Evaluation of Colicin Effect on the Induction of Treated Mice in Prevention of Infection Caused by Escherichia coli K99

Copyright © 2016 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Background The neonatal calf, pig, lamb, and kid are mostly involved with many diarrheal diseases.1 Epidemiologic studies have indicated enterotoxigenic Escherichia coli (ETEC) K99 as the major cause of neonatal diarrhea occurring in the first four days of life.2 The disease has been estimated to be responsible for as much as 30% of the economic losses in animal husbandry.3 The use of antibiotics for diarrheal diseases has been blamed for the emergence of multidrug resistance in food animals and human medicine.4,5 The high risk of antimicrobial resistance and increasing antibiotic residues in environment have compelled to look for alternative antibiotics.4,6 The potential use of bacteriocin-producing bacteria as probiotic and bio-protective agents has recently received increasing attention.7,8 Colicins are narrow-spectrum bacteriocins produced by colicinogenic E. coli (CEC) that are toxic only to bacteria closely related to the producing strain.9 They are produced by strains of E. coli that carry a colicinogenic plasmid, bearing the genetic determinant for colicin synthesis, immunity, and release.3,5 Surveys have shown that approximately 30% of all E. coli strains can produce colicin.10 Pore-forming colicin, such as colicin V (ColV), binds to its target bacteria, disrupts the ionic gradient of the cell, and then kills it.5 Although many studies have investigated the inhibitory effect of colicin against intestinal E. coli pathogen,8,10,11 none of them has explained an experimental in vivo study against E. coli K99. However, the study of interaction between pathogenic bacteria and the host needs a simple and cheap animal model.


Escherichia coli K99 Preparation
The E. coli k99 was grown in Minca broth medium and incubated overnight (O/N) at 37°C.Cells were washed with phosphate buffer saline (PBS) (Kimiateb Co., Iran) by centrifugation at 3000×g for 4 minutes and the number of bacteria was adjusted to 1×10 8 CFU/mL.

Colicin Solution Preparation
The method of Pugsley and Oudega, 16 with some modifications, was used to prepare colicin solution.Briefly, CEC strains were grown in Luria-Bertani (LB) (HiMedia, India) broth and incubated O/N at 37ºC.Five milliliters of the O/N culture was inoculated into 50 mL of fresh LB broth and incubated at 37°C for 3 to 4 hours with shaking (160 rpm).When the optical density at 600 nm reached 0.4, mitomycin C solution (250 µg/mL) was added to attain a volume of 500 ng/mL at final concentration (mitomycin induces Colicin production).After 1 hour additional incubation, the broth was disrupted by sonication (UP200H; Hielscher Co. Germany), then centrifuged, and the supernatant was filtrated through low protein-binding 0.22-µm-pore-size membrane filters (Spritzenfilter, Denmark) and stored at -80°C as colicin solution.The production of colicin was proved in previous experiment, before we examine the antibacterial effect of some colicinogenic E. coli by Spot method.

Mice Treatment
The experiment was conducted on 2 mice groups of 2-week-old (30 in each group).Prior to bacterial inoculation, all mice were administered 10 mg/L streptomycin sulfate (Jaber-E-BneHayyan Pharmaceutical Mfg.Co., Iran) in their drinking water to eliminate resident E. coli facultative bacteria.Antibiotic treated mice were screened for considering fecal E. coli by plating on eosin methylene blue (EMB) agar (HiMedia, India) plates.The mice were divided equally into 2 groups.The first group was maintained in a separate cage as control inoculated with oral PBS.All mice in the second group were fed with colicin solution twice a day, with 2-day intervals.

Treated Mice Comparison
Pathogenic E. coli K99 was used for challenge.The LD 50 was estimated according to Yousif et al. 17 Both control and treated groups were challenged by 3 LD 50 of E. coli K99 and followed up for 1 week.Fecal samples from both groups were taken 7 days after challenge.One gram of feces was separately transferred onto 1 mL of PBS.They were serially diluted tenfold (1:10) in PBS (pH 7.2), 200 µL of each dilution was plated onto nutrient agar, and the plates were incubated for 24 hours at 37°C.Colony forming units (CFUs) were monitored per gram feces.

Data Analysis
The results were declared as means ± standard deviation (SD).Analysis of variance (ANOVA) was used to distinguish statistical significant differences (P < .05)among various groups.A 2-tailed, paired Student's t test was carried out to characterize statistical variation between groups.

Bacterial Detection
PCR was used for determination of the genes encoding the colicins for CEC and virulence factors for E. coli K99.Multiplex PCR identified 2 fimbrial (F 5 and F 41) and one STa toxin genes of E. coli K99 and CEC harbored Col V (Figure 1).

Experimental Challenge
Treated mice group showed mild signs of illness and depression for 2-3 days including loss of appetite and weight, dehydration, ruffled fur, lethargy, and hunched posture.Diarrhea may or may not be seen.They went back to normal condition at the middle of the experiment (4 days after the experiment was started).As noted, E. coli k99 was established in GI tract of control group and watery diarrhea was observed with different sings of colibacillosis post challenge.The infected mice could be fatal if not treated within 3-4 days (Figure 2).

Colony Count
The excretion rate of E. coli K99 in control and treated mice groups are shown in Table 2.The levels of E. coli K99 recovered in the feces of treated group was significantly lower than those in the feces of control group (P < .05).However, except for a difference observed at day 1 (less than 1.2 log 10 CFU g -1 ), none of these differences were statistically significant.There were observed significant differences between 2 groups after day 4 (where the differences were more than 1.2 log 10 CFU g -1 ) (P < .05).A maximum of 1.262 log 10 CFU g -1 reduction in E. coli K99 was obtained 5 days after colicin solution intake (P < .05).

Discussion
We examined the efficacy of ColV in preventing diarrhea caused by E. coli K99.Treated mice with colicin solution prevented colonization of E. coli K99 in GI tract.An in vitro previous study by Shirazi et al proposed that colicin production by CEC is responsible for the inhibition of E. coli k99.Using Spot method, we showed colicin inhibited the growth of E. coli K99. 12 The finding of Shirazi et al was comparable with the results of the present study, in which ColV could abrogate lethal activity of E. coli K99 in mice.
The use of CEC 9 to colonize in the GI tracts of lamb and kid who suffer from clinical diarrhea is currently considered .Some experiments revealed that colicin removed E. coli from GI tract and inhibited the growth of E. coli K88, the causative agent of swine diarrhea. 18Lema et al fed lambs with a mixture of CEC to reduce the incidence of diarrhea, and observed a decrease in pathogen levels. 19The results indicated that oral administration of E. coli K99 after colicin inoculums is more effective than vice versa.This might refer to competitive aspect of CEC.Colicin was primarily replaced in infant GI tract, all receptor binding sites were blocked, and thereafter, E. coli    K99 attachment was inhibited. 9he differences between control and test groups in fecal shedding was 1.2 and 1.4 log CFU g -1 feces after days 4 and 5, respectively.These differences among other studies were varied, as 1.75, 1.52, 1.6, and 1.8 log 10 CFU g -1 feces per days 16, 18, 21, and 18, respectively. 10The mechanism of action of CEC in the inhibition of E. coli K99 was not fully understood.It seems that the competitive inhibition of pathogenic bacteria from adhering to respective host receptors, production of anti-bacterial substances such as colicin, and probiotic diversity should be the inhibitory mechanisms of CEC in deterrence of E. coli K99 colonization. 20The rate of the colicin solution that reached the GI tract may have been the ascertaining factor in the appearance of diarrhea. 12olicin significantly decreased the fecal shedding level of the E. coli K99 at early hours post challenge.Not all E. coli K99 would be killed by colicin solution; in fact, fewer of the E. coli K99 was able to cause inflammatory response and produce diarrhea in the treated mice group.Fairbrother et al 21 and Gyles 22 verified this may be due to the low expression of interleukin-1β (IL-1β) in the GI tract tissue treated with colicin solution.During the inflammatory response, IL-1β is firstly secreted by macrophages and active lymphocytes.IL-1β release has been associated with E. coli K99 toxin production.Girard et al 23 demonstrated in high dose of colicin intake, the concentration of IL-1β in GI tract was significantly decreased.They also indicated low-dose colicin solution increased IL-1β levels to over six-fold, higher than those in the control group.
In conclusion, this result indicated the potential use of CEC to protect colonization of E. coli K99 in GI tract of murine model and reduce fecal shedding and therefore prevent diarrhea.Colicin and biotherapy are good alternative antibacterial agents instead of antibiotic.Investigation on livestock for applying colicin in animal farms is recommended.

Figure 2 .
Figure 2. Number of Alive and Dead Mice in Treated and Control Challenge.

Table 1 .
Oligoneucleotide Primers Used in Multiplex PCR

Table 2 .
Fecal Shedding of Escherichia coli K99 by Adult Mice During 7 Days of Experiment E.

coli K99 Count (log 10 CFU g -1 ) diff 3 P Control Group (n = 30) Treated Group (n = 30)
1ariables with P < .05, in different alphabetical letters, are considered significant.1Average; 2 Diarrhea; 3 Differences (diff) between the mean of control group and treated group in each day.