Detection of Vibrio cholerae and Acanthamoeba species from same natural water samples collected from different cholera endemic areas in Sudan

Background Vibrio cholerae O1 and V. cholerae O139 infect humans, causing the diarrheal and waterborne disease cholera, which is a worldwide health problem. V. cholerae and the free-living amoebae Acanthamoeba species are present in aquatic environments, including drinking water and it has shown that Acanthamoebae support bacterial growth and survival. Recently it has shown that Acanthamoeba species enhanced growth and survival of V. cholerae O1 and O139. Water samples from different cholera endemic areas in Sudan were collected with the aim to detect both V. cholerae and Acanthamoeba species from same natural water samples by polymerase chain reaction (PCR). Findings For the first time both V. cholerae and Acanthamoeba species were detected in same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected V. cholerae was found with Acanthamoeba in same water samples. Conclusions The current findings disclose Acanthamoedae as a biological factor enhancing survival of V. cholerae in nature.


Background
Vibrio cholerae species are widely distributed in aquatic environments [1]. They comprise nearly 200 serogroups based on the O antigenic structures [2]. V. cholerae O1 and V. cholerae O139 infect humans, causing the diarrheal and waterborne disease cholera [3], which is a worldwide health problem.
V. cholerae inhabits aquatic environments and human intestines [4], and cholera outbreaks are associated with contaminated food and water supplies. The seasonality of cholera has been associated with physical and biological factors [5]; however, many factors affect the survival of V. cholerae in aquatic environments such as attachment to plankton, loss to predators [6].
Acanthamoebae are free-living protozoa distributed worldwide in nature [7,8] and may affect the survival of V. cholerae. It is well known that V. cholerae and Acanthamoeba species are present in aquatic environments, including drinking water [9][10][11] and the use of water with poor microbiological quality increases the risk of human illness since acanthamoebae and bacteria are involved in complex interactions important to medical and environmental microbiology. It is known that acanthamoebae benefit from extracellular bacteria as food, which may enhance survival of the amoebae in different environments. In contrast, the role of Acanthamoebae as hosts for bacteria has been proposed for many pathogenic bacteria [12][13][14][15][16][17][18][19][20][21][22][23][24].
Recent publications [25][26][27][28] showed that Acanthamoeba species enhanced growth and survival of V. cholerae O1 and O139 in laboratory microcosm co-culture experiments, but no study has so far been published on the detection of both V. cholerae and Acanthamoeba from similar sites. In this context, cholera is endemic and poses  [29].
In this paper, water samples from different cholera endemic areas in Sudan were collected with the aim to detect both V. cholerae and Acanthamoeba species from similar sites of collected natural water samples by polymerase chain reaction (PCR).

Sample collection
Four hundred water samples collected from 4 states in Sudan previously known as foci of V. cholerae. The states are Gadarif, Juba, Kordofan and Khartoum. 128 samples were from zeers (home pots), 167 from hafirs (a hafir is an underground reservoir designed for storing rain water carried by streams and used for domestic water supply and for agricultural purposes in rural areas in the Sudan), and 66 from water tanks and 39 from lakes.
DNA extraction 50 ml water was centrifuged for 10 min at 4000 rpm and the pellets were used for DNA extraction using Qiagen DNA mini kit (Qiagen, Valencia, CA, USA).

DNA amplification
In the first reaction two primers sets were used. One set, referred to as the AcU primer 5'-GGC CCA GAT CGT TTA CCG TGA A-3' and the Ac L primer 5'-TCT CAC AAG CTG CTA GGG GAG TCA-3' and in the second reaction also two primers sets were used, one set referred to as the VCT-1 primer Gel analysis of PCR product PCR condition were: 32 cycles of 95°C (denaturation) for 4 min, 55°C (annealing) for 20 sec, and 72°C for 10 sec (extension). PCR products were analyzed by electrophoresis on agarose gel in 1× TBE buffer (Tri base, boric acid and EDTA (pH 8.0). The gel was stained in 0.1% SYBR Green bath, visualized by UV translumination, and photographed using Polaroid films. DNA fragment 487 bp for Acanthamoeba was obtained in the first reaction and DNA fragment 308 bp for Vibrio cholerae toxin was obtained in the second reaction.

Statistical analysis
χ2 test was performed for comparative statistical analysis of together-and alone-detected microorganisms to show the significant existence of alone V. cholerae or that found with Acanthamoeba.

Results and discussion
V. cholerae O1 and V. cholerae O139 are widely distributed in aquatic environments [17] causing the diarrheal and waterborne disease cholera [22]. V. cholerae and Acanthamoeba species are present in aquatic environments, including drinking water [9,16,19]. A number of studies report that free-living amoebae (FLA) support survival of pathogenic bacteria [19] and more studies are still needed on distribution of V. cholerae and FLA in nature [30]. In the current study we collected water samples from endemic areas in Sudan to detect V. cholerae and Acanthamoeba species in same natural water samples by PCR targeting cholera toxin gene (toxA) and Acanthamoeba 18 S RNA gene.
Analyzing presence of detected microorganisms showed that the detected number of together-and alone-identified microorganisms (amoebae and bacteria) (table 2) was significantly differed (p value of χ2 was < 0.05). V. cholerae needs to be found with other microorganisms such as Acanthamoebae a finding disclosed by this study since 89% of detected V. cholerae was found with Acanthamoebae compared to 11% V. cholerae, which was found alone. As regards amoebae 38% of Acanthamoebae was found with V. cholerae and 62% was found alone. Moreover, prevalence of V. cholerae alone was 0.25% and that of Acanthamoeba alone was 3.25%, while prevalence of both Acanthamoeba and V. cholerae was 2% (table 2). Taken together, this clearly shows that Acanthamoeba and V. cholerae can be isolated at similar sites but it does not disclose interaction between them. However, in previous studies it has been found that Acanthamoeba and V. cholerae interact in beneficial ways for both microorganisms and it could thus be speculated that such interaction is important for the microorganisms also in nature. [12][13][14][15][16][17][18][19][20][21][22][23][24].
For the first time this study show that both V. cholerae and Acanthamoeba species can be detected in the same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected V. cholerae was found with Acanthamoeba and 11% was found alone. Taken together role of Acanthamoeba species in survival of V. cholerae [18,25,26,31] may strongly disclose Acanthamoedae as a biological factor enhancing survival of V. cholerae in nature.
Acanthamoebae support bacterial survival and growth [12,25,26,31] and save the bacteria from the effects of chlorination [5], antibodies [5] and antibiotics [12,25,26,31], increasing the risk of human illness caused by bacteria or Acanthamoebae. Accordingly, a need to find an effective means of detection and killing of both Acanthamoeba and bacteria is warranted to reduce the risk of spread of V. cholerae.

Conclusions
89% of detected V. cholerae was found with Acanthamoeba disclosing Acanthamoedae as a biological factor enhancing survival of V. cholerae in nature.