Association of Vibrio cholerae with fresh water amoebae - Journal of ...

J. Med. Microbiol. - Vol. 36 (1992), 303-306
0 1992 The Pathological Society of Great Britain and Ireland
Association of Vibrio cholerae with fresh water amoebae
SUSAN THOM*, D. WARHURSTt and B. S. DRASAR
Departments of Clinical Sciences and t Medical Parasitolog y, London School of Hygiene and Tropical Medicine, Keppel
Street, London WC 1 E 7HT and *Department of Microbiology, The Medical School, Framlington Place, Newcastle upon
Tyne NE2 4HH
Introduction
Summary. An investigation was undertaken to determine whether Acanthamoeba polyphaga
SHI and Naegleria gruberi 1518/le could affect the survival of various strains of Vibrio
cholerae in laboratory microcosms. In microcosms pre-inoculated with trophozoites of
amoebae, all six strains of V. choZerae tested survived and multiplied during 24 h. In control
microcosms without trophozoites of amoebae, survival of the V. cholerae strains was much
decreased. Two strains of V. cholerae were used to determine whether V. cholerae might
survive ingestion within amoebae and subsequent encystment. Strain 152 was re-isolated
from excysting N. gruberi 15 1 8/ 1 e but not from A. polyphaga SHI. Strain 9 1 12 could not be
isolated from cysts of either species of amoebae.
Evidence for an aquatic reservoir of Vibrio cholerae
is derived from laboratory based investigations of the
effects of physico-chemical stresses on the survival of
V. cholerael and from field studies in which V. cholerae
was isolated from water without any evident association
with cases of clinical cholera.293 Furthermore, it
has been shown that V. cholerae can survive long
periods of low nutrient stress as coccoid ultramicrobacteria,
a possible resting stage4 formed by
reductive division and size reduction.' Despite the
isolation of V. cholerae from multiple environmental
sites and living organisms, including plants,6 plankton,7
shellfish' and fish,' the precise identity of the
ecological niche favouring its survival in an aquatic
environment remains obscure. However, other bacteria
are known to survive in association with amoebae
and symbiotic relationships between infecting bacteria
and host amoebae have been demonstrated. ''-I2
Legionella pneumophila has been shown to persist
within various species of amoebae. The fastidious
nature of L. pneumophiza in laboratory culture combined
with evidence of legionellae infecting macrophages
in man led some to postulate that it is not a
free-living aquatic organism' and to examine whether
it might be associated with free-living amoebae.14
Subsequently, it was shown that L. pneumophila could
infect and multiply within amoebae of Acanthamoeba
and Naegleria ~pp.'~-'~ This suggests that amoebae
may be the primary reservoir of L. pneumophila in the
environment.
However, there has been no similar investigation of
Received 5 Oct. 1990; revised version received 13 May 1991;
accepted 4 July 1991.
303
a potential association between V. cholerae in the
aquatic environment and free-living amoebae. If V.
cholerae could survive ingestion by amoebae, the
controlled local micro-environment would afford pro-
tection from external fluctuations. Furthermore, amoe-
bae encyst in response to environmental stress. The
cyst form protects the amoebae from desiccation and
food deprivation and it might also protect the vibrio.
Here the potential for survival of V. cholerae in
association with amoebic trophozoites and within
amoebic cysts was examined.
Materials and methods
Bacteria and amoebae
Suspensions of V. cholerae strains 17 (clinical isolate,
Tanzania), 102 (Tanzania), 152 (water, Dacca), 164
(Bangladesh), 91 12 (clinical isolate, Australia) and
9351 (water, Australia) were prepared in Page's
saline17 containing NaClO.l% (MPS), from overnight
cultures incubated at 25°C or 37°C on Trypticase Soy
Agar (TSA; Oxoid). The suspensions were adjusted to
an OD6'' of 0.04 and 3 ml were inoculated on to pure
adherent cultures of N. gruberi 1518/le and A.
polyphaga SHI trophozoites seeded at 105/ml in plastic
flat-sided tissue-culture tubes (Nunc Ltd). The amoe-
bae had been grown as axenic cultures at 30°C in
SCGYEM medium as modified by Aufy et al. ' ' Tubes
inoculated with bacteria alone served as controls and
were taken through the whole experimental procedure.
The amoebae culture tubes and controls were incu-
bated at 25", 30" and 37°C for 1 h in the trophozoite
survival experiment and for 1 or 3 h in the encystment
survival experiment. All experiments were performed
in duplicate.

304 S. THOM, D. WARHURST AND B. S. DRASAR
Survival u f V. cholerae in truphozuites
Amoebae were exposed to V. cholerae as outlined
above. After incubation for I h, unattached bacteria
were decanted and the tubes were rinsed with MPS.
Tubes were placed in an ice-bath to detach the
amoebae which were then resuspended in MPS.
Suspended amoebae (0.1 ml) were inoculated on to
non-nutrient agar (“A) overlaid with heat inacti-
vated Escherichia coli and incubated at 30°C for up to
7 days to confirm the presence of viable trophozoites.
Ten-fold serial dilutions were prepared from the
suspensions of amoebae, and from suspensions that
had been freeze-thawed once, and 0-1-ml portions
spread on to TSA at pH 8-6. Plates were incubated
overnight at 37°C and colonies were counted.
In a second series of experiments, following the
rinse after incubation for 1 h, the tubes were refilled
with MPS and re-incubated for 24h before further
processing as above.
Surfiival of V. cholerae in cysts
After challenge of amoebae with V. cholerae,
unattached bacteria were decanted and the tubes were
rinsed in MPS. Encystment medium” was added to
each tube and the tubes were incubated at room
temperature for 18 h. After centrifugation at 890 g for
2min, the supernate was discarded, the tubes were
washed twice in MPS containing HCl 0.5% v/v and
centrifuged at 890g for 2 min; the pellet was
resuspended in fresh MPS containing HCl 0.5% v/v
and incubated at room temperature for 18 h. The
centrifugation and washing procedures were then
repeated but with MPS as the wash medium. Finally,
the cysts were resuspended in MPS containing heat-
inactivated E. coli and incubated for up to 96 h at
room temperature. Samples (0- 1 ml) of the suspension
were plated on to NNA overlaid with E. coli, TSA and
Thiosulphate Citrate Bile Salts Agar (TCBS; Oxoid)
plates. The suspensions remaining were freeze-thawed
once and additional samples were spread on to TSA
4
1 24
a b
Time (h)
1 24
Fig. 1. Survival of V. cholerue strain 17, from inoculum grown at (a)
25-C or (b) 37‘C, with A. pofyphugu SHT trophozoites ( 105/ml) over
24 h. Amoeba co-cultures (X, 0, Q) and V. cholerue control
suspensions (H, 0, A) were incubated at 25°C (X, m), 30°C
(0.0) or 37°C (A, A).
and TCBS plates. All the plates were incubated
overnight at 37°C and colonies were counted.
Results and discussion
All six strains of V. cholerae survived in association
with trophozoites of both species of amoebae for 24 h.
In most co-cultures with amoebae, the viable count of
vibrios at the end of the 24-h period was greater than
in the original inoculum, whereas control cultures of
V. cholerae incubated in the absence of amoebae
showed a decrease in viable count. Typical results are
presented in figs. 1-4. Of the two strains of V. cholerae
tested for survival in amoebic cysts, strain 152 was
recovered from cysts of N. gruberi 1518/le (fig. 5) but
not from cysts of A. polyphaga SHI, whereas strain 17
could not be isolated from either species of amoeba
following encystation. Longer exposure of amoebae to
the inoculating dose of V. cholerae resulted in a greater
recovery of viable bacteria from amoebic cysts. No
viable V. cholerae was isolated from the control
cultures incubated without amoebic cysts (fig. 5).
V. cholerae has not previously been associated with
a
1 24
Time (h)
b
1 24
Fig. 2. Survival of V. cholerue strain 17, from inoculum grown at (a)
25°C or (b) 37°C with N. gruberi 1518/le trophozoites (105/ml)over
24 h. Amoeba co-cultures (X, 0, Q) and V. cholerue control
suspensions (m, 0, A) were incubated at 25°C (X, m), 30°C
(0,O) A).
or 37°C (A,
a
1 24 1
Time (hf
Fig. 3. Survival of V. cholerae strain 91 12, from an inoculum grown
at (a) 25°C or (b) 37”C, with A. pofyphugu SHT trophozoites (lo5/
ml) over 24 h. Amoeba co-cultures (X, 0, A) and V. cholerue control
suspensions (m, 0, A) were incubated at 25°C (X, m), 30°C
(0,O)
or 37°C Cn, A).
b
24

6.
2-
'/
.". . . . . . . .
1- .
a b
0 .
1 24 1 24
Time (h)
Fig. 4. Survival of V. cholerue strain 91 12, from inoculum grown at
(a) 25°C or (b) 37"C, with N. gruberi 1518/le trophozoites (105/ml)
over 24 h. Amoeba co-cultures (X, 0, A) and V. cholerae control
suspensions (R, 0, A) were incubated at 25°C (X, U), 30°C
(0,O) or 37°C (A, A).
6 1
a
Temperature PC)
Fig. 5. Survival of V. cholerae 152 in N. gruberi 1518/le cysts: (a)
before encystment ; (b) after encystment (1 8 h), treatment with HC1
0.5% v/v (18 h) and excystment (96 h); 0 V. cholerae control, V.
cholerue + N. gruberi co-culture.
an intracellular mode of existence. Unlike a number
of other enteric pathogens of man, such as shigellae,
entero-invasive E. coli, salmonellae and Yersinia
enterocolitica2' the pathogenesis of V. cholerae does
not involve cellular invasion. The results of this
investigation have shown that V. cholerae is capable
of survival within amoebae. The presence of A.
polyphaga or N. gruberi trophozoites increased the
survival of V. cholerae when compared to that in
control microcosms lacking amoebae. Different strains
of V. cholerae isolated from environmental and clinical
sources not only survived, but multiplied over 24 h in
microcosms containing amoebic trophozoites. Control
cultures of V. cholerae incubated alone declined in
viability over the same period,
Furthermore, it was shown that V. cholerae could
survive encystment within N. gruberi cysts. The
introduction of an acid wash following encystment
ensured that all extra-amoeba1 V. cholerae and remain-
ing trophozoites were killed. All control cultures of V.
cholerae were sterile after this treatment. The re-
isolation of viable V. cholerae after excysting of
b
V. CHOLERAE AND WATER AMOEBAE 305
Table. Number of experiments in which viable V. cholerae
152 was isolated from N. gruberi 15 18/le cysts
Vibrio Experimental Number of
growth incubation isolations
temperature ("C) temperature ("C) (total expts)
25
25
25
37
37
37
25
30
37
25
30
31
amoebic cultures demonstrated that the vibrio was
ingested by the trophozoites and that it could survive
within amoebic cysts (table).
No single test condition exclusively favoured sur-
vival of V. cholerae within amoebae. It is interesting
to note that of the two strains of V. cholerae tested for
survival in cysts, it was the environmental isolate and
not the clinical isolate that survived ingestion and
subsequent encystment by the environmental species
of amoebae (N. gruberi). However, as only two species
of amoebae and two strains of V. cholerae were used
in the encystment experiments, no firm conclusions
on inter-strain variations can yet be drawn.
The results of this study are compatible with those
of King et a1.21 who investigated the susceptibility of
coliform bacteria and bacterial pathogens to free
chlorine residuels in the presence or absence of
amoebae and ciliate protozoa. Their results led to the
proposal that resistance to digestion by predatory
protozoa was an evolutionary precursor of bacterial
pathogenicity and a survival mechanism for bacteria
in aquatic environments.
In the context of this hypothesis,21 a study of the
phylogenetic relationship between Chlamydia (obli-
gate intracellular parasites) and other bacteria gave
interesting results in that ribosomal RNA from
Chlamydia hybridised preferentially with DNA from
V. cholerae.
The combination of these results and those from the
current study indicate that V. cholerae may have an
intra-cellular/amoebal habitat. An intra-amoeba1 hab-
itat would not necessitate an invasive capacity since
internalisation of the vibrio would be effected by the
amoebae. That no single combination of conditions
tested particularly favoured an association with amoe-
bae does not detract from the significance of the
association. The survival of V. cholerae within cysts of
certain species of amoebae for long periods would
provide a protected niche under unfavourable condi-
tions and a means of dispersal. An alteration in
conditions could result in the emergence of the
trophozoite from the cyst, the intra-amoeba1 multipli-
cation of the vibrio, and its subsequent release into
the environment.
This work was supported by the Wellcome Trust. We thank S.
Kilvington (Public Health Laboratory, Bath) for supplying axenic
cultures of the two free-living amoebae.

306 S. THOM. D. WARHURST AND B. S. DRASAR
References
1. Miller CJ, Drasar BS, Feachem RG. Response of toxigenic
Vibrio cholerae 01 to physico-chemical stresses in aquatic
environments. J H-vg 1984; 93: 475-495.
2. Bashford DJ, Donovan TJ, Furniss AL, Lee JV. Vibrio cholerae
in Kent. Lancef 1979; 1 : 436-437.
3. Colwell RR, Kaper J, Joseph SW. Vibrio cholerae, Vibrio
parahuemoi-vticus, and other vibrios: Occurrence and distribution
in Chesapeake Bay. Science 1977; 198: 394-396.
4. Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ,
Colwell RR. Survival and viability of nonculturable
Escherichia coli and Vibrio cholerae in the estuarine and
marine environment. Microb Ecoil982; 8: 313-323.
5. Hood MA, Guckert JB, White DC, Deck F. Effect of nutrient
deprivation on lipid, carbohydrate, DNA, RNA and
protein levels in Vibrio cholerae. Appl Enuiron Microbiol
1986; 52: 788-793.
6. Spira WM, Huq A, Ahmed QS, Saeed YA. Uptake of Vibrio
cholerae biotype eltor from contaminated water by water
hyacinth (Eichornia crassips). Appl Environ Microbioll98 1 ;
42: 550--553.
7. Colwell RR, Kaper J, Seidler R et al. Isolation of 01 and non-
01 Vibriu cholerae from estuaries and brackish water
environments. In: Proceedings of the fifteenth joint
conference on cholera. The US-Japan Cooperative Medical
Science Program. 1980 : 44-60.
8. Blake PA, Rosenberg ML, Bandeira Costa J, Soares Ferriera
P, Levy Guimaraes C, Gangarosa EJ. Cholera in Portugal,
1974. I. Modes of transmission. Am J Epidemioll977; 105:
337-343.
9. Gyobu Y, Kodama H, Uetake H, Katsuda S. Studies on the
enteropathogenic mechanism of non-0 1 Vibrio cholerae
isolated from the environment and fish in Toyama
perfecture. Microbiol Immun 1984; 28: 735-745.
10. Jeon K W. Development of cellular dependence on infective
organisms: micrurgical studies in amoebas. Science 1972;
176: 1122-1123.
11. Hall J, Voelz H. Bacterial endosymbionts of Acanfhamoeba sp.
J Parasitoll985; 71 : 89-95.
12. Mirelman D. Ameba-bacterium relationship in amebiasis.
Microbiol Rev 1987; 51 : 272-284.
13. Tison DL, Hope DH, Cherry WB, Fliermans CB. Growth of
Legionellapneumophila in association with blue-green algae
(Cyanobacteria). Appl Enuiron Microbiol 1980 ; 39 : 456-
459.
14. Rowbotham TJ. Preliminary report on the pathogenicity of
Legwnella pneumophila for freshwater and soil amoebae. J
CIin Patholl980; 33: 1179-1 183.
15. Anand CM, Skinner AR, Malic A, Kurtz JB. Interaction of L.
pneumophila and a free living amoeba (Acanthamoeba
palestinensis). J Hyg 1983; 91 : 167-178.
16. Holden EP, Winkler HW, Wood DO, Leinbach ED. Intracellular
growth of Legionellapneumophila within Acanthamoeba
castellanii Neff. Infect Immun 1984; 45: 18-24.
17. Page FC. Taxonomic criteria for limax amoebae, with descriptionsof
3 new speciesof Hartmannella and 3 of Vahlkampjia.
J Protozooll967 ; 14: 499-52 1.
18. Aufy F, Kilvington S, Mann PG, Warhurst DC. Improved
selective isolation of Naegleria fowleri from the environment.
Trans R Soc Trop Med Hyg 1986; 80: 350-351.
19. Neff RJ, Ray SA, Benton WF, Wilborn M. Induction of
synchronous encystment (differentiation) in Acanthamoeba
sp. Methods Cell Physioll964; 1 : 53-83.
20. Formal SB, Hale TL, Sansonetti PJ. Invasiveenteric pathogens.
Rev Infect Dis 1983; 5: S702-S707.
21. King CH, Shotts EB, Wooley RE, Porter KG. Survival of
coliforms and bacterial pathogens within protozoa during
chlorination. Appl Enuiron Microbioll988; 54: 3023-3033.
22. Palme L, Falkow S. Characterization of cloned genes from
Chlamydia trachomatis. In : Lieve L, Bonventre PF, Morello
JA, Silver SD, Wu HC (eds) Microbiology--1986. Washington
DC, American Society for Microbiology. 1986: 91-
95.