1. Introduction
n the microbial contamination of water and food, identifying small amounts of contaminating bacteria has always been a concern of researchers [7, 6, 5, 4]. Accordingly, it is necessary to concentrate the bacteria and increase its number. The most prevalent approach to solve this problem is to grow bacteria to elevate their number, which increases the time of bacterial detection to several hours or even days [9, 8]. An effective and smart solution to solve this problem is to concentrate bacteria by physical and non-cultured methods [17, 18, 19]. The present study aimed to enrich Vibrio cholerae (V. cholerae), as the most crucial microbial contaminant in water; thus, we used physical filtration and evaluated this yield by culture method and the diagnostic methods of ATP and PCR.
2. Materials and Methods
 V. cholerae bacterium obtained from the reference laboratory (Bouali Hospital) was initially confirmed using specific tests (specific culture medium & molecular PCR methods). Then, a certain concentration of bacteria was artificially transferred in a certain volume of sterile water. Next, with the help of Watman’s 0.45-micron filters, which contain detachable preservatives, bacteria were extracted from the environment and concentrated on the filter. Finally, the performance of the method after and before filtration using cell culture (determining & counting colony, CFU), ATP assay (using the leading Nuragen company kit & Hygina luminometer), and molecular PCR method (with specific primers of Vibrio cholerae ompW gene) were compared.
3. Results
To confirm the filtration method, V. cholerae was filtered 3 times with different concentrations. The present research results indicated that the physical filtration method in concentrating V. cholerae bacteria presents high efficiency and recycling performance. The sample, before filtration, provided no positive result in the ATP and PCR assay methods; however, after filtration, the presence of bacteria in both methods was observed and proven in isolated and recycled samples (Figures 1 & 2). The sensitivity of the filter method was also evaluated by PCR test, i.e. estimated according to Figure 3 and by comparing Figures 3a and b; accordingly, by revealing the PCR reaction results of concentrated and non-concentrated samples, the sensitivity of CFU 10¹ filter technique was estimated. 4. Discussion and Conclusion
 According to the obtained data, the filtration method in concentrating V. cholerae can be introduced as reliable and practical in removing contaminants, concentrating, and isolating bacteria. The main problem of the filter-based concentration method is the recycling of bacteria from the filter. With the method used, the bacterial recycling efficiency reached 100% (Figure 1). The same efficiency was observed in previous investigations. For example, in 1996, Hug et al. used the filter method to remove V. cholerae contamination from contaminated water, which also achieved 100% filtration separation efficiency [17]. Other methods of concentrating bacteria include the adsorption approach using magnetic nanoparticles Immunomagnetic Separation (IMS). Accordingly, in 2001, Hudson et al. could use this method to separate Listeria bacteria from meat samples and in <24 hours, the bacteria were isolated and identified by PCR [23]. 
The current research findings suggested that using a physical concentration strategy with filtration, V. cholerae can be detected in contaminated water samples in the shortest time without the need for culture.

Ethical Considerations
Compliance with ethical guidelines
This article is a meta-analysis with no human or animal sample.

Funding
This study was extracted from MSc. thesis of the first author at the Department of Biology, Faculty of Chemical Engineering, Malek-e-Ashtar University of Technology, Shahin Shahr. Also, this study was supported by the Research Institute of Biological Sciences and Technology of the Malek Ashtar University of Technology.

Authors' contributions
Conceptualization, methodology, writing – original draft, and writing – review & editing: Mehdi Zeinoddini; Investigation: Abolfazl Moradi.

Conflicts of interest
The authors disclosed no conflicts of interest.

Acknowledgements
We would like to thank the Research Institute of Biological Sciences and Technology of the Malek Ashtar University of Technology for their support.


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