E. coli is a gram negative, rod shaped, non-spore forming, and motile bacteria. This is a commensal bacterium that resides as the most common and predominant inhabitant in the intestinal microflora of human and other mammal.1 It is transmitted via fecal–oral route. Its versatility and adaptability makes it very commonly found in water, soil, and food.2 E. coli are usually nonpathogenic and may serve as indicators of faecal contamination of food and water.3
Probiotic is a Latin derived word that means ‘for life’.4 Nonpathogenic micro-organisms, used to control the growth of other micro-organisms are called probiotics and worldwide studies have shown that they constitute a significant group of bio therapeutics.5 Humans live in close association with vast numbers of micro-organisms that can be present on the skin, in the mouth and in the gastro-intestinal tract where gastro-intestinal tract consists of a rich flora of more than 500 different bacterial species, some of which have important health functions.6 These nonpathogenic microorganisms known to have beneficial effects on the digestive ecosystem as they colonize in the bowel and confer resistance to infections as demonstrated by studies in animal models as well as by clinical trials.7 Bacteria and yeast are the most common microorganisms used as probiotics. They differ in their mechanisms of action, metabolism and resistance to antibiotics. Essentially, four bacterial genera and one yeast genus are the basis for most preparations: Enterococcus, Bifidobacterium, Escherichia, Lactobacillus and Saccharomyces. E. coli Nissle 1917 (Mutaflor) is one of the most broadly studied and used probiotic bacteria worldwide.8
Species of the genus Shigella are among the bacterial pathogens that are most frequently isolated from patients suffering diarrhea. Every year around 165 million Shigella induced diarrheal cases are reported and among that 99% of cases occur in Lower to middle income countries mainly in children (69%) because of overcrowding and poor sanitation.9 Antibiotic is prescribed in shigellosis to speed recovery, reduce the seriousness of disease and reduce the length of time patients are infective,10 however, some antimicrobial drugs can have serious side effects while others may not be effective against the Shigella bacteria. With the continuing use of antibiotics, the bacterial population will soon amplify the resistant type replacing the sensitive predecessor and thereby abolishing clinical value of the antibiotic.11 Resistance to antimicrobials is creating hindrance to the healthcare system worldwide. This complicate treatment results, increases treatment cost, and limits the therapeutic options that contribute to the loss of efficiency of antimicrobial drugs.12 Therefore, to overcome this antibiotic associated problems, probiotics especially E. coli might be a suitable choice of treatment against Shigella infection in developing countries.
This research will try to understand and hypothesize that some people who live in poor hygienic environmental condition may remain unaffected from shigellosis because they might contain E. coli in their gut which inhibits Shigella infection. In addition, in poor sanitation condition there should be competition among microorganisms. To establish this phenomenon, this study was designed to isolate novel E. coli strain as probiotic from the poor sanitation areas of Chattogram City, Bangladesh.
Materials and Methods
This study focused on poor hygienic regions firstly for the isolation of E. coli as probiotic strains against common pathogenic organism and secondly to evaluate misuse of antibiotics in a vastly populated city of Bangladesh. One of the hypothesis of this study presume that in the unhygienic areas E. coli from human sources might has the capacity to inhibit pathogenic organism like Shigella by which most of the people remain protected though their living and sanitation condition is very poor. So, sample that is associated with human gut such human stool was our primary target. To select sample location a survey was carried out for choosing appropriate sample location in the Chattogram city area. The survey took almost two weeks. The study population was drawn from four poor areas of Chattogram Figure 1. The locations were visited and the situation was observed. These locations are very crowded. Huge number of people lives in these small areas. Most of them are day laborer. Most of the people use common latrines and the sanitation system is not developed. These areas are: Chittagong railway station, JhautolaTpp colony, Shahid lane Akbarshah, and Karnaphuli market Figure 1. Several points in these areas were chosen for sample collection where collective stool samples of these people can be found. We targeted conduits that came out from latrines of common people.
Sample collection and processing
Aseptic condition was maintained while collecting sewage samples and after collection the samples were brought to molecular biology lab of the Department of Genetic Engineering and Biotechnology at University of Chittagong, Bnagladesh. The EMB agar media was prepared into a sterile conical flask and sterilized by autoclaving. After that the media was cooled to 45ºC and then was poured into sterile petridishes. The dishes were allowed to solidify. After solidification, a sterile micro wire loop for the semi-quantitative method was used for the plating and it has a 4.0 mm diameter designed to deliver 0.01 ml. A loopful of the uniformly mixed stool sample was inoculated into EMB agar plate (EMB contains dyes that are toxic to gram positive bacteria. It is a specialized media for gram negative bacteria. In EMB agar plate typical E. coli colony is usually characterized by green metallic sheen). The loop was sterilized using bunsen burner. After inoculation, all the plates were kept in the incubator in an inverted position at 37ºC for 24 hours. All the steps were done in laminar air flow that was previously swiped with 70% ethanol. There is always a bunsen burner light up while working in the laminar air flow. To maintain aseptic condition, hands were washed with 70% ethanol. From each sample spot 3 stool samples were collected (with in close proximity) in 3 separate tubes. Three stool samples instead of one stool sample were collected from same position to avoid experimental error as well as to increase the probability getting positive result for the target organism. After collection, samples carried to the lab in ice bag on the same day and perform streak plating (as explain above) in 3 separate EMB plate to get the single colonies of E. coli. The next day EMB agar plates were prepared and the previous plates were observed for bacterial growth. From the previous 3 plates the plate that shown perfect streaking pattern with characteristics single E. coli colony was selected for next step. Only one single colony that showed green metallic sheen were picked up with sterile inoculating loop and further inoculated in EMB agar plates. After inoculation, the plates were kept in the incubator in an inverted position at 37ºC for 24 hours. At 24 hours after incubation the plates were observed. On the same day, MacConkey agar media was prepared and sterilized and cooled to 45ºC-50ºC and poured into sterile petri plates. After solidification, these plates were also streaked by single colonies from previous EMB agar plates and then kept in the incubator for 24 hours. MacConkey is an indicator, a selective and differential culture medium that is used for the isolation of gram negative enteric bacteria and the differentiation of lactose fermenting gram negative bacteria. Lactose fermenting strains grow as pink colony and lactose non fermenting strains are colorless and transparent. The sample that showed positive results E. coli both in EMB and MacConkey agar was selected, coded and stored in slant for biochemical and molecular identification. According to above protocol, Eight single colonies were picked from 24 stool samples ((3 samples x 8 sample spots = 24 samples).of different sample spots and coded as Ec-CRS1 (E. coli Chittagong Railway Station 1), Ec-RS2 (E. coli Chittagong Railway Station 2), Ec-JHT3 (E. coli Jhautola station 3), Ec-AKS4 (E. coli Akbarshah 4),Ec-AKS5(E. coli Akbarshah 5). Ec-AKS6 (E. coli Akbarshah 6), Ec-KPM7(E. coli Karnaphuli market 7), Ec-KPM8 (E. coli Karnaphuli market 8).
Primarily selected E.coli colonies were confirmed by performing Catalase test, Indole test, Methyle red test, Vogues-praskeur test and citrate utilization test according to the procedure describe in Cowan and steel, 2004 and also by gram staining.13, 14
Extraction of the genomic DNA from the isolated E. coli strains were conducted by boiling method.15 DNA concentration was measured using Nanodrop 2000 spectrophotometer (Thermo Scientific, USA).
PCR assay for the identification of bacteria, coliform and faecal coliform
In this study, molecular detection of organism was carried out by PCR using the previously published primers and targeted gene.16 Primer specificity was determined by searching for similar sequences in microbial genome using the Basic Local Alignment Search Tool (BLAST). A PCR thermal cycler (NyxTechnik) was used for amplification and the PCR products were analyzed by 1.5% agarose gel electrophoresis. In each experiment, positive control (Previously identified E. coli,16 was carried out as the standard genomic DNA along with negative control (PCR mixtures except genomic DNA). Target gene, primer sequence, cycling parameters, amplicon size are shown in the supplementary Table 1.
Probiotic activity test
Probiotic activity test was performed by co culturing Shigella and E. coli on the same plate. Shigella strain was provided from Microbiology lab of Department of Microbiology, University of Chittagong, Bangladesh. Nutrient broth was taken in eight tubes and was inoculated with freshly prepared E. coli culture. Eight test tubes were inoculated with both Shigella and E. coli culture. One test tube was inoculated with Shigella culture. The tubes were incubated at 37ºC for overnight at shaking condition. The next day, each culture was serially diluted with in test tubes containing sterile distilled water up to 10-7 times. From each diluted solution, 1ml of solution was transferred into plate containing MacConkey agar media. The plates were stirred with hand gently clockwise and anti-clockwise so that sample was mixed thoroughly with the media. The plates were allowed to stand steady to solidify the media. After solidification, the plates were incubated in inverted position for 24 hours at 37ºC. The next day, colony counting was done by total viable count (TVC) method.
As mention in the methodology part, all the stool samples from different sample location were streaked on EMB agar plates for characteristic E. coli colony (metallic green sheen). EMB positive E. coli were further culture on MacConkey agar to observe characteristic E. coli colony (pink) on MacConkey agar. Finally, 8 single colonies from 24 stool samples that were collected from eight sample points of four different locations around Chattogram city were selected and coded (explained in detail in the methodology section) as E. coli according to selective plating result Table 2.
First Gram staining experiment were done for each selected isolate. Gram staining result revealed that all the isolates were Gram negative. Five biochemical tests were carried out for the identification of selected E. coli. All of them found positive to Indole test, Methyl red test, Catalase test and negative to Voges-Proskauer test, Citrate utilization test. The biochemical test results are summarized in the Table 2. The biochemical test results confirmed the identification of all the primarily selected isolates as E. coli Table 2.
Molecular identification E. coli as a bacteria, coliform and faecal coliform was done by amplifying 16srDNA, lacZ and uidA gene, respectively. The PCR analysis of these genes resulted in 100% positive for all the eight selected isolates Figure 2. These were identified by observing the band size with respect to DNA marker on 1.5% agarose gel on the basis of 800 bp, 874 bp and 147bp for the genes 16srDNA, LacZ and uidA, respectively (Figure 2 B, C and D).
Probiotic activity test
The antagonism of E. coli for Shigella was perceived by co-culturing Shigella with E. coli and observing their growth on the same plate. The result showed that in the co-culture E. coli effectively decreased the number of Shigella colony. On MacConkey agar plates, all the isolates of E. coli except Ec-AKS6 inhibited Shigella growth in co-culture Figure 3. Shigella and E. coli colonies were also counted separately on MacConkey agar plates for comparison as control to evaluate culture condition Figure 3. The test was done in triplicate for statistical analysis.
The normal resident gastrointestinal microbiotas are the major factor protecting animals and humans against intestinal colonization by pathogenic bacteria.17 Microbes being such a large physical part of the gastrointestinal tract, so it is vitally important that specialists appreciate their existence, and consider what role they might have in health and disease. Recent understanding of the functions of intestinal microflora and the use of probiotic microorganisms is a novel concept for the improvement of human health and an innovative approach for new food product development in functional foods for specific diseases.18 E. coli and its human host usually coexist with mutual benefit for decades.1 The probiotic effect of E. coli is well established and proven by several in vitro and in vivo experiments19 reported that exoproducts of the E. coli strain H22 inhibits some enteric pathogens both in vitro and in vivo by agar overlay method (in vitro) and observing inhibition zone (ex vivo)20 published that E. coli strain Nissle 1971 combat lamdoid bacteriophage stx and λ thus opening a new window for the treatment of infections caused by shiga toxin producing pathogens.
In this study, eight E. coli strains were isolated and identified through conventional microbiological analysis in order to examine probiotic activity against Shigella one of the causing agent of diarrhea in Bangladesh. The results of microbiological detection Table 2 of E. coli were similar as.21, 22 Along with culture based detection, molecular identification of the selected isolates were done by PCR amplification of the three genes; 16s rDNA for bacterial identification Figure 2 B, lacZ gene for coliform identification Figure 2 C and uidA gene for fecal coliform identification16 Figure 3. All the amplified products showed bands on agarose gel electrophoresis showing positive results for identification. Bands of around 800 bp, 874 bp, and 147 bp were found respectively for the three genes in all sample isolates. Through molecular identification it was confirm that all the samples are coliform bacteria and they are from the intestines of warm blood animal. As the study areas of this research project were mainly the slum areas, it can be assumed that all the isolates are from human gut.23
The antagonism of E. coli for Shigella was perceived by co-culturing Shigella with E. coli and observing their growth on same plate. The statistical analysis of the result showed that in the co-culture, all the E. coli samples except Ec-AKS6 (E. coli Akbarshah 6), caused significant inhibition of Shigella Figure 3. According to this result Ec-CRS1 (E. coli Chittagong Railway Station 1), Ec-RS2 (E. coli Chittagong Railway Station 2), Ec-JHS3 (E. coli Jhautola station 3), Ec-AKS4 (E. coli Akbarshah 4), Ec-AKS5 (E. coli Akbar Shah5), Ec-KPM7(E. coli Karnaphuli Market 7), Ec-KPM8 (E. coli Karnaphuli market 8) are considered as effective probiotic strain Figure 3. The exact mechanism how E. coli inhibit intestinal pathogen is still not clearly understood. The inhibition activity might be due to the production of specific antimicrobial substances, such as microcins. However, the microcin negative isogenic mutant of E. coli has been shown to be as effective as the wild strain in competing with pathogenic bacteria. In fact, because of the narrow spectrum of bacteriocin activity, it is unlikely to be responsible for the inhibitory effect of E. coli.24 Effective adherence of E. coli to intestinal epithelial cells may block necessary receptors for attachment of invasive bacteria thereby inhibiting them. E. coli adheres strongly to the intestinal cell wall that results in a biofilm formation of nonpathogenic bacteria thus restricts the pathogenic bacteria. According to some other studies, E. coli Nissle 1917 and other probiotic strains may stimulate the synthesis of endogenous epithelial antimicrobial peptides such as human Beta Defensin–2 which helps to exert the beneficial effects of the probiotic strain. The growth and metabolic activity of E. coli may also cause changes in the pH or chemical composition of the colonic lumen that make the surface unfavorable to the pathogenic bacteria.25 In this study, the sample isolates showed positive results by inhibiting growth of the pathogenic Shigella strain. This inhibition might be the result of competitive exclusion of the pathogen by E. coli by creating hostile micro ecology and competitive reduction of essential nutrients Figure 4.
Production and secretion of antimicrobial substances and selective metabolites can also be the reason of inhibition. To check this, toxicity test has been done to check whether E. coli supernatant has any effect in Shigella inhibition.26 This experiment was performed but due to lack of proper equipment facilities E. coli supernatant could not properly separated (result not shown).
Probiotics may help not only in disease reduction but also in health improvement and can be included in other sectors like poultry, agriculture where extensive antibiotic use is harmful because of emergence of multi drug resistance bacteria. This study demonstrated that E. coli strains from environmental source can act as a potent antagonist against enteric pathogen Shigella. This study will serve as a background to find a friendly alternative to this massacre. In future, this study can be done on a bigger platform targeting larger regions and sample numbers for the isolation of novel probiotic strains. There is an urge to think of a substitute of the conventional treatment of infectious disease in Bangladesh, so more studies on probiotic should be enrolled.
Source of Funding
Research and Publication Offices, University of Chittagong, Bangladesh, for research grant. Research Grant number; Memo no- 6429/2018.