European International Journal of Science and Technology Vol. 4 No. 4 April, 2015

65

BDELLOVIBRIO-LIKE BACTERIA PARASITIZING IN THE

CELLS OF CANDIDA SPECIES

Sevda Muradova 1, Zakir Garaev

2 and Akif Gurbanov

3

1,2,3

Department of Microbiology and Immunology Azerbaijan Medical University,

Baku - AZ 1022, Baku, 23 Bakikhanov Street

Corresponding author:

Akif Gurbanov

Doctor of medical sciences, professor of department of Microbiology and Immunology,

Azerbaijan Medical University,

Baku – AZ 1022, Baku city, 23 Bakikhanov Street

Email: Akifgurbanov@mail.ru

Abstract

Bacteria-predators such as Bdellovibrio and similar microorganisms are widely encountered in

nature. They parasitize inside of bacteria especially in different species of gram-negative bacteria. However,

the relationship of similar bacteria with Candida spp. has not been reported. For the first time we have

found Bdellovibrio-like bacteria (BLB), which parasitizes inside of the Candida cells. The BLB were found

in all isolated Candida strains, in all nutrient media and their growth depends on the nutrient media used

for cultivation. By using an electron microscopy we have revealed that the BLB have two stages of life cycle:

the extracellular and intracellular, but unlike the Bdellovibrio, they multiply in intracellular vacuoles. As

Bdellovibrio, they dissolve and digest the host cells. We suggest that the relationship between BLB and

Candida cells is another example of microbial parasitism and deserves special attention for the treatment

and prevention of diseases caused by Candida spp.

Key words: Bdellovibrio-like bacteria, Candida spp., an intracellular parasitism.

European International Journal of Science and Technology ISSN: 2304-9693 www.eijst.org.uk

66

I. Background

Bacteria-predators such as Bdellovibrio and similar microorganisms are found in soil, freshwater and

marine waters. It is known that these microorganisms play an important role in the elimination of pathogens

in the environment. They dissolve and absorb both living and dead cells of bacteria, especially different

species of gram-negative bacteria such as Salmonella spp., Escherichia coli, etc. (1, 2).

There are a limited number of fungi that may contain intracellular bacteria-predators. It was

described that the bacteria localized within fungi cells vacuoles is associated with the membrane.

Presumably, the bacteria provide fungal cells with nutrients and protect them from adverse conditions (3, 4).

However, the relationship of similar bacteria with Candida spp. has not been reported and studied yet.

For the first time we have found Bdellovibrio-like bacteria (BLB), which parasitizes inside of the

cells of Candida spp. Based on the above, the aim of this investigation was a detailed study of the

relationship of BLB with Candida cells.

II. Materials and methods

2.1. Culture of Candida spp. It was used culture of Candida strains isolated from the patient. The

strains were incubated for 48 hours at a temperature of 370C on Sabouraud medium and were kept at 4

0C

temperature during 10 days.

2.2. The samples for electron microscopy investigation. The samples for electron microscopy are

prepared in accordance with known protocols (5). Thick suspension of Candida cells was centrifuged for 5

minutes at 5000 rev/min. After addition of the fixer (2.5% glutaraldehyde (pH 7.2) in 0.1m buffer solution),

the obtained deposit was kept in the fridge at 40C. To remove from the fixer the sediment was washed 3

times with 1M phosphate buffer and for secondary fixation was kept for 90 minutes in 1% osmium tetroxide

(OsO4). Then, in order to remove from osmium tetroxide, the sediment was washed 3 times with 1M

phosphate buffer. The sediment was kept in each different concentration (50%, 70% and 95%) of ethanol

during 10 minutes for dehydration. For the preparation of blocks, the samples are initially processed with a

mixture of different proportions of araldite epon and acetone. At the end, after addition of epon araldite for a

certain period of time, the sample was poured into the molds.

The semithin sections with a thickness of 1 micron from the obtained blocks were prepared using EM

UC 7 - Leica microtome and stained with 1% toluidine blue. The ultrathin sections with a thickness of 60

nm were prepared by using the microtome with a diamond knife. The obtained samples were stained in the

uranyl acetate and the plumbum citrate and examined under transmission electron microscope (JEM 1400,

Japan).

Investigations were carried out in the Electron Microscopy Laboratory of the Azerbaijan Medical

University.

III. Results and discussions

First of all, we observed the bacteria-predators in pure culture of Candida spp. obtained from vaginal

discharge, grown on Sabouraud agar as a lawn culture. Some sterile zones (“negative” colonies) were

observed on the surface of nutrient medium where Candida fungi had been cultivated. In the study of this

phenomenon it was revealed that the cell lysis of Candida spp. was associated with bacteria-predators. Like

Bdellovibrio, they expose the host cell to significant structural changes that lead to their death. 167 strains of

Candida spp. obtained from patients were used in the further investigation. The BLB were found in all

isolated Candida strains, in all nutrient media and their growth depended on the nutrient media used for

cultivation. Similarly, in the saline and distilled water having no nutrients for the growth of fungi, these

bacteria multiplied inside fungi cells with high intensity. Their growth was also possible to observe in old

European International Journal of Science and Technology Vol. 4 No. 4 April, 2015

67

cultures where nutrients in the medium get decreased. It should be noted that extracellular forms of bacteria

were found more frequently in liquid nutrient media than in the solid nutrient media.

We found that unlike the Bdellovibrio, the BLBs multiplied inside of intracellular vacuoles and had

the extracellular and intracellular stages of life cycle. These bacteria have active (motile) and inactive (non

motile) forms in the both stage. They multiply inside of the vacuole of Candida cell. This time they are

actively motile. When were fixed on the vacuole’s wall they become a dormant (non motile) form. After the

destruction of the vacuole they are trying to leave fungal cell membrane and through the defect of membrane

they are leaving the cell. Parasites leaving the cell have an active motility. Then they stick to any substrates

and become of round shape and dormant. As Bdellovibrio, they dissolved and digested the host cells. Figure

1 shows the Candida cells, which were observed under a light microscope for 35 days, where was a gradual

decrease in Candida cells up to their disappearance. Simultaneously, it was observed that the number of

extracellular forms of BLB was gradually increased. At the same time there were also met actively motile

forms of parasites.

In the initial investigations there were observed relationships of BLB with Candida spp. under light

microscopy in wet mount preparations. In the early days, there was formed vacuole in the cells of Candida,

and after a few days, motile microorganisms (1-2 individuals) were clearly observed inside of them. In the

following days the number of vacuoles became larger (2-4 and above), and they had different sizes, then

confluence with each other formed a single large vacuole, covering the whole cell. As a result of active

motility BLBs were fixed on the vacuole membrane of the host cell. Located on the vacuole membranes,

they were clearly visible on both stained and unstained preparations (Fig.2).

Release of mature parasites from the fungi cells, was accompanied with profound changing in the

host cell, especially violation of the integrity and strength of the cell membrane. Changes in the shape of

cell, led to formation a spherical forms. Despite the change in the fungal cells, they still remained viable and

could multiply considerably. In this case, parasites were transmitted to daughter cells through the vacuole.

At the end, destroyed components of the host cells were used as nutrition substrates by BLB.

The study of the relationship of BLBs with Candida cells under an electron microscope confirmed

our assumptions. Reproduction of BLBs caused significant changes in the cells of Candida spp. First of all,

there was a change in the spatial reorganization organelles with homogeneous structure formation. After

these, it was observed that the changes formed "empty" cells, and scarring formation in the leaving place of

parasites on the cell membrane (Fig. 3). At the end, an extracellular BLB formed cells with different sizes,

around which there was a capsule-like layer (Fig. 4).

The results of studing ultrastructural changes of Candida cells proved that BLBs parasitized in fungi

cells. As stated above, there are no available reports of such bacteria infecting Candida cells in the literature.

However, in some sources (6) the symbiotic relationship of Candida spp. with Helicobacter pylori is

described. The properties (small size, fast motility, nonculturablity, etc.) of these microorganisms

parasitizing in Candida cells are similar to bdellovibrions. But unlike bdellovibrions they multiply only in

the vacuole of the host cells. It is discussed the importance of these vacuoles in virulence and stability of

fungi, inside which we have found parasitic bacteria. In particular, there are sources that point to the

importance of these vacuoles in stress, in normal hyphal branching of Candida spp. (7, 8). But there is no

information about multiplication of the parasite-bacteria inside these vacuoles yet.

The dates about similar structural changes in Candida cells are also described in the literature. It is

described the degradation of eukaryotic cells with vacuol formation and its association with autophagy. A

group of scientists note the role of Golgi complex in the autophagy formation (9, 10). According to the

results of our study such changes of Candida cells are due to the parasite-bacteria but not with the autophagy

formation.

European International Journal of Scien

The multiplication of BLBs in va

their death. However, our investigations

"eat", and remains fragments of fungal

"negative" colonies of the fungi on the

idea.

Figure 1. Study of Candida cells in the

Figure 2. Bdellovibrio-like bacteria

membranes (methylene blue stain and

Figure 3. The phased struct

Bdellovibrio-like bacteria. A – ultr

reorganization of organelles with ho

consecutive destruction of the fungi c

comparison of the parasite’s leaving

mother cell.

A B

E F

ence and Technology ISSN: 2304-9693

vacuole of Candida cell leads to ultrastructural ch

ns show that they are not trying to kill fungal cell

cells used as nutrition substrates. Observation

e surface of a solid nutrition medium is another

he wet mount preparation during 35 days.

ia inside of Candida cells, fixed on the va

d wet mount preparation, x1000).

ctural changes in Candida cells, caused by

tra structure of Candida cells; B - the ce

omogeneous structure formation, C-F - stru

cell membrane; G - the parasite’s leaving fro

g from the host cell with the blastospore’s s

C D

G H

www.eijst.org.uk

changes and ultimately

ells, but gradually they

n of the sterile areas -

er confirmation of this

vacuole and the cell

by multiplication of

cell where is spatial

uctural changes and

rom the host cell; H -

separation from the

European International Journal of Scien

Figure 4. Electron microscopic feature

A - intracellular motile forms inside o

non motile forms.

IV. Conclusion

At the end we would like to no

microbial parasitism and it deserves spec

Candida cells by BLBs may be prospect

spp.

Acknowledgement

We thank to head of the Electr

professor Eldar Gasimov for direct assist

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