1. Introduction
Diarrhea is one of the most common infectious diseases among humans globally [1] . It causes significant health risk particularly among pediatrics with most vulnerable age group affected is children less than 5 years of age, which is also, accounted for the high mortality rates in this age group [2] . ORS (oral rehydrating solution) has been a mainstay treatment in managing 90% of children with mild to moderate diarrhea [3] . Along with ORS zinc supplements are also found helpful in reducing the duration and volume of stools [4] . However, in pediatric infectious diarrhea antibiotics including Ampicillin, Cefexime, Ceftriaxone, Amikacin, Nalidixic acid and Ciprofloxacin are also required as their immunity is not sufficient to clear the infections [5] . Antibiotics on account of their toxicity, have limited use in pediatric age group [6] as well as their resistance is a rising threat to the human worldwide these days [7] .
In Pakistan irrational and repeated use of antibiotics for infectious diarrhea in children below 5 years of age has resulted in increased resistance and several toxicities of broad spectrum antibiotics, including alteration of the normal gut flora; which could lead to potential risk for future serious infections among children [8] .
Evidence from the literature suggested the clinical use of probiotics for the treatment of bacterial gastroenteritis [9] . Probiotics are defined as the living microorganisms which when dispensed in the body at appropriate amounts prove to be favorable in promoting the health benefits, and at the same time it also increases the stability of the intestinal flora [10] . The probiotics used for the treatment of diarrhea in children are of bacterial or fungal origin. The bacterial strains of commonly used probiotics are Bifidobacterium and Lactobacillus, whereas, Saccharomyces boulardii is a yeast, which is a type of fungus [11] . The strains of Lactobacilli and Saccharomyces boulardii have proven its efficacy in reducing diarrhea [12] .
Currently the probiotics are used along with antibiotics for infectious diarrhea but their direct bactericidal/bacteriostatic effects have not been tested or proved on the basis of any laboratory data or in-vitro culture and sensitivity tests. To the best of our knowledge, the current study is innovative across the globe. This study will help us to know the efficacy of probiotics Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii in comparison to the standard antibiotics against microbial organisms causing infectious diarrhea in children less than 5 years of age on the basis of stool culture and sensitivity.
2. Material and Methods
This prospective quasi experimental study was carried out in a pediatric unit and the microbiology laboratory of a tertiary care hospital, Dr. Ruth. K. M. Pfau Civil Hospital Karachi from December 2017 till May, 2018. The eligibility criteria for the recruitment in this clinical study were; children aged six months to five years, clinical diagnosis of acute diarrhea as per World Health Organization (WHO) criteria (i.e. having at least four liquid stools in the past 24 hours along with clinical signs and symptoms of dehydration on clinical examination). Children having systemic infection, malabsorption syndrome, severe acute malnutrition, blood in stool, have received antibiotics in last fourteen days or immediately require antibiotic for current infection were excluded.
The research was conducted following the ethical guidelines of Helenski declaration and Pakistan Medical and Research Council. Written informed consent was obtained from the parents or guardian prior to the recruitment of children with diagnosis of acute diarrhea. Moreover, the guardian or parents were comprehensively briefed about the research purpose and procedures involved. Importantly, the anonymity and confidentiality of the study participant’s data was maintained throughout the research with no unauthorized person having access to the data. The research is approved by the institutional Ethical Review Committee (ERC), and the research was initiated after the approval was granted by the ERC.
Parents or guardian were informed and briefed to collect at least 5 gram of faeces of eligible study participants in a sterilized stool culture bottle. The data related to basic demographics was also collected for each eligible participants being recruited in this research.
Routine laboratory examination and stool culture were performed within four hours after collection of specimen. Initially, the stool samples were grossly examined for color and consistency. Later, the microscopic examinations of stool samples were performed to identify the presence of any cellular elements (i.e. red blood cells, white blood cells, pus cells), eggs, protozoa, cysts of parasites etc.
The stool culture was performed to identify the enteric pathogen causing acute diarrhea i.e. E. coli and Klebsiella. Standard procedures and steps (i.e. collecting in sterilized container and immediate processing within four hours) were followed for the stool culture process. At day 1 the stool sample was inoculated with Salmonella Shigella agar (SS agar), MacConkeys agar, and selenite enrichment broth being incubated aerobically at 37˚C overnight. Following day, subculture from selenite F broth on Salmonella Shigella agar was performed. At day 3, the stool cultures were re-examined for the presence of organisms i.e. E. coli, Klebsiella and Salmonella. The biochemical identification was later done for the confirmation of pathogenic strain by using; Simon citrate agar, SIM medium agar, Urea agar and TSI agar. The serological analysis was carried out by using E.coli and Salmonella and Shigella Antisera.
On each of the stool sample that showed positive growth of E. coli, Klebsiella and Salmonella, all standard antibiotics and two probiotics, Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii were applied. The standard antibiotics applied were Amikacin, Amoxicillin, Gentamycin, Cefuroxime, Ceftazidime, Aztreonam, Ampicillin, Cefotaxime, Ciprofloxacin, Nalidixic Acid, Levofloxacin, Ceftriaxone, Imipenim, Trimethoprim-Sulfamethoxazole, Cefoperazone/Sulbactam and Piperacillin/Tazobactam, according to the Clinical Laboratory Science Institute (CLSI) 2018 guidelines [13] . The Zones of Inhibition (ZOI) of each antibiotic were measured. The calibrated vernier caliper was used to measure the diameters in millimeters (mm) of each antibiotic disc along with the clear surrounding clear area till the edges of the clear zone (showing no bacterial growth).
While the two probiotics, Lactobacilli and Saccharomyces boulardii were analyzed by dried modification method [14] . Standard procedure and protocol were followed where initially 2 µL of overnight culture was spotted on MRS agar. Later plates were dried at room temperature for half an hour and incubated an aerobically on 37˚C for 18 hours. Plates were overlaid with 10 ml of specific microorganism specific medium. Once the colonies were developed, the plates were again overlaid with 10 ml of soft microorganism specific medium and later laid for overnight culture of the target pathogenic strain. Following, 48 hours of incubation the ZOI was measured and interpreted as (ZOI > 20 mm as sensitive and less than 10 mm as resistant).
3. Statistical Analysis
The data was analyzed using statistical software SPSS version 19.0. Initially, the data was validated twice for incorrect entries by checking with the study proforma. The categorical variables (i.e. gender, age categories and culture isolates) were presented as frequency/percentage while for their significance Chi square was applied.
4. Results
In the present research, stool sample from 325 children with confirmed diagnosis of acute diarrhea were collected and among those, 152 stool samples showed positive bacterial growth. Figure 1 gives details of the age categories (months) of 152 children with positive stool culture. Among all 152 positive culture isolates majority, 53 (34.9%) were found to be in the age group of 13 - 24 months, around eight percent in less than or equal to 12 months and 37 - 48 months of age categories. Figure 2 gives details of gender distribution. Majority, 57% were males while forty three 43% percent were females.
Table 1 shows the mean ZOI (mm) for individual organisms isolated from the stool samples of children with diarrhea for all antibiotics and the two probiotics Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii.
Figures 3-5 show the sensitivity pattern of different antibiotics and Probiotics, Lactobacilli and Saccharomyces boulardii for different organisms isolated. For E. coli the highest sensitivity was observed for Amikacin (96.7%), Gentamycin
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Table 1. Mean Zones of Inhibition (mm) of antibiotics and probiotics Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii for all three Organisms isolated from the stool samples of children with diarrhea, total N = 152.
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Figure 3. Sensitivity patterns of Antibiotics and Probiotics, Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii for E. coli (n = 92).
AMK; Amikacin, AMC; Amoxicillin-clavulanic acid, CN; Gentamycin, CXM; Cefuroxime, CAZ; Ceftazidime, ATM; Aztreonam, AMP; ampicillin, CTX; cefotaxime, CIP; Ciprofloxacin, NA; Nalidixic acid, LEV; Levofloxacin, CRO; Ceftriaxone, IMP; Imipenem, SXT; Trimethoprim-Sulfamethoxazole, CEF/SUL; Cefoperazone/Sulbactam, PIP/TAZO; Piperacillin/tazobactam, LACT; Lactobacillus paracasei/Lactobacillus acidophilus, SACC; Saccharomyces boulardii.
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Figure 4. Sensitivity patterns of Antibiotics and Probiotics, Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii for Klebsiella (n = 56).
AMK; Amikacin, AMC; Amoxicillin-clavulanic acid, CN; Gentamycin, CXM; Cefuroxime, CAZ; Ceftazidime, ATM; Aztreonam, AMP; ampicillin, CTX; cefotaxime, CIP; Ciprofloxacin, NA; Nalidixic acid, LEV; Levofloxacin, CRO; Ceftriaxone, IMP; Imipenem, SXT; Trimethoprim-Sulfamethoxazole, CEF/SUL; Cefoperazone/Sulbactam, PIP/TAZO; Piperacillin/tazobactam, LACT; Lactobacillus paracasei/Lactobacillus acidophilus, SACC; Saccharomyces boulardii.
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Figure 5. Sensitivity patterns of Antibiotics and Probiotics, Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii for Salmonella typhi (n = 4)
AMK; Amikacin, AMC; Amoxicillin-clavulanic acid, CN; Gentamycin, CXM; Cefuroxime, CAZ; Ceftazidime, ATM; Aztreonam, AMP; ampicillin, CTX; cefotaxime, CIP; Ciprofloxacin, NA; Nalidixic acid, LEV; Levofloxacin, CRO; Ceftriaxone, IMP; Imipenem, SXT; Trimethoprim-Sulfamethoxazole, CEF/SUL; Cefoperazone/Sulbactam, PIP/TAZO; Piperacillin/tazobactam, LACT; Lactobacillus paracasei/Lactobacillus acidophilus, SACC; Saccharomyces boulardii.
(95.7%) Imipenim (95.7%) and Piperacillin/Tazobactam (84.8%). While, the other antibiotics which showed lower sensitivity are Amoxicillin/Clavulanic acid (17.4%), Ceftazidime (13%), Ampicillin (6.5%), Cefotaxime (13%) and Nalidixic acid (26.1%). Moreover, for Klebsiella the highest sensitivity was observed for Imipenim (98.2%), Amikacin (94.6%), Piperacillin/Tazobactam (92.9%) and Gentamycin (89.3%). Although, lower sensitivity is observed for Amoxicillin/Clavulanic acid (10.7%), Cefuroxime (17.9%), Ampicillin (7.1%) and Ceftriaxone (25%), for Salmonella typhi the highest sensitivity (i.e. 100%) was observed for Amikacin, Gentamycin, Ciprofloxacin, Levofloxacin, Cefoperazone/Sulbactam and Piperacillin/Tazobactam. Whereas, some of the antibiotics such as Cefuroxime, Ceftriaxone, Imipenim, Trimethoprim/Sulfmethoxazole revealed moderate sensitivity (i.e. 50%). Few of the antibiotics had shown no sensitivity. However, for Lactobacilli the sensitivity for E. coli, Klebsiella and Salmonella typhi were 28.3%, 25% and 25% respectively. While for the second probiotic, Saccharomyces boulardii the sensitivity for E. coli, Klebsiella and Salmonella typhi were 37%, 32.1% and 25% respectively.
5. Discussion
Though, different antibiotics have been used for the treatment of bacterial diarrhea, but considering growing resistance of antibiotics and harmful effects, [7] researchers are more focused towards exploring the alternative means of treatment. Probiotics were first explored in 1907 by a Russian scientist Ellie Metchnikoff who linked the wellbeing and longevity of Bulgarian workers with the substantial ingestion of yogurt which contained large amounts of Lactobacillus species [15] . The other probiotic Saccharomyces boulardii was discovered in 1920 by a French microbiologist Henri Boulard, during a visit to Indochina, where he found that some people did not develop infectious diarrhea during the outbreak because they were already consuming a special type of tea made up of the outer skin of the tropical fruits lychee and mangosteens [9] . Generally, probiotics are preferred for the treatment of acute diarrhea by clinicians [8] and have been hypothesized in promoting the health benefits including the stability of the intestinal flora [16] .
The possible mechanisms of action of Saccharomyces boulardii comprises, in vivo antimicrobial activity, immune system activation, antitoxin activity which helps to reduce the enterotoxin, enhances the enzymatic activity and favors the absorption and nutrition [17] . Locally, it has shown to secrete a heat-labile factor which decreases bacterial adherence [18] . One of the previous studies also revealed the immunomodulating effects on the intestine of the rats orally treated with Saccharomyces boulardii, attributed to increase the levels of secretory IgA and components of crypt cells of the small intestine [19] . Moreover, the probable mechanism of action of Lactobacillus species is to increase the mucin expression in human intestinal epithelial cells which blocks the invasion and adherence of pathogenic E. coli [20] . Additionally it is able to inhibit tumor necrosis factor (TNF) mediated inflammation and apoptosis in intestinal epithelial cells [21] . It has demonstrated mitogenic effects and has enhanced regeneration of mucosal lining [22] . However their direct antimicrobial effects are largely unknown due to lack of scientific evidence on the basis of in-vitro or culture and sensitivity tests. Hence their role as alternatives to antibiotics in bacterial gastroenteritis is not very much convincing as a single agent and used along with antibiotics.
The current study was aimed to evaluate the direct antimicrobial effects of probiotics on the basis of sensitivity testing of cultured stool samples of pediatric patients with diarrhea. This is evident from Table 1 that probiotics, Lactobacilli and Saccharomyces boulardii had shown overall low zone of inhibitions for all organisms in comparison to majority of antibiotics. However, the zones of inhibition of Saccharomyces boulardii for E. coli were equivalent to trimethoprim/sulfamethoxazole and ampicillin, 11.16± 4.74 mm and 11.84 ± 1.82 mm respectively. No parallel or comparable data is available reporting the ZOIs for probiotics as our study is the first to assess these on isolated cultured samples of stool in pediatric patients with infectious diarrhea. The results of our study highlighted that the sensitivity of Lactobacilli for E. coli, was 28.3%, which is higher than majority of the commonly prescribed antibiotics including Amoxicillin/Clavulanic acid (17.4%), Ceftazidime (13%), Ampicillin (6.5%), Cefotaxime (13%) and Nalidixic acid (26.1%). Similarly, for Klebsiella the sensitivity of Lactobacilli was 25% and was slightly better in comparison to commonly prescribed antibiotics including Amoxicillin/Clavulanic acid (10.7%), Cefuroxime (17.9%), Ampicillin (7.1%) and was equivalent in comparison to Ceftriaxone (25%).
Whereas, the sensitivity of Saccharomyces boulardii for E. coli, was 37%, slightly higher in contrast to most commonly prescribed antibiotics used for pediatric diarrhea including, Amoxicillin/Clavulanic acid (17.4%), Ceftazidime (13%), Ampicillin (6.5%), Cefotaxime (13%), Nalidixic acid (26.1%), and approximately equivalent to Cefuroxime (34.8%), Aztreonam (30.4%), Trimethoprim/Sulfmethoxazole (34.8%) and Ceftriaxone (37%). The sensitivity of Saccharomyces boulardii for Klebsiella was displayed to be 32.1%, better than the widely prescribed antibiotics, Amoxicillin/Clavulanic acid (10.7%), Cefuroxime (17.9%) and Ceftriaxone (25%). The lower sensitivity of commonly prescribed antibiotics for E. coli and Klebsiella in our study revealed that these antibiotics are prescribed irrationally and often misused because of their over the counter availability [23] . However the sensitivity of the two probiotics, Lactobacilli (25%) and Saccharomyces boulardii (25%) were lower as compared to all antibiotics in case of Salmonella typhi.
Furthermore our study also highlighted that two tested probiotics, Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii had lower sensitivity against all of the three organisms isolated in our stool samples, when compared to more effective antibiotics which are not prescribed routinely for the treatment of infectious diarrhea in adults as well as in pediatrics. Overall for E. coli, the highest sensitivity was shown for Amikacin (96.7%), followed by Gentamycin (95.7%) Imipenim (95.7%) and Piperacillin/Tazobactam (84.8%). For Klebsiella the highest sensitivity was observed for Imipenim (98.2%), after that Amikacin (94.6%), Piperacillin/Tazobactam (92.9%) and Gentamycin (89.3). Nevertheless the use of majority of the above mentioned antibiotics is limited because of potential toxicities such as nephrotoxicity, ototoxicity, neurotoxicity and elevated levels of sodium, potassium and magnesium. Hence, these antibiotics should not be considered as first-line agents in the treatment of diarrhea in children and should be reserved as a final resort for serious and life-threatening infections. WHO has also warned that frequent use of these highly sensitive antibiotics may result in rise of the resistance of many of the gram negative organisms including E. coli, Klebsiella and Salmonella typhi [24] [25] .
Although several studies have documented the beneficial effects of probiotics in diarrhea but their outcome was on the basis of clinical follow up of the patients with improvement or decrease in the duration of diarrhea. However the magnitude of therapeutic effects of probiotics was neither assessed nor compared with antibiotics, hence no related studies are available.
Besides, a couple of studies revealed that probiotics have become extensively popular and have rapidly achieved high level of use in Europe and Asia for the cure of diarrhea [26] [27] [28] . The results of a meta-analysis, reported that Saccharomyces boulardii significantly reduced the duration of diarrhea (mean difference, −19.7 hours; 95% confidence interval, −26.05 to −13.34), stool frequency on day 2 (mean difference, −0.74; 95% confidence interval, −1.38 to −0.10) and day 3 (mean difference, −1.24; 95% confidence interval, −2.13 to −0.35), the risk for diarrhea on day 3 (risk ratio, 0.41; 95% confidence interval, 0.27 to 0.60) and day 4 (risk ratio, 0.38; 95% confidence interval, 0.24 to 0.59) after intervention compared with control [29] .
The National Institute for Health and Clinical Excellence established in England also suggested the use of probiotics along with ORS for the treatment of acute diarrhea in children. The evidence-based clinical practice guidelines based on systematic reviews of randomized controlled trials displayed that Lactobacilli and Saccharomyces boulardii, were the efficacious agents in reducing the duration of diarrhea by one day [30] [31] .
To the best of our knowledge the current study is innovative as for the first time, antimicrobial activity of the two most commonly prescribed probiotics, Lactobacilli and Saccharomyces boulardii were evaluated and compared with the standard antibiotics used against infectious diarrhea in children on the basis of stool culture and sensitivity. Furthermore the results of the current study also provided the scientific evidence of direct antimicrobial effects of probiotics which is an addition to their already described mechanisms of action. Even though in current study in-vitro testing of both probiotics clearly revealed that overall their sensitivities against organisms causing diarrhea were on lower side and not remarkably better in comparison to the majority of the standard antibiotics for infectious diarrhea in our pediatric clinical set up.
6. Conclusions
The results of the present study highlighted that both probiotics, Lactobacilli (Lactobacillus paracasei/Lactobacillus acidophilus) and Saccharomyces boulardii possess direct antimicrobial or bactericidal action but have overall low sensitivities against microbial organisms causing infectious diarrhea in our clinical set-up. Although both probiotics were found to have either equivalent or slightly higher sensitivities when compared to most commonly prescribed antibiotics (Amoxicillin/Clavulanic acid, Ceftazidime, Ampicillin, Cefotaxime, Cefuroxime, Ceftriaxone Nalidixic acid and Trimethoprim/Sulfmethoxazole). On the contrary, both of the probiotics had lower sensitivities in comparison to more effective and less commonly prescribed antibiotics (Amikacin, Gentamycin, Imipenim and Piperacillin/Tazobactam). Therefore, probiotics cannot be considered as sole agents or alternative treatments to antibiotics and hence antibiotics remain the mainstay of treatment for pediatric infectious diarrhea in our clinical set-ups.
However, the results of current study should be further validated by other multicenter studies conducted on diverse population.
7. Limitations of the Study
The study had certain limitations. Firstly, the study was conducted at only one hospital, where majority of patients are with low socio economic class and low education visits. Secondly, limited sample size and selection of only study site had restricted the generalization and external validity of the study findings. Thirdly, as the study was in-vitro where the laboratory analysis was undertaken to compare the sensitivity of antibiotics with probiotics with no follow-up involved.
Future Recommendations
Further similar experimental studies should be carried out on a large sample size in various clinical settings in order to validate the results of current study. Probiotics other than Lactobacilli and Saccharomyces boulardii should also be assessed and compared with antibiotics and with other probiotics used in the management of pediatric diarrhea.