Indian Journal of Microbiology Research 2020;7(4):313–321

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Indian Journal of Microbiology Research

Journal homepage: https://www.ijmronline.org/

Original Research Article

Isolation, identification and functional characterization of Escherichia coli as
probiotic against Shigella in Bangladesh
A M Masudul Azad Chowdhury1 , Sanjida Akter1 , Sohana Akter Mina1, *
1 Dept. of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong, Bangladesh

ARTICLE INFO ABSTRACT

Article history: Background: Antibiotic resistance due to misuses of antibiotics is currently became potent threat for
Received 13-10-2020 treating patients suffering from infectious diseases in Bangladesh. It is the high time to develop some
Accepted 28-11-2020 alternative placebo for the treatment of infectious diseases especially in Bangladesh. Probiotic especially
Available online 25-12-2020 E. coli could be that alternative choice of treatment against Shigella infection.
Aims: The present study was undertaken to examine the potentiality of Escherichia coli as probiotic against
Shigella.
Keywords: Materials and Methods: During this study, four poor hygienic areas around Chattogram City, Bangladesh
Antibiotic resistance
were selected through survey and then human stool samples were collected. Isolation and identification of
Probiotic Escherichia coli were done with a combination of microbiological and PCR analysis. Probiotic activity of
Escherichia coli isolated E. coli against Shigella was determined by co-culture test.
Shigella
Results: Total eight isolates were identified as Escherichia coli by microbiological and biochemical test.
PCR
All the isolates were also confirmed as bacteria, coliform as well as fecal coliform through PCR. In the
probiotic activity test, all the identified isolates except one showed significant result as probiotic.
Conclusion: This research identified the potentiality of Escherichia coli as probiotic to treat shigellosis in
Bangladesh. The outcomes of this study might function as a strong background to develop Escherichia coli
as probiotic against Shigella infection in Bangladesh.

© This is an open access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.

1. Introduction significant group of bio therapeutics. 5 Humans live in close

association with vast numbers of micro-organisms that can
E. coli is a gram negative, rod shaped, non-spore forming, be present on the skin, in the mouth and in the gastro-
and motile bacteria. This is a commensal bacterium that intestinal tract where gastro-intestinal tract consists of a
resides as the most common and predominant inhabitant rich flora of more than 500 different bacterial species,
in the intestinal microflora of human and other mammal. 1 some of which have important health functions. 6 These
It is transmitted via fecal–oral route. Its versatility and nonpathogenic microorganisms known to have beneficial
adaptability makes it very commonly found in water, soil, effects on the digestive ecosystem as they colonize in the
and food. 2 E. coli are usually nonpathogenic and may serve bowel and confer resistance to infections as demonstrated
as indicators of faecal contamination of food and water. 3 by studies in animal models as well as by clinical trials. 7
Probiotic is a Latin derived word that means ‘for Bacteria and yeast are the most common microorganisms
life’. 4 Nonpathogenic micro-organisms, used to control used as probiotics. They differ in their mechanisms of
the growth of other micro-organisms are called probiotics action, metabolism and resistance to antibiotics. Essentially,
and worldwide studies have shown that they constitute a four bacterial genera and one yeast genus are the basis
for most preparations: Enterococcus, Bifidobacterium,
* Corresponding author. Escherichia, Lactobacillus and Saccharomyces. E. coli
E-mail address: sohanageb@cu.ac.bd (S. A. Mina).

https://doi.org/10.18231/j.ijmr.2020.056
2394-546X/© 2020 Innovative Publication, All rights reserved. 313
314 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321

Nissle 1917 (Mutaflor) is one of the most broadly studied of them are day laborer. Most of the people use common
and used probiotic bacteria worldwide. 8 latrines and the sanitation system is not developed. These
Species of the genus Shigella are among the bacterial areas are: Chittagong railway station, JhautolaTpp colony,
pathogens that are most frequently isolated from patients Shahid lane Akbarshah, and Karnaphuli market Figure 1.
suffering diarrhea. Every year around 165 million Shigella Several points in these areas were chosen for sample
induced diarrheal cases are reported and among that 99% collection where collective stool samples of these people
of cases occur in Lower to middle income countries can be found. We targeted conduits that came out from
mainly in children (69%) because of overcrowding and latrines of common people.
poor sanitation. 9 Antibiotic is prescribed in shigellosis
to speed recovery, reduce the seriousness of disease and 2.2. Sample collection and processing
reduce the length of time patients are infective, 10 however,
some antimicrobial drugs can have serious side effects Aseptic condition was maintained while collecting sewage
while others may not be effective against the Shigella samples and after collection the samples were brought
bacteria. With the continuing use of antibiotics, the bacterial to molecular biology lab of the Department of Genetic
population will soon amplify the resistant type replacing Engineering and Biotechnology at University of Chittagong,
the sensitive predecessor and thereby abolishing clinical Bnagladesh. The EMB agar media was prepared into a
value of the antibiotic. 11 Resistance to antimicrobials is sterile conical flask and sterilized by autoclaving. After
creating hindrance to the healthcare system worldwide. This that the media was cooled to 45ºC and then was poured
complicate treatment results, increases treatment cost, and into sterile petridishes. The dishes were allowed to solidify.
limits the therapeutic options that contribute to the loss of After solidification, a sterile micro wire loop for the semi-
efficiency of antimicrobial drugs. 12 Therefore, to overcome quantitative method was used for the plating and it has a
this antibiotic associated problems, probiotics especially E. 4.0 mm diameter designed to deliver 0.01 ml. A loopful
coli might be a suitable choice of treatment against Shigella of the uniformly mixed stool sample was inoculated into
infection in developing countries. EMB agar plate (EMB contains dyes that are toxic to
This research will try to understand and hypothesize gram positive bacteria. It is a specialized media for gram
that some people who live in poor hygienic environmental negative bacteria. In EMB agar plate typical E. coli colony
condition may remain unaffected from shigellosis because is usually characterized by green metallic sheen). The
they might contain E. coli in their gut which inhibits loop was sterilized using bunsen burner. After inoculation,
Shigella infection. In addition, in poor sanitation condition all the plates were kept in the incubator in an inverted
there should be competition among microorganisms. To position at 37ºC for 24 hours. All the steps were done
establish this phenomenon, this study was designed to in laminar air flow that was previously swiped with 70%
isolate novel E. coli strain as probiotic from the poor ethanol. There is always a bunsen burner light up while
sanitation areas of Chattogram City, Bangladesh. 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
2. Materials and Methods
proximity) in 3 separate tubes. Three stool samples instead
2.1. Sample survey of one stool sample were collected from same position
to avoid experimental error as well as to increase the
This study focused on poor hygienic regions firstly for the probability getting positive result for the target organism.
isolation of E. coli as probiotic strains against common After collection, samples carried to the lab in ice bag on
pathogenic organism and secondly to evaluate misuse of the same day and perform streak plating (as explain above)
antibiotics in a vastly populated city of Bangladesh. One of in 3 separate EMB plate to get the single colonies of E.
the hypothesis of this study presume that in the unhygienic coli. The next day EMB agar plates were prepared and
areas E. coli from human sources might has the capacity the previous plates were observed for bacterial growth.
to inhibit pathogenic organism like Shigella by which From the previous 3 plates the plate that shown perfect
most of the people remain protected though their living streaking pattern with characteristics single E. coli colony
and sanitation condition is very poor. So, sample that is was selected for next step. Only one single colony that
associated with human gut such human stool was our showed green metallic sheen were picked up with sterile
primary target. To select sample location a survey was inoculating loop and further inoculated in EMB agar plates.
carried out for choosing appropriate sample location in the After inoculation, the plates were kept in the incubator in
Chattogram city area. The survey took almost two weeks. an inverted position at 37ºC for 24 hours. At 24 hours
The study population was drawn from four poor areas of after incubation the plates were observed. On the same day,
Chattogram Figure 1. The locations were visited and the MacConkey agar media was prepared and sterilized and
situation was observed. These locations are very crowded. cooled to 45ºC-50ºC and poured into sterile petri plates.
Huge number of people lives in these small areas. Most After solidification, these plates were also streaked by single
 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321 315

Fig. 1: Location of samples in Bangladesh. As mention in the above section, four poor hygienic areas were selected around Chattogram
city, Chittagong, Bangladesh. Samples were collected from eight spots from those locations. Each red mark indicates the position of
sample spot in the map

colonies from previous EMB agar plates and then kept in above protocol, Eight single colonies were picked from 24
the incubator for 24 hours. MacConkey is an indicator, stool samples ((3 samples x 8 sample spots = 24 samples).of
a selective and differential culture medium that is used different sample spots and coded as Ec-CRS1 (E. coli
for the isolation of gram negative enteric bacteria and the Chittagong Railway Station 1), Ec-RS2 (E. coli Chittagong
differentiation of lactose fermenting gram negative bacteria. Railway Station 2), Ec-JHT3 (E. coli Jhautola station 3), Ec-
Lactose fermenting strains grow as pink colony and lactose AKS4 (E. coli Akbarshah 4),Ec-AKS5(E. coli Akbarshah
non fermenting strains are colorless and transparent. The 5). Ec-AKS6 (E. coli Akbarshah 6), Ec-KPM7(E. coli
sample that showed positive results E. coli both in EMB Karnaphuli market 7), Ec-KPM8 (E. coli Karnaphuli market
and MacConkey agar was selected, coded and stored in slant 8).
for biochemical and molecular identification. According to
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2.3. Biochemical tests 2.6. Statistical analysis

Primarily selected E.coli colonies were confirmed by Statistical significance was evaluated with Student’s t-test
performing Catalase test, Indole test, Methyle red test, for repeated measurements. All values are represented
Vogues-praskeur test and citrate utilization test according to as the means ± standard deviation for three-independent
the procedure describe in Cowan and steel, 2004 and also by experiments.
gram staining. 13,14
3. Results

2.4. Molecular identification 3.1. Selective plating

As mention in the methodology part, all the stool samples
2.4.1. DNA extraction
from different sample location were streaked on EMB
Extraction of the genomic DNA from the isolated agar plates for characteristic E. coli colony (metallic
E. coli strains were conducted by boiling method. 15 green sheen). EMB positive E. coli were further culture on
DNA concentration was measured using Nanodrop 2000 MacConkey agar to observe characteristic E. coli colony
spectrophotometer (Thermo Scientific, USA). (pink) on MacConkey agar. Finally, 8 single colonies
from 24 stool samples that were collected from eight
2.4.2. PCR assay for the identification of bacteria, sample points of four different locations around Chattogram
coliform and faecal coliform city were selected and coded (explained in detail in the
methodology section) as E. coli according to selective
In this study, molecular detection of organism was carried plating result Table 2.
out by PCR using the previously published primers and
targeted gene. 16 Primer specificity was determined by
3.2. Biochemical test
searching for similar sequences in microbial genome using
the Basic Local Alignment Search Tool (BLAST). A PCR First Gram staining experiment were done for each selected
thermal cycler (NyxTechnik) was used for amplification isolate. Gram staining result revealed that all the isolates
and the PCR products were analyzed by 1.5% agarose were Gram negative. Five biochemical tests were carried
gel electrophoresis. In each experiment, positive control out for the identification of selected E. coli. All of them
(Previously identified E. coli, 16 was carried out as the found positive to Indole test, Methyl red test, Catalase test
standard genomic DNA along with negative control (PCR and negative to Voges-Proskauer test, Citrate utilization test.
mixtures except genomic DNA). Target gene, primer The biochemical test results are summarized in the Table 2.
sequence, cycling parameters, amplicon size are shown in The biochemical test results confirmed the identification of
the supplementary Table 1. all the primarily selected isolates as E. coliTable 2.

2.5. Probiotic activity test 3.3. Molecular identification

Molecular identification E. coli as a bacteria, coliform and
Probiotic activity test was performed by co culturing faecal coliform was done by amplifying 16srDNA, lacZ and
Shigella and E. coli on the same plate. Shigella strain uidA gene, respectively. The PCR analysis of these genes
was provided from Microbiology lab of Department resulted in 100% positive for all the eight selected isolates
of Microbiology, University of Chittagong, Bangladesh. Figure 2. These were identified by observing the band size
Nutrient broth was taken in eight tubes and was inoculated with respect to DNA marker on 1.5% agarose gel on the
with freshly prepared E. coli culture. Eight test tubes were basis of 800 bp, 874 bp and 147bp for the genes 16srDNA,
inoculated with both Shigella and E. coli culture. One LacZ and uidA, respectively (Figure 2 B, C and D).
test tube was inoculated with Shigella culture. The tubes
were incubated at 37ºC for overnight at shaking condition.
3.4. Probiotic activity test
The next day, each culture was serially diluted with in test
tubes containing sterile distilled water up to 10−7 times. The antagonism of E. coli for Shigella was perceived by co-
From each diluted solution, 1ml of solution was transferred culturing Shigella with E. coli and observing their growth
into plate containing MacConkey agar media. The plates on the same plate. The result showed that in the co-culture
were stirred with hand gently clockwise and anti-clockwise E. coli effectively decreased the number of Shigella colony.
so that sample was mixed thoroughly with the media. The On MacConkey agar plates, all the isolates of E. coli except
plates were allowed to stand steady to solidify the media. Ec-AKS6 inhibited Shigella growth in co-culture Figure 3.
After solidification, the plates were incubated in inverted Shigella and E. coli colonies were also counted separately
position for 24 hours at 37ºC. The next day, colony counting on MacConkey agar plates for comparison as control to
was done by total viable count (TVC) method. evaluate culture condition Figure 3. The test was done in
 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321 317

Table 1: Target genes, primers, cyclic condition,composition of PCR mixture and amplicon size
Target genes Primer sequence 5´-3´ Cycling parameters Composition of Amplicon Size
PCR mixtures (bp)
Bacterial: 16srDNA AGAGTTGATCCTGGCTCAGa 5 min at 95◦ C, 35 For 20 µ l: 10 µ l 800
cycles of 95◦ C for 40s,
GACTACCAGGGTATCTAATb master mix, 4µ l
57◦ C for 72◦ C for 1 template, 2 µ la ,
min 2µ lb , 3µ l water
Coliform: lacZ ATGAAAGGCTGGCTACAGGAAGGCCa 5 min at 95◦ C, 25 For 20 µ l: 10 µ l 874
CACCATGCCGTGGGTTTCAATATTb cycles of 95◦ C for 1 master mix, 4µ l
min and 72◦ C for 1 min template, 2 µ la ,
2µ lb , 3µ l water
Faecalcoliform: uidA TGGTAATTACCGACGAAAACGGa 5 min at 95◦ C,30 cycles For 20 µ l: 10 µ l 147
ACGCGTGGTTACAGTCTTGCGb of 95◦ C for 50s, 62◦ C master mix, 4µ l
for 50s and 72◦ C for 1 template, 2 µ la ,
min 2µ lb , 3µ l water
a Forward primer; b Reverse primer

Table 2: Summarized results of microbiological analysis

Sample Colony character Gram Biochemical tests
Comments
ID EMB MCK staining IT MRT CUT VPT CT
Ec-CRS1 GMS Pink G− + + - - + E. coli
Ec-CRS2 GMS Pink G− + + - - + E. coli
Ec-JHT3 GMS Pink G− + + - - + E. coli
Ec-AKS4 GMS Pink G− + + - - + E. coli
Ec-AKS5 GMS Pink G− + + - - + E. coli
Ec-AKS6 GMS Pink G− + + - - + E. coli
Ec- GMS Pink G− + + - - + E. coli
KPM7
Ec- GMS Pink G− + + - - + E. coli
KPM8
GMS = Green metallic sheen, G− = Gram negative, IT= Indole test, MRT= Methyl red test, CUT= Citrate utilization test, VPT= VogesPraskauer Test, CT=
Catalase test.
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 Akbarshah 5). Ec-AKS6 (E. coli Akbarshah 6), Ec-KPM7 (E. coli Karnaphuli market 7), Ec-KPM8 (E. coli Karnaphuli
market 8).

triplicate for statistical analysis. observing inhibition zone (ex vivo) 20 published that E. coli
strain Nissle 1971 combat lamdoid bacteriophage stx and λ
4. Discussion thus opening a new window for the treatment of infections
caused by shiga toxin producing pathogens.
The normal resident gastrointestinal microbiotas are the In this study, eight E. coli strains were isolated and
major factor protecting animals and humans against identified through conventional microbiological analysis
intestinal colonization by pathogenic bacteria. 17 Microbes in order to examine probiotic activity against Shigella
being such a large physical part of the gastrointestinal one of the causing agent of diarrhea in Bangladesh. The
tract, so it is vitally important that specialists appreciate results of microbiological detection Table 2 of E. coli
their existence, and consider what role they might have were similar as. 21,22 Along with culture based detection,
in health and disease. Recent understanding of the molecular identification of the selected isolates were done
functions of intestinal microflora and the use of probiotic by PCR amplification of the three genes; 16s rDNA for
microorganisms is a novel concept for the improvement bacterial identification Figure 2 B, lacZ gene for coliform
of human health and an innovative approach for new identification Figure 2 C and uidA gene for fecal coliform
food product development in functional foods for specific identification 16 Figure 3. All the amplified products showed
diseases. 18 E. coli and its human host usually coexist with bands on agarose gel electrophoresis showing positive
mutual benefit for decades. 1 The probiotic effect of E. results for identification. Bands of around 800 bp, 874 bp,
coli is well established and proven by several in vitro and and 147 bp were found respectively for the three genes in
in vivo experiments 19 reported that exoproducts of the E. all sample isolates. Through molecular identification it was
coli strain H22 inhibits some enteric pathogens both in confirm that all the samples are coliform bacteria and they
vitro and in vivo by agar overlay method (in vitro) and are from the intestines of warm blood animal. As the study
318 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321

Fig. 2: Molecular identification isolates as E. coli. through electrophoretic (1.5% agarose) separation of 16 srDNA, LacZ and uidA gene.
(A) Thermo Scientific 1 kb DNA ladder. (B) Detection 16srDNA gene to confirm the isolates as bacteria. (C) Detection of coliform by
lacZ gene. (D) Detection of faecal coliform by uidA gene. Here, EC-CRS1, EC-CRS2, EC-CRS1JHT3, EC-AKS4, EC-AKS5, EC-AKS6,
EC-KPM7 and EC-KPM8 are isolates; PC: Positive Control; NC: Negative Control

areas of this research project were mainly the slum areas, it AKS5 (E. coli Akbar Shah5), Ec-KPM7(E. coli Karnaphuli
can be assumed that all the isolates are from human gut. 23 Market 7), Ec-KPM8 (E. coli Karnaphuli market 8) are
considered as effective probiotic strain Figure 3. The exact
The antagonism of E. coli for Shigella was perceived mechanism how E. coli inhibit intestinal pathogen is still
by co-culturing Shigella with E. coli and observing their not clearly understood. The inhibition activity might be
growth on same plate. The statistical analysis of the result due to the production of specific antimicrobial substances,
showed that in the co-culture, all the E. coli samples such as microcins. However, the microcin negative isogenic
except Ec-AKS6 (E. coli Akbarshah 6), caused significant mutant of E. coli has been shown to be as effective
inhibition of Shigella Figure 3. According to this result as the wild strain in competing with pathogenic bacteria.
Ec-CRS1 (E. coli Chittagong Railway Station 1), Ec-RS2 In fact, because of the narrow spectrum of bacteriocin
(E. coli Chittagong Railway Station 2), Ec-JHS3 (E. coli activity, it is unlikely to be responsible for the inhibitory
Jhautola station 3), Ec-AKS4 (E. coli Akbarshah 4), Ec-
 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321 319

Fig. 3: Probiotic activity of E. coli against Shigella., E. coli with Shigella were cultivated in test tube containing equal volume of
Nutrient broth. At 18 hours after incubation probiotic activity of E. coli against Shigella were measured by Total viable count experiment
through pour plate method on MacConkey agar plates. The error bars represent data from three independent experiments (mean± standard
deviation). The two-tailed Student’s t-test was used for the statistical analysis. * P< 0.05, ns: No significance
320 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321

Fig. 4: Schematic model of probiotic activity of E. coli. In this study, the E. coli isolates inhibiting growth of the pathogenic Shigella
strain. This inhibition might be the result of competitive exclusion of the pathogen by E. coli strain

effect of E. coli. 24 Effective adherence of E. coli to pathogenic Shigella strain. This inhibition might be the
intestinal epithelial cells may block necessary receptors for result of competitive exclusion of the pathogen by E. coli
attachment of invasive bacteria thereby inhibiting them. by creating hostile micro ecology and competitive reduction
E. coli adheres strongly to the intestinal cell wall that of essential nutrients Figure 4.
results in a biofilm formation of nonpathogenic bacteria Production and secretion of antimicrobial substances and
thus restricts the pathogenic bacteria. According to some selective metabolites can also be the reason of inhibition.
other studies, E. coli Nissle 1917 and other probiotic To check this, toxicity test has been done to check whether
strains may stimulate the synthesis of endogenous epithelial E. coli supernatant has any effect in Shigella inhibition. 26
antimicrobial peptides such as human Beta Defensin–2 This experiment was performed but due to lack of proper
which helps to exert the beneficial effects of the probiotic equipment facilities E. coli supernatant could not properly
strain. The growth and metabolic activity of E. coli may separated (result not shown).
also cause changes in the pH or chemical composition
of the colonic lumen that make the surface unfavorable 5. Conclusion
to the pathogenic bacteria. 25 In this study, the sample
isolates showed positive results by inhibiting growth of the Probiotics may help not only in disease reduction but also
in health improvement and can be included in other sectors
 Chowdhury, Akter and Mina / Indian Journal of Microbiology Research 2020;7(4):313–321 321

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