phage-resistant through lysogeny
while simultaneously providing the lysogenized bacterium with
a ‘‘vibriocide’’ (i.e., phage) that could destroy nonlysogenic
competing strains. Jouravleva et al. (13) speculated that such
territorialism might be in play, given the differences in the
lysogeny and sensitivity of clinical isolates to V. cholerae fila-
mentous vibriophage 493. Our data showing that disease inci-
dence tracks inversely with the prevalence of lytic phages in
the environment also suggests that V. cholerae cells lysogenic
for phages might modulate epidemics through phage-related
territorialism.
Other than CTX?, the most common phage released by
toxigenic El Tor O1 strains are ?-like (7,10). The ?-like JSF2
phage was frequently isolated from water samples in this study,
together with a V. cholerae O1 strain that was lysogenized with
this phage (Table 3). In the case of ? phage lysogens, territori-
alism would be a highly successful strategy if ? lysogens could
also become resistant to other O1 phages. Indeed, several
non-O1serogroupsofV.cholerae(amongthem,O139andO141)
curiously carry ? prophages and produce particles that lyse only
O1 strains (8, 14). It was previously hypothesized that these
non-O1 strains were derived from precursor O1 strains that
happenedtobelysogenizedby?beforetheirconversiontoanew
non-O1 serogroup. In light of the data presented in this report,
we propose that these precursor O1 ? lysogens might have
switched to non-O1 serogroups to escape other non-?, O1-
specific phages amplified during O1 epidemics (e.g., JSF1, JSF4,
JSF5, and JSF6). This dynamic could have led to the emergence
of globally distributed pathogenic strains of both the O139 and
O141 serogroups as well as numerous other pathogenic non-
O1?non-O139 strains that carry prophages with O1 specificity
(Fig.4).Finally,phagesJSF1,JSF4,andJSF5lysed9of10strains
of the classical biotype examined. It is well known that the
seventh pandemic O1 clone of the El Tor biotype globally
displaced O1 strains of the classical biotype within a decade of
its introduction into South Asia (1). The sensitivity of O1
classical strains to phages currently prevalent in the Bangladesh
aquatic environment suggests that these phage types may have
played a role in the elimination of the previously highly success-
ful classical biotype.
Similarly, phage JSF6 grows on several non-O1?non-O139
hosts but also lyses O1 strains. It is apparent that the concen-
tration of permissive hosts for JSF6 in the aquatic environment
might lead to higher concentrations of JSF6 particles and render
the water unsuitable for propagation of an O1 epidemic. Thus,
environmental non-O1?non-O139 V. cholerae strains could in-
fluence the epidemiology of O1 and O139 strains by seeding the
environment with phages that kill O1 and O139 strains (Fig. 4).
The low incidence of cholera in the interepidemic period could
be explained by such an ecological role for non-O1?non-O139
environmental V. cholerae strains as lytic or lysogenic hosts for
phages that also kill O1 and O139 strains.
It has been previously reported that the abundance of other
aquatically adapted bacterial genera, such as Prochlorococcus
Fig. 4.
occur in waves, with different serogroups (e.g., O1 or O139) dominating. Corresponding conditions that exist in the aquatic environment are diagramed in the
circlesshown.Theabsenceofyellowphagespecificforyellowovalcellsprovidesanopportunityforthatserogrouptobegintheseasonalepidemicandtransmit
efficiently.However,yellowphageseventuallyamplifyintheenvironmentandattackthisserogroup,endingthatepidemic.Adifferentserogroup(redovalcells)
isresistanttotheyellowphage.Inthecaseshown,redcellsactuallycarryaprophage(yellowintracellularcircle)andthusshedyellowphage.Asecondepidemic
waveduetotheredserogroupfollowsandrunsitscourseuntilredphagesbloomandpreventenvironmentaltransmissionofthisserogroup.Theinterepidemic
period is dominated by sporadic disease due to other serogroups (blue cells) resistant to both phages. These strains usually lack typical virulence factors but are
more environmentally adapted than virulent strains. However, blue cells may harbor prophages that kill virulent serogroups and may acquire virulence
determinants by horizontal gene transfer. Thus, blue cells may eventually emerge to become a new epidemic serogroup.
Schematic model for the influence of phage on cholera seasonality and serogroup emergence. Seasonal cholera epidemics (increased cases over time)
1706 ?
www.pnas.org?cgi?doi?10.1073?pnas.0408992102 Faruque et al.