|Year : 2015 | Volume
| Issue : 5 | Page : 67-72
Screening of the novel colicinogenic gram-negative rods against pathogenic Escherichia coli O157:H7
H Mushtaq1, J Bakht2, N Bacha1
1 Centre of Biotechnology and Microbiology, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan
2 Institute of Biotechnology and Genetic Engineering, University of Agriculture Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan
|Date of Submission||11-Jul-2013|
|Date of Acceptance||10-Oct-2013|
|Date of Web Publication||6-Feb-2015|
Centre of Biotechnology and Microbiology, University of Peshawar, Peshawar, Khyber Pakhtunkhwa
Source of Support: None, Conflict of Interest: None
Purpose: Escherichia coli (E. coli) O157:H7 is gram-negative enteric pathogen producing different types of Shiga toxin. This bacterium is the most corporate cause of haemorrhagic colitis in human. Administration of antibiotics (particularly sulfa drugs) against this pathogen is a debatable topic as this may increase the risk of uremic syndrome; especially in children and aged people. Around the world, microbiologists are in search of alternative therapeutic methods specially probiotics against this pathogen. In the present study, we have focused on the investigation of alternate bio-therapeutics (probiotics) for the treatment of patients infected with E. coli O157:H7. This study is based on the identification of colicin-producing gram-negative bacteria (particularly enterobacteriaceae) which can competently exclude E. coli O157:H7 from the gut of the infected individual. Materials and Methods: Hundred samples from human, animal faeces and septic tank water were analysed for nonpathogenic gram-negative rods (GNRs). Results: Out of these samples, 175 isolates of GNRs were checked for their activity against E. coli O157:H7. Only 47 isolates inhibited the growth of E. coli O157:H7, among which majority were identified as E. coli. These E. coli strains were found to be the efficient producers of colicin. Some of the closely related species i. e., Citrobacter sp, Pantoea sp. and Kluyvera sp. also showed considerable colicinogenic activity. Moreover, colicinogenic species were found to be nonhaemolytic, tolerant to acidic environment (pH 3) and sensitive to commonly used antibiotics. Conclusion: Nonhaemolytic, acid tolerant and sensitive to antibiotics suggests the possible use of these circulating endothelial cells (CEC) as inexpensive and inoffensive therapeutic agent (probiotics) in E. coli O157:H7 infections.
Keywords: Bacteriocins, colicin, E. coli O157:H7, human uremic syndrome, probiotics
|How to cite this article:|
Mushtaq H, Bakht J, Bacha N. Screening of the novel colicinogenic gram-negative rods against pathogenic Escherichia coli O157:H7. Indian J Med Microbiol 2015;33, Suppl S1:67-72
|How to cite this URL:|
Mushtaq H, Bakht J, Bacha N. Screening of the novel colicinogenic gram-negative rods against pathogenic Escherichia coli O157:H7. Indian J Med Microbiol [serial online] 2015 [cited 2019 Mar 26];33, Suppl S1:67-72. Available from: http://www.ijmm.org/text.asp?2015/33/5/67/150895
| ~ Introduction|| |
Food-borne pathogens entail several noxious agents among which Shiga toxin producing E. coli O157: H7 is the most virulent one. Humans procure infection by eating raw meat, taking raw milk, person-to-person contact and use of sewage-contaminated water may predispose healthy individuals to this pathogen.  Several E. coli strains have been known to cause haemorrhagic colitis in humans; however, the highest outbreak of this disease has been reported in relation to O157:H7.  Primarily, the patients display conjoint symptoms of gastroenteritis such as watery diarrhoea, vomiting and abdominal cramps, which become severe if left unrestrained. The later complications include bloody diarrhoea and kidney failure eventually leading to Human Uremic Syndrome (HUS), especially in children and aging people.  Periodic studies have materialised the examination of risk factors predisposing children and elderly people to HUS. , Of these risk factors, the most controversial one is the antibiotic administration for the treatment of acute E. coli O157:H7 enteritis.  Some in vitro studies have proclaimed that sub-inhibitory concentrations of trimethoprim-sulfomethoxazale upsurge the release of cytotoxins by the pathogen that may worsen the clinical outcome of the patient. , As prophylactic measures to inhibit severity of E. coli O157:H7 infections, alternate methods have been proposed such as the use of commensal nonpathogenic bacteria (probiotics) and bio-preservatives including bacteriocins. ,
The mammalian gut is flooded with different useful bacteria among which E. coli compromise 1% of the whole population. , These commensal bacteria use special surveillance mechanism in intestine to compete with one another and with invading micro-organism for nutrition and space. One of the tools for competitive exclusion is the extracellular production of bacteriocins by these species.  Bacteriocins are small size relatively narrow killing spectrum proteins of different types which are produced by both gram-positive and gram-negative bacteria. Colicins, classical bacteriocins, are manufactured by E. coli strains and other closely related species. ,, More than 30 colicins have been identified in several studies among which colicin A, B, D, E1-E9, Ia, Ib, Js, K, M, U, 5 and 10 are more conspicuous groups that inhibit the target strains. 
In humans and domesticated animals, probiotic therapy is a disease-precluding strategy used to ensure that 'good' bacteria (probiotics) complex in the gastrointestinal tract and prevent the accumulation of pathogenic bacteria. The probiotic strain's ability to produce antimicrobial compounds (such as bacteriocins, colicins, etc.) is the most important attribute that is extensively used.  Nonpathogenic colicin-producing strains that can kill, exclude or inhibit the sensitive target strain in its affinity have been added as probiotic, especially in cattle and calves feed.  Exclusion of E. coli O157:H7 in cattle with the administration of probiotic colicinogenic E. coli has been reported. ,, The acuteness of E. coli O157:H7 to colicins, in particular E2 and V has been reported which showed inhibitory activity against various diarrhoeagenic E. coli including O157: H7. ,
| ~ Materials and Methods|| |
Collection of samples
Proper approval has been taken from the 'Centre of Biotechnology and Microbiology Ethical Committee', University of Peshawar, Pakistan, for the conduction of the subject research work. In sterile glass vials with 15% glycerin, 35 uninfected human faeces samples were collected from local hospitals while 35 animal samples from colon of slaughtered animals were collected in local animal market. Septic tank water samples i. e., 30 was directly collected in falcon tubes containing 15% from homemade septic tanks of different houses.
Isolation and characterisation of gram-negative rods
A direct inoculum from faeces and septic tank water samples was cultured on MacConkey Agar (MAC) plates. The lactose-positive and lactose-negative colonies were randomly picked and were characterised morphologically by Gram's staining. Various isolates of gram-negative rods (GNR) were stocked in Nutrient Agar slants at 4°C and sub-cultured monthly.
Food-borne pathogen used for testing for antimicrobial sensitivity i.e., E. coli O157:H7 was received from National Institute of Biotechnology and Genetic Engineering, Faisalabad. The strain was cultured three times on Sorbitol MacConkey agar plates to refresh the culture before experimental purpose.
Antimicrobial activity of isolated strains was carried out by a well-diffusion method. The indicator strains was seeded in Lauria agar plates (LA) by mixing 3 ml of precultured Lauria broth (LB) into semisoft LA and wells of 20 mm were bored at proper distance. The wells were filled with 20 μl of LB media in which producer strains were grown at 37°C for 48 hours on constant shaking. The antibacterial activity of the producers strain appeared in the form of zone of inhibition form around the wells.
Biochemical characterisations of active strains
The genera- and specie-level identification of all isolates against E. coli O157:H7 was carried out by using standard API 20 E strips (Version 2.0, Bio-Merieux).
Detection of colicinogenic bacteria
E. coli strains and some closely related species including Citrobacter sp., Pantoea sp., Klyuvera sp and Shigella sonnei were tested for their ability to produce colicins. Single colonies from pure cultures of specie were inoculated in LB containing Mitomycin C 0.25 μg at 37°C for 24 hours. Broth cultures were centrifuged to separate cell mass and supernatant and supernatant LB was given exposure to chloroform vapour for 30 minutes. The cell mass was left untreated. The indicator organism was seeded in LA plates and chloroform treated supernatant (20 μl) was poured in three wells while untreated cell mass was poured in other three wells. The plates were allowed to stand for 1-2 hours at room temperature, and then incubated for 24 hours at 37°C. Following incubation, inhibitory zones formed by antibacterial activity of supernatant and cell mass was measured and compared.
Preliminary test for detection of disease causing ability of isolates was checked. Haemolysis caused by pathogens for the uptake of haemoglobin was determined on Blood agar media (BA). Beta or alpha haemolysis caused by active strains was determined.
The ability of active species to grow on alternate pH ranges (pH 1 to pH 6) was analysed by successive culturing on NA media.
Antibiotic sensitivity of the colicinogenic isolates to various antibiotics including Penicillin G, Streptomycin, Augmentin, Amikacin, Gentamicin, Ciprofloxacin, Cefixime, Cefotaxime, Imepenam, Chloramphenicol and Cefoperazone was determined on MH media.
| ~ Results|| |
The data revealed that 100% of human adult, 80% of human children faeces, 100% cow/buffalo faeces samples, 90% sheep faeces samples and 76% of septic tank water contained GNR. From total 88 samples, 175 isolates were taken randomly and sub-cultured to obtain pure cultures. Considering overall recovery rate, highest percentage of recovery was (94%) showed by animal faeces samples [Table 1]. In preliminary screening, the antimicrobial activity of pure isolates was determined by disc-diffusion method. Isolates which were active in inhibiting growth of indicator strain produced clear zones of inhibition around the wells [Figure 1]. Out of 175, 47 isolates displayed antibacterial activity against the indicator strain i. e., E. coli O157:H7. Among these active isolates, 15 human adult isolates, 5 human children isolates, 8 cow/buffalo isolates, 8 sheep isolates and 11 septic tank isolates were antagonistic towards E. coli O157:H7 [Figure 2]. Biochemical identification of 47 active isolates confirmed the presence of different species of enterobacteriaceae. Overall 16 E. coli, 9 Citrobacter freundii, 5 Klyuvera sp., 3 Pantoea sp., 1 Shigella sonnei, 5 Pseudomonas putida/fluorescence, 2 Raoultella terrigena, 1 Klebsilla oxytoca, 1 Aeromonas hyrophila 2, 1 pasteurella pneumotropica, 1 Salmonella More Details ser pollurum and 1 Serratia plymuthica strain were obtained [Figure 3].
|Figure 1: The plate is overlaid with indicator strain (E. coli O157:H7) while wells are filled with producer strain (inoculum size 20 μl). The zone of inhibition around wells indicates that producer strain (HA14 and HA15) released an antibacterial agent which diffused through the media and spread around wells, thus inhibiting the growth of indicator strain. The difference in the diameter of zones shows the diffusion ability of antibacterial agent and inhibitory activity of the producer strain|
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|Figure 2: The graphs show frequency of occurrence of gram‑negative rods (GNR) with antibacterial activity against E. coli O157:H7. The portion of bars marked with red color shows number of isolates which are inactive while blue color of the bars indicates total number of active isolates with inhibitory activity against the pathogen|
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|Figure 3: The figures display all of active isolates which were identified through biochemical tests. The highest numbers of isolates obtained are E. coli (blue area) and Citrobacter species (maroon area). The next to most common species are Pseudomonas flourescences/putida (5) and Klyuvera species (5). Three species of Pantoea sp., two strains of Raoultella terrigena, two Pasteurella pneumotropica, one Shigella sonnei, one Klebsiella oxytoca and one Aeromonas hyrophila 2 are found antagonistic in nature|
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The data revealed that out of 47 active species against O157: H7, only above-mentioned species were analysed for their ability to produce colicins. In total, 8 isolates including 4 out of 16 E. coli, 2 out of 9 Citrobacter freundeii, 1 out of 3 Pantoea sp. and 1 out of 5 Klyuvera sp. were found to be colicinogenic. Shigella sonnei and Salmonella strains did not show any inhibitory activity against the indicator strain [Figure 4]. In the present study, adult humans and bovine faeces yielded 64% of E. coli, whereas 50% colicinogenic E. coli were found. It was also specified that 26.8% of the total gram-negative isolates attained antibacterial activity to exclude O157:H7 while 17% of the active isolates produced colicins against it. In this study, a preliminary test based on blood cell lysis was carried out. The data indicated that with an exception of Aeromonas hydrophila 2, all active isolates with antagonistic activity i. e., E. coli, Citrobacter freundii, Klyuvera sp., Pantoea sp., Shigella sonnei, Pseudomonas putida/fluorescence, Raoultella terrigena, Klebsilla oxytoca Pasteurella pneumotropica, Salmonella ser pollurum and Serratia plymuthica did not produce any type of haemolysis (Alpha or Beta haemolysis) on BA media [Figure 5]. Our results showed that the colicinogenic species i. e., E. coli, Citrobacter freundii, Pantoea and Klyuvera spp. isolated from gut of animal and humans were grown on lowest pH of 3. Below this pH, negligible growth was obtained on the NA media plates [Table 2].
|Figure 4: The overlaid culture is the indicator strain (E. coli 0157:H7) seeded in soft lightweight authentication module (LAM) and wells contain 20 μl broth cultures of colicinogenic strains i.e., E. coli, Pantoea sp. and Shigella sonnei. E. coli and Pantoea sp. produced inhibitory zones against indicator strain while Shigella had no effect|
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|Figure 5: Colicinogenic isolates are cultured on blood agar media. The control specie i. e., Staphylococcus aureus has produced clear zone (Beta‑haemolysis) due to RBC haemolysis. GNR strains i.e., E. coli, Pantoea sp. and Citrobacter freundii did not produce zone of haemolysis confirming that the colicinongenic strains are nonhaemolytic and nonpathogenic to mammalian cells|
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| ~ Discussion|| |
Our results indicated that various samples collected from adult and children faeces, animal faeces and septic tank water were contaminated with GNR. The data further suggested that the highest percentage of recovery was noted in animal faeces samples. On the other hand, highest percentage of GNR (E. coli) has been reported from human adult samples.  Probiotic bacilli active against E. coli O157:H7 and Salmonella sp. have been isolated from human breast milk.  The antimicrobial activity of pure isolates was also determined by disc-diffusion method. One-third of the isolates (47 out of 175 isolates) showed antibacterial activity against E. coli O157:H7. Among these, human adult isolates were more antagonistic towards E. coli O157:H7. High number of antagonistic bacteria in human adults is the indication of strong immunity towards invading opportunistic GNR. Commensal bacteria in animals and humans have been found to exhibit significant antibacterial activity against invading pathogens.  However, only few strains isolated from faecal samples have been found capable of inhibiting common human pathogens in vitro, including E. coli O157:H7.  E. coli O157:H7 is difficult to be controlled with antibiotic like other strains and also develop resistance with antibiotic treatment.
Biochemical identification of 47 active isolates confirmed the presence of different species of enterobacteriaceae including E. coli, C. freundii, Klyuvera sp., Pantoea sp., S. sonnei, P. putida/fluorescence, R. terrigena, K. oxytoca, A. hyrophila and P. pneumotropica, S. ser pollurum and S. plymuthica strain. The diversity of microbial population in the intestine of humans and animals depends upon antibiotic uptake, diet and infection by opportunistic pathogens.  Out of 47 active species against O157: H7, only above-mentioned species produced colicins. S. sonnei and Salmonella strains did not show any inhibitory activity against the indicator strain. Addition of Mitomycin C enhanced the induction of colicin by these isolates when added to the broth media.  Moreover, competitive exclusion of E. coli O157: H7 by colicinogenic E. coli in cattle which were isolated from human and other animal faeces has been reported.  In the present study, adult humans and bovine faeces yielded 50% colicinogenic E. coli. It was also specified that 26.8% of the total gram-negative isolates attained antibacterial activity to exclude O157:H7 and majority of the active isolates produced colicins against it. Isolates to be used as probiotic were also examined for their pathogenic character. Aeromonas hydrophila strains produced slight beta haemolytic zones around its colony.  As the isolates were taken from normal human and animal micro-flora, therefore, the risks associated with the presence of any opportunistic pathogen were partially reduced.
The colicinogenic species i. e., E. coli, C. freundii, Pantoea and Klyuvera spp. isolated from the gut of animal and humans were able to grow on lowest pH of 3. Below this pH, negligible growth was obtained on the NA media plates. Acid tolerance is a critical requirement for probiotic bacteria, especially for those which are taken as oral supplements. These isolates have to pass highly acidic environment of stomach and then colonise colon region which also maintains pH 5.5 to pH 6. Colicinogenic strains with antagonistic activity against E. coli O157:H7 were found highly sensitive to Imepenam, Ciprofloxacin, Gentamicin and Amakacin while moderate sensitivity to Streptomycin and Erythromycin and Chloramphenicol. Whereas, consecutive highly resistant to Cefperazone, Augmentin and Penicillin G was shown by E. coli, Citrobacter, Klyuvera and Pantoea sp.
The presented research and its findings do not have any financial, nonfinancial or other competing interests with any organisation or individual. Furthermore, none of the authors hold and have applied for any patent related to the content of the manuscript.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]