Cholera remains a major risk in developing countries like Bangladesh, particularly after natural or man-made disasters and becoming increasingly resistant to antibiotics. Effective prevention strategies will be essential in reducing the disease burden of these bacterial infections. Here, we used the specificity and rapid-acting properties of bacteriophages as a potential prophylaxis therapy for cholera, a severely dehydrating disease caused by Vibrio cholerae 01 or 0139 serogroup. In this study, a single bacteriophage, JSF4 specific for V. cholerae 01 serogroup, was used to reduce the severity of cholera therapeutically in the infant mice model. Bacterial counts were decreased up to 106 times in the intestines of bacteriophage-treated animals and increased up to 24 times in the untreated control mice intestines. This is the first report that a single bacteriophage JSF4 might be useful to treat cholera caused by V. cholerae 01 serogroup strains and could be an alternative to antibiotics. In the future, JSF4 bacteriophages may also have profound implications in phage therapy for controlling cholera caused by pathogenic V. cholerae 01 serogroup strains.
The causative organism Vibrio cholerae is a facultative anaerobe, gram-negative, non-spore forming curved rod, about 1.04-1.06 µm long flagellated bacterium (Lekshmi et al., 2018; Maheshwari et al., 2011). Cholera (meaning being ‘a gutter) is the correct name for the disease, caused by the entry of contaminated water (Lekshmi et al., 2018). Due to poor infra-structure, sanitation, and access to clean drinking water in developing countries like Bangladesh cholera has become an emerging disease for centuries. It also flourishes at the time of societal disruption, such as natural calamities like the 2010 earthquake in Haiti or the current refugee crisis in Yemen (Orata et al., 2014; Rabaan et al., 2019).
V. cholerae is classified based on its somatic antigens (O antigens) into serovers or serogroups. Out of 200 serogroups of V. cholerae, only the serogroups O1 and O139 were the causative agent of current epidemics, and V. cholerae 01 is the major infectious agent (Orata et al., 2014; Rabaan et al., 2019). The non-O1/non-O139 biotypes do not cause cholera, because they do not contain TCP/CT genes, but in some cases where they may show diarrheal symptoms (Hasan et al., 2015). The O1 and O139 strains are prevalent in several endemic regions, including Yemen, parts of Africa, Southeast Asia, and Haiti (Orata et al.,2014; Kühn et al., 2014; Hsueh et al., 2019; Kirigia et al., 2009; Hossain et al., 2018; Khan et al., 2018; Leitner et al., 2015).
The first 6 pandemics (1816-1923) were caused by the Classical O1 biotype, whereas the seventh (1961 to the present) was caused by the El Tor biotype (Hu et al., 2016). The current pandemic affects 3-5 million persons per annum, causing 21000-143000 deaths (Hu et al., 2016; Ali et al., 2015). A healthy person within an hour of the onset symptoms may become hypotensive and may die between 2-3 hours if proper treatment is not provided (Todar et al., 2004). However, the World Health Organization gave the advice that only the severe cases of cholera should be treated with antibiotics due to the spread of antimicrobial resistance. Alternative approaches to control cholera are emergency needed. Biological control using bacteriophages is a good alternative, particularly where antibiotic resistance may be a problem (Czaplewski et al., 2016 ).
In this study, a novel V. cholerae 01 serogroup-specific bacteriophage JSF4 is used. This bacteriophage is very efficient to lyse both the clinical and environmental pathogenic V. cholerae 01 serogroup strains, and one of our previous reports showed that the seasonal epidemics of cholera caused by V. cholerae 01 serogroup strains in Bangladesh is mostly regulated by this bacteriophage (Faruque et al., 2005). So, we assumed that these bacteriophages could be an effective therapeutic agent for controlling cholera caused by V.cholerae 01 serogroup strains. In the current study, we showed that the oral administration of JSF4 bacteriophages could reduce V. cholerae 01 concentration in infant mice. Given the well-documented challenges associated with the emergence of antibiotic-resistant bacteria, bacteriophages might yet provide a viable alternative to antibiotics. So, in future, we can use JSF4 bacteriophages for the development of a new type of therapeutic agent against cholera.
Media preparation: Taurocholate tellurite gelatin agar (TTGA), a highly selective medium for the isolation of V. cholerae was used (Monsur et al.,1961). To prepare TTGA plates, 14 gm of TTGA powder (Hi-Media, India) was added in 1 litre of distilled water and autoclaved at 121°C for 15 minutes and pour 25 mL of media into each 10-cm Petri plate. To prepare Luria Agar (LA) plates, 14 gm of Luria Agar powder (Biomark Laboratories, India) was added in 500 mL of distilled water and sterilization was done by autoclaving at 121°C for 15 minutes.
To prepare liquid media, Nutrient Broth (NB), 13 gms of Nutrient Broth powder (Hi-Media, India) and 10 gms of NaCl were added in 1 litre of distilled water and autoclaved at 121°C for 15 minutes. To prepare semi-solid media (0.8% soft-agar for bacteriophage growth), 0.8 gm agar was added in 100 mL of NB and autoclaved it.
Growth of bacterial culture: Mainly V. cholerae 01 serogroup strain named N-16961 was used in this study. A single colony of V. cholerae 01 strain was inoculated into the nutrient broth and incubated at 37°C for overnight. To get a single colony of V. cholerae 01 strain, V. cholerae 01 cultured was streaked onto TTGA plate and incubated at 37°C for overnight.
Antibiotic sensitivity test (AST) through the disc diffusion method: Bacterial growth inhibition was determined by standard disc diffusion method (Bauer et al., 1966). Briefly, Petri plates were prepared by pouring 25 mL of Nutrient Agar (Biomark Laboratories, India) and allowed to solidify. Plates were dried and 100 µL of bacterial suspension containing 1x106 cells/mL was poured in each plate and spread uniformly. The plates were allowed to dry for 5 minutes. The antibiotic discs (Oxoid Ltd., UK) were then placed on the surface of the plates (Bauer et al.,1966). After that, the plates were incubated at 37°C for 24 h and the zone of inhibition was observed and measured in millimeters.
Collection, cultivation and harvest of JSF4 bacteriophages: We have collected V. cholerae 01 serogroup-specific bacteriophage JSF4 from Molecular Genetics Laboratory, International Centre for Diar-rhoeal Disease Research, Bangladesh (ICDDR, B) (as a kind gift from Dr Shah Mohammad Faruque, a former director of the Centre for Food and Water Borne Diseases in ICDDR, B). Faruque et al. isolated and purified this bacteriophage from environmental waters in Bangladesh (Faruque et al., 2005). After collection, we cultivated the phage on its host strain by the method described previously (Faruque et al., 2005). Briefly, the phage was inoculated in nutrient broth containing the host strain and incubated at 37°C for overnight with shake, then filtered the broth with 0.22µ pore size of filters (Millipore Corporation, Bedford, Mass.) to remove the bacteria and bacterial debris. The filtrate contained the phages and maintained the phage stock in normal saline at 4°C. We then detected and quantified the bacteriophages by the standard double-layer plaque assay method as described previously (Faruque et al., 2005) and below.
Laboratory animals: Swiss albino mice (5 days old) of either sex obtained from the Department of Pharmacy, Jahangirnagar University, Savar, Dhaka, Bangladesh were used in this study. The animals were kept in well-ventilated and hygienic compartments, maintained under standard environmental conditions, fed with standard rodent pellet and water.
Quantification of host bacterial strain at the feeding time and after incubation in mice intestines: The ability of JSF4 bacteriophages to reduce the severity of cholera was determined by using batches of ten 5-day old mice with modifications previously described method (Nishibuchi et al.,1983). We divided twenty 5-day-old infant mice into two batches (control batch and experimental batch). Each batch contained 10 mice. In control batch, each mouse was given 50 µL of overnight cultured bacterial suspension containing 3.94±0.54×108 cfu/mL orally. In mice of the experi-mental batch, we tried to feed the equal number of bacteria to each mouse like the control batch, but in practice, each mouse of the experimental batch was inoculated 3.96±0.42×108 cfu/mL orally. After 1 hour incubation, each mouse of the experimental batch was given 50 µL of JSF4 phage from the stock containing 2.04±0.31×109 pfu/mL orally. All mice were kept at RT for 18 hrs from initial inoculation time. After 18 hrs incubation, the mice were sacrificed, and the small intestines were removed and homogenized. The homogenates were serial diluted, plated on TTGA plates and incubated at 37°C for overnight. The colonies were counted and compared with the controls and initial input.
Quantification of JSF4 bacteriophages at the feeding time and after incubation in mice intestines: To determine how much JSF4 bacteriophages inoculated and how much increase after incubation in mice intestine, we performed it by plaque assay method described previously (Faruque et al., 2005). Briefly, logarithmic-phase cells (500µL) of host V. cholerae 01 strain (N-16961) in nutrient broth were mixed with 3.5-ml aliquots of soft agar (nutrient broth containing 0.8% agar), and the mixtures were overlayed on LA plates. Dilutions of bacteriophages were inoculated on the plates, and the plates were incubated for 16 h at 37°C. A sample was scored positive for bacteriophages when a plaque was observed on the bacterial lawn. To estimate the concentration of bacteriophage particles the plaques were counted in the sample and expressed as pfu/mL.
Antibiogram profile of V.cholerae 01 serogroup strain N-16961: This study needed a multi-drug resistant, pathogenic V.cholerae 01 serogroup strain as a host for JSF4 bacteriophages. The strain N-16961 was highly pathogenic and belonged to V.cholerae 01 serogroup (Islam et al., 2013), so we choose this strain as a host and determined its antibiogram profile through the disc diffusion method. From antibiotic sensitivity test, we have found that this strain is resistant to Ampicillin, Ceftriaxone, Methicilline and Trimethoprim. So, it is a multiple drug-resistant strain (Table 1). These types of multi-drug resistant strains are frequently emerged and make the treatment predicament through antibiotics. So, it is imperative to develop alternative prophylaxis therapy against such kinds of multiple drug-resistant pathogenic strains and phage therapy can be a suitable alternative.
Host specificity of JSF4 bacteriophages: Before inoculation of JSF4 bacteriophages into experimental mice, we determined its host specificity through the standard double layer plaque assay method (Faruque et al., 2005). From this assay, we have found that the bacteriophage JSF4 was 100% host specific and very efficient to lyse pathogenic V.cholerae 01 serogroup strain N-16961 (Fig 1A). Through its efficient lysis activity, we used this bacteriophage as a therapeutic agent in our current in vivo experiment.
Phage therapeutic activity of JSF4 bacteriophages inside mice intestines: To determine whether bacteriophage JSF4 acts as a therapeutic agent, we inoculated the pathogenic host V. cholerae 01 strain and its specific bacteriophage JSF4 in the experimental batch of 5 days old mice. In control mice, we fed only the bacteria. After 18h incubation, the mice were sacrificed, and the small intestines were removed and homogenized. From the homogenates, the bacteria were enumerated through the standard serial dilution plate count method. From this in vivo experiment, we have found that the bacterial counts in the intestines of phage-treated animals were reduced by up to 106 times (Fig 3) and increased up to 24 times in the untreated control mice intestines (Fig 2). This result indicated that JSF4 bacteriophages reduce the severity of cholera caused by pathogenic V. cholerae 01 strains. At the same time, we have enumerated JSF4 phages in the intestines of phage-treated animals through the plaque assay method (Fig 1B) and found that the number of phages increased up to 45 times (Fig 4) compared to the initial input. The increasing number of phage indicated that it reduces the severity of disease through its efficient lysing activity the host.
A novel treatment for cholera involves the therapeutic use of lytic bacteriophages. Phages are able to kill antibiotic-resistant bacteria and amount of phages increases proportionally to the number of infecting bacteria. This treatment strategy is inspired by the natural life cycle of V. cholerae in which blooms of the bacteria during outbreaks are followed by the expansion of lytic bacteriophage (Reyes-Robles et al., 2018; Silva-Valenzuela et al., 2019), The micro-organism we examined in this study was Gram-negative bacteria. It was suggested that the lipo-polysaccharides present in the outer membrane of Gram-negative bacteria gave them more resistance towards antibacterial agents than Gram-positive bacteria. Nevertheless, we demonstrated here potent antibacterial activity of JSF4 phages against Gram-negative enteric pathogen V. Cholerae 01 serogroup. In the present study, we show that the oral administration of JSF4 bacteriophages could reduce V. cholerae concentration in infant mice models. Given the well-documented challenges associated with the emergence of antibiotic-resistant bacteria, bacteriophages can yet provide a viable alternative to antibiotics. Moreover, this bacteriophage JSF4 can not only able to reduce the population of viable V. Cholerae 01 strain but also can survive a wide range of temperature, pH and salinity (Islam et al., 2020).
We thank Dr. Shah Mohammad Faruque, a professor in the School of Life Sciences at Independent Uni-versity Bangladesh for providing us JSF4 bacterio-phage and its pathogenic host strain N-16961 as a kind gift. We also thank the Research Cell and the Dept. of Biochemistry and Molecular Biology, Mawlana Bhashani Science and Technology University, Tangail, Bangladesh for providing us some funding and logistic support, lab facilities respectively.
All animal experimental procedures were conducted according to the NIH Guide for the Care and Use of Laboratory Animals.
All authors of this manuscript declare no conflict of interest to publish it.
Dr. Phelipe Magalhães Duarte, Professor, Department of Veterinary, Faculty of Biological and Health Sciences, University of Cuiabá, Mato Grosso, Brazil.
Department of Biochemistry and Molecular Biology, Mawlana Bhashani Science and Technology University (MBSTU), Santosh, Tangail-1902, Bangladesh.
Islam MJ, Khatun MM, Yesmin S, Ghagra S, Hasan S, Nobel FA, Mozibullah M, Sohel M, Mogal MR, Hossain MA, Hasan MM, and Islam MK. (2020). Bacteriophage JSF4 can be a potential prophylaxis therapy for Cholera: an alternative approach to antibiotics. Am. J. Pure Appl. Sci., 2(5), 121-128. https://doi.org/10.34104/ajpab.020.01210128m