1. Introduction
Clostridium difficile is a Gram-positive, spore-forming, toxin-producing anaerobic bacillus and is a component of the intestinal flora in healthy infants. It is also a known cause of pseudomembranous colitis and diarrhea in patients under antibiotic therapy [1, 2]. The causative agent of Clostridium Difficile Infection (CDI) is exotoxins A and B produced by this bacterium [3, 4].
Probiotics are micronutrients (bacteria and yeast) that settle in different parts of the body, especially in the intestine, modify the microbial flora and exert beneficial effects on the health of the host [6]. A significant percentage of patients admitted to infectious disease wards are older people who need long-term antibiotic therapy, and increased colonization of Clostridium difficile in them is associated with a higher risk of CDI [8-12]. The use of probiotic yogurt can prevent the growth of Clostridium difficile and reduce morbidity and even mortality in these patients [7]. Of the studies on the effect of probiotics on CDI, few studies have investigated the impact of probiotics on the frequency of C. difficile in older people. In this regard, this study investigates the effect of probiotic yogurt use on the rate of Clostridium difficile in hospitalized elderly patients.
2. Materials and Methods
In this double-blind, randomized clinical trial, 132 patients over 60 years old who were admitted to the infectious Ward of Vali-e-Asr Hospital in Arak, Iran were selected through convenience sampling method. They were diagnosed with acquired pneumonia and under antibiotic therapy using ceftriaxone and azithromycin. After obtaining informed consent from them, they were randomly divided into two groups of intervention (n=66) and control (n=66).
The patients in the intervention group received 200 mL probiotic yogurt from day 1 to day 8 of hospitalization twice per day (lunch and dinner). The control group received 200 mL of regular yogurt for the same period, both at lunch and dinner. In the first and eighth days of the study, all stool samples were taken, and their DNA extracted, and the extract was used in a Polymerase Chain Reaction (PCR) with 16S RNA and srRNA Universal primers. The PCR product was sent for nucleotide sequencing, and the results underwent BCAST analysis. Mean and Standard Deviation (SD) were used to describe the quantitative data and the number and percentage were used to describe the qualitative data.
3. Results
At the beginning of the study, the prevalence of Clostridium difficile in the intervention and control groups was 4 (6.1%) and 1 (1.5%), respectively. After the intervention, the number of stool samples with CDI and the number of cases with antibiotic-associated colitis symptoms were not significantly different between the intervention and control groups (P <0.05). The comparison of symptoms between the two groups showed that the prevalence of fever (P=0.039) and constipation (P=0.046) was higher in the intervention group at the beginning of the study, and the difference was statistically significant (Table 1).

Furthermore, the study showed that the likelihood of developing CDI in people with a history of cerebrovascular diseases was 1.69 times higher than those with no such history. The history of antibiotic use in the past 6 months also increased the risk of developing CDI by 1.97 times.
4. Discussion
This randomized clinical trial study was performed to determine the effect of probiotic yogurt on the incidence of C. difficile in 132 patients (mean age: 72 y) admitted to the Infectious Disease Ward of Vali-e-Asr Hospital. At the beginning of the study, the prevalence of C. difficile was 3.8%. Also, in positive cases, the relative frequency of fever was higher. Based on the results of this study, a history of corticosteroid use, antibiotic use in the past 6 months, proton pump inhibitors, history of contact with a child at home, history of hospitalization, and history of brain injury in patients with positive C. difficile were higher. This difference was statistically significant According to the Fisher exact test. By considering positive C. difficile as the dependent variable, history of cerebrovascular disease, antibiotic use in the past 2 to 6 months, using proton pump inhibitors, history of contact with child at home, history of hospitalization in the past 6 months, history of corticosteroid use, and fever were risk factors of CDI. In our study, there was a decrease in the incidence of positive C. difficile on the 8th day of the study compared to the first day, but it was not statistically significant between the two groups.
Thus, the use of probiotic yogurt has no significant effect on reducing the prevalence of C. difficile and colitis symptoms in comparison with regular yogurt. For more accurate results, it is recommended to conduct studies with a larger sample size in several different health centers.
Ethical Considerations
Compliance with ethical guidelines
This is a registered clinical trial (Code: IRCT20160 92229915N1) approved by the Research Ethics Committee of Arak University of Medical Sciences (Code: 10.165.93).
Funding
The present paper was extracted from a PhD. thesis approved by Elahe Eqbal in the Department of Infectious Diseases at Arak University of Medical Sciences.
Authors' contributions
Conceptualization by Masoomeh Sofian, Elaheh Eghbal, and Pegah Mohaghegh; investigation by Elaheh Eghbal, Ehsanollah Ghaznavi-Rad, Amitis Ramezani, and Masoomeh Sofian; editing and final draft preparation by Masoomeh Sofian and Pegah Mohaghegh. All authors met standard writing standards based on the recommendations of the International Committee of Medical Journal Publishers (ICMJE).
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the authorities of infectious disease ward and the laboratory staff of Vali-e-Asr Hospital for their valuable cooperation.


References