EFFECT OF PROBIOTICS IN THE PREVENTION OF ANTIBIOTIC ASSOCIATED DIARRHOEA AND CLOSTRIDIUM DIFFICILE INFECTION

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Maheswari et al. SJIF Impact Factor Volume 4, Issue 08, Research Article ISSN EFFECT OF PROBIOTICS IN THE PREVENTION OF ANTIBIOTIC ASSOCIATED DIARRHOEA AND CLOSTRIDIUM DIFFICILE INFECTION N. Uma Maheswari* and S. Abinaya PG and Research Department of Microbiology S.T.E.T.Women s College, Mannargudi, Thiruvarur District, Tamil Nadu, India. Article Received on 06 June 2015, Revised on 29 June 2015, Accepted on 18 July 2015 *Correspondence for Author N. Uma Maheswari PG and Research Department of Microbiology S.T.E.T.Women s College, Mannargudi, Thiruvarur District, Tamil Nadu, India. ABSTRACT Diarrhoea as a common side effect of antibiotics, causes increased treatment costs and extended length of stay in acute health care facilities. Clostridium difficile as a cause of diarrhea has become a major issue in many countries, resulting in a search for the best way to prevent its occurrence. Prevention primarily revolves around control of antibiotic use, followed by comprehensive infection control procedures once outbreaks occur. C.difficile have become more frequent, more severe, more difficult to treat and more likely to recur after initial treatment. Although most people become infected with C.difficile in the hospital, the infection has also become more common in patients who have not been hospitalized or through community- acquired infection. Cefotaxime, amikacin and ampicillin were mainly responsible for causing AAD. Therefore conservative use of antibiotics would be beneficial to decrease the occurrence of AAD. Hence the present study was planned as C.difficile infected samples were collected from 50 patients, out of fifty, 10 male, 28 female and 12 children. The results of our study were clearly showed that 7 samples are frequently associated with children s 5-12 years age group. Commercial antibiotics and probiotics were administrated for the control of AAD; Lactobacillus casein was effectively control the diarrhea than antibiotics. KEYWORDS: C.difficile, Probiotics, antibiotics, diarrhea, Lactobacillus, Vancomycin. Vol 4, Issue 08,

2 INTRODUCTION Diarrhoea is a common symptom of gastrointestinal disease, characterized by increased frequency and fluid consistency of stools. It is a common side effect of antibiotics, causes increased treatment costs and extended length of stay in acute healthcare facilities. [1] Clostridium difficile infection as a cause of diarrhea has become a major issue in many countries, resulting in a search for the best way to prevent its occurrence. Prevention primarily revolves around control of antibiotic use, followed by comprehensive infection control procedures once outbreaks occur. Nevertheless, other potential factors have been explored, including the use of probiotic bacteria and yeast. The potential mechanisms by which probiotics may work; their safety; what future research is required; and recommendations for their use in clinical practice. [2] C.difficile associated diarrhea is severe foem of diarrhea caused by the bacteria. It is contagious and spreads easily from patient to patient, thus initial outbreaks can rapidly spread to other patients unless strict and comprehensive infection control measures are put in place immediately. [3] Probiotics are defined as live microorganisms which when administrated in adequate amounts, confer a health benefit on host. They are live microbial feed supplements that beneficial affects the host by removing or improving its intestinal balance. Most probiotic microorganisms are lactic acid bacteria such as Lactobacillus and Staphlococcus. [4] Therefore the present study was carried out to analyse the effect of probiotics on the diarrhoeal pathogens isolated from the infected patients stool samples collected from Government Hospital, Mannargudi, Thiruvarur District, Tamil Nadu, India. MATERIALS AND METHODS Collection of sample: Stool sample were collected from the patients with the help of containers. The stool specimen should be collected in clean, wide-mouthed containers that can be covered with tight-fitting lid. In some instances, collection of rectal swab rather than stool specimen may be necessary, particularly in neonates or in severely debilitated adults. The sample must contain the name and address, sample number. All stool samples were processed within 2 hrs of collection. Alcoholic shock treatment: Faecal materials were mixed with equal parts of industrial methylated spirit or absolute alcohol. Homogenize using a vortex mixer. Leave at room temperature for 60 mins at 20 0 C. [5] Vol 4, Issue 08,

3 Culture Procedure The stool samples were inoculated in Robertson s cooked meat (RCM) broth (Hi Media, Mumbai) for enrichment, and incubated at 37 0 C for h. Direct plating of each sample were done on Cycloserine cefoxitin fructose egg yolk agar (CCFA) Dehydrated culture media; all the plates were incubated anaerobically in Anaerobic jar for hrs. Suspected colonies were identified as C. difficile by Gram stain and standard biochemical tests like sugar fermentations, gelatin hydrolysis, hydrogen sulphide production, etc. After 48 hrs of incubation on CCFA, colonies of C. difficile were 4 mm or larger, flat to slightly raised yellowish rhizoid colonies which had a speckled opalescence and strong horse manure-like odour. [6] Idenificaion of Bacteria The colonies were subjected to Gram s staining in order to identify the morphology and gram s staining reaction of bacteria and also the motility test were performed. After the biochemical tests were performed. [7] Probiotics Sensitivity Test The anti-bacterial activity of probiotic was done by Agar Well Diffusion method. Sterilized MHA medium was poured into sterile petriplates and allowed to solidify. After solidification of the medium, the inoculums were vortexed and 1.00 ml of the inoculum were spread evenly over the surface o the agar plates using a glass spreader. A well of 5 mm diameter was made in each plate with a sterile cup borer. The probiotic was introduced into the separate plate and in a single well. The antagonistic test was performed in triplicate. The efficiency was tested against pathogens. After incubation, the diameter of the inhibition zone around the well was measured in mm, this clear zone was used as indication o the ability of probiotic antagonism to the tested pathogen. [8,9] The following probiotics were used in this sensitivity test. Lactobacillus bulgaricus, Lactobacillus cassei, Bifidobacterium bifidum, Saccharomyces boulardii. Antibiotic Sensitivity Test This sensitivity test was carried out by Disc Diffusion method. Two great scientists William Kirby and A.M.Bauer developed it during the year This test is done to determine which antibiotic effective against the particular pathogen. In this method sterilized MHA medium was prepared and sterilized and poured into the sterile petriplates and allowed to solidify. After solidification of the medium the inoculams were vortexed and 1.00 ml of Vol 4, Issue 08,

4 the inoculam were spread evenly over the surface o the agar plates using a glass spreader. Both test and control organisms were used in two separate plates. Maximum of six discs may be place in a plate. The plates were incubated. After incubation Width of the zone of inhibition is usually measured. [10] The following antibiotic discs were used in this sensitivity test. Vancomycin, Metronidazole, Amikasin, Ceflotoxin, Cephalosporins, Clindamycin. Statistical Analysis The values are expressed as mean±standard error mean from antibiotics and probiotics zone of inhibition. [11] RESULTS Isolation and identification of Bacteria The C.difficile was grown on dehydrated culture media, the organisms nearly 4mm or larger, flat to slightly raised yellowish rhizoid colonies, and strong horse manure like odour will be produced. The CDAD is treated with live organisms to prevent diarrhea associated with antibiotics shows that probiotics may be effective in preventing antibiotic associated diarrhoea. Hence it was confirmed as C.difficile gram positive rod shaped bacterium. The biochemical test was highly used for the confirmation of the bacterium. Here mostly the bacterium dose not ferment sugar, and some utilize the sugar, carbohydrate, etc., Hence the organism was positive to urease, citrate. Antibiotic Sensitivity Test The sensitivity of vancomycin were showed the zone of inhibition against C.difficile 21±5.07 mm. Metronidazole was showed 14 ± 2.2 mm zone and Amickasin were showed 12 ± 1.4 mm zone against C.difficile. Followed by Cephalosporin, Clindamycin, Ceflotoxin was effectively resistant to C.difficile. The resistant of Clindamycin were showed the zone of inhibition against C.difficile (2 ±2.678 mm), cephalosporin (1.6 ± 2.84 mm), and ceflotoxin (0.8 ± 3.16 mm). (Table 2). Probiotic Sensitivity Test: Probiotic susceptibility of the isolated organisms was determined by agar diffusion method. Among them C.difficile was sensitive to Lactobacillus casein (24 ± 6.75 mm), Bifidobacterium bifidum (21± 5.23 mm) and Saccharomyces boulardii (18 ± 4.87 mm) (Table 1) Lactobacillus casein was showed better response on C.difficile. Hence our study clearly highlighted that probiotics are effectively control the diarrhea than commercial antibiotics. Vol 4, Issue 08,

5 Table 1: Probiotic sensitivity S.No Probiotics Result Zone of inhibition (mm) 1 Lactobacillus cassei S 24 ± Bifidobacterium bifidum S 21± Saccharomyces boulardii S 18 ± 4.87 Table 2: Antibiotic sensitivity S.No Antibiotics Result Zone of inhibition (mm) 1 Vancomycin S 21 ± Metronidazole S 14 ± Amikasin S 12 ± Ceflotoxim R 0.8 ± Cephalosporins R 1.6 ± Clindamycin R 2 ±2.678 DISCUSSION The C.difficile was isolated from the patient ward was more than that of outpatient ward. The C.difficile is easily spread through various ways. This may be due to the following factors; The antibiotics like Clindamycin, Cephalosporins and Fluoroquinolones are disturbs the colonic microbiota. The C. difficile can be found on beside tables, door knobs, counters, lavatories, and sinks in the hospital. It also spread between patients via contact with contaminated objects and hands of healthcare workers. Our results are in agreement with Diagnostic role of stool culture and toxin detection in antibiotics associated diarrhea due to C.difficile in children. [12] Among positive cases, 68,9 percent responded to discontinuation of antibiotics and 31.1 per cent to metronidazole therapy. In the study combination of cefotaxime and amickacin was responsible for diarrhea in 28.9 per cent positive cases. The major cost implications associated with C.difficile infection/ outbreaks related to increased hospital stay, antibiotic treatment, possible ward closure and loss of bed days together with infection control requirements. If with a larger study, are confirmed the justification for the use of probiotic therapy for all patients receiving antibiotic therapy for all patients receiving antibiotic therapy on admission to hospital would be clearly evident. Our study report clearly highlighted the controlled effect of probiotics such as Lactobacillus casein, Bifidobacterium bifidum and Saccharomyces boulardii. [13] Vol 4, Issue 08,

6 CONCLUSION From our study concluded that probiotics are effectively control of AAD, such as L.caseiin by well diffusion method, and then commercial antibiotic such as Vancomycin by disc diffusion method. Avoidance of unnecessary use of antibiotics is of paramount importance. Hospital antibiotic use should be limited and regulated. Medical personnel should be enforced to follow simple enteric precautions and educated about the disease and its epidemiology. REFERENCES 1. Bartlett JG, Narrative review: The new epidemic of Clostridium difficile-associated enteric disease. Ann Intern Med. 2006; 145: Fekety R, Kim KH, Brown D. Epidemiology of antibiotic-associated colitis: isolation of Clostridium difficile from the hospital environment. Am J Med. 1981; 70: Pandey C, Kelly CP, Joshi MA, Gao N, O Keane CJ, Castagliuolo L. Saccharomyces boulardii inhibits Clostidium difficle toxin A binding and enterotoxicity in rat ileum Gastroenterology. 2007; 104: Sindhu GY, Khetarpaul L. Probiotics as prophylactic agents against antibiotic associated diarrhoeae in hospitalized patients. Harefuah. 2001; 146: Goldenberg JZ, Ma SS, Saxton JD. Probiotics for the prevention of Clostridium difficileassociated diarrhea in adults and children. Cochrane Database Syst Rev. 2013; 5: McFarland LV. Alternative treatments for Clostridium difficile disease: what really works? J Med Microbiol. 2005; 54: Miller MA. Clinical management of Clostridium difficile-associated disease. Clin Infect Dis. Suppl. 2007; 2: Ng SC, Hart AL, Kamm MA, Stagg AJ, Knight SC. Mechanisms of action of probiotics: Recent advances. Inflamm Bowel Dis. 2008; 15: Surawicz CM, Brandt LJ, Binion DG. Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. Am J Gastroenterol. 2013; 108: Wistrom J, Norrby SR, Myhre EB, Eriksson S, Granstrom G, Lagergren L. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: A prospective study. J Antimicrob Chemother. 2013; 47: Vol 4, Issue 08,

7 11. Wullt M, Hagslatt ML, Odenholt I. Lactobacillus plantarum 299v for the treatment of recurrent Clostridium difficile-associated diarrhoea: A double-blind, placebo-controlled trial. Scand J Infect Dis. 2003; 35: Young GP, Ward PB, Bayley N. Antibiotic-associated colitis due to Clostridium difficile: double-blind comparison of vancomycin with bacitracin. Gastroenterology. 1985; 89: Zar HK. statistical methods for the biological people Am J Clin Nutr. 1984; 91: Vol 4, Issue 08,