INTRODUCTION
A decade ago, probiotics were considered as those viable microorganisms that exhibited a beneficial effect on the health of the host by improving its intestinal microbial balance.[1,2] There are previous studies about the preventive effect of probiotics for enteric bacteria and it was conducted invivo.[3] Bacteria tend to produce toxins that damage the tissue and create severe infection situations thus play the main role in pathogenesis.[4] Several definitions of probiotics can be found in literature, but the most recent and comprehensive definition is provided by the World Health Organization and Food and Agricultural Organization “Probiotics are live microorganisms when administered in adequate amounts confer a health benefit on the host.”[5] The probiotic microorganisms mainly consist of the strains of the genera Lactobacillus and Bifidobacterium, Streptococcus, and some Enterococcus species.[6] These bacteria are gram-positive, rod-shaped, nonspore-forming, and nonaerobic as well as acid tolerant, aero tolerant, and fastidious, with lactic acid as the major end product of sugar fermentation.[7] It is believed that an optimum “microbial population balance” in our digestive tract is associated with good nutrition and health.[8] Several research has shown that the addition of probiotics to food provides several health benefits including a reduction in the level of serum cholesterol, improved gastrointestinal function, enhanced immune system, and lower risk of colon cancer.[9–13] Lactic acid bacteria are also commercially used as starter cultures for the manufacture of dairy-based probiotic foods.[14,15] Probiotic products consist of diverse enzymes, vitamins, capsules, or tablets and some fermented foods comprise microorganisms which have valuable effects on the health of the host.[16,17] They can contain one or several species of probiotic bacteria. Most of the products which destine human consumption are produced in fermented milk or given in powders or tablets. These capsules and tablets are not used for medical applications. These probiotics are just used as health-supporting products. The oral consumption of probiotic microorganisms produces a defensive effect on the gut flora.[18–20] Lots of studies propose that probiotics have beneficial effects on microbial disorders of the gut, but it is difficult to show the clinical effects of such products. The probiotic preparations have positive therapeutic effects and are used for the remedy from traveler’s diarrhea, diarrhea resulting from antibiotic-resistant pathogens, and acute diarrhea.[21] Various studies have indicated that probiotics may alleviate lactose intolerance; have a positive influence on the intestinal flora of the host; stimulate/modulate mucosal immunity; reduce inflammatory or allergic reactions; reduce blood cholesterol; possess anti-colon cancer effects; reduce the clinical manifestations of atopic dermatitis, Crohn’s disease, diarrhea, constipation, candidiasis, and urinary tract infections; and competitively exclude pathogens considering this impressive list of potential health-promoting benefits, it is not surprising that there continues to be considerable interest in the use of probiotics as biotherapeutic agents. Furthermore, given a heightened awareness among consumers of the link between diet and health and the fact that probiotics-containing foods are generally perceived as “safe” and “natural,” the global market for such foods is on the increase, particularly dairy based products marketed for the prophylaxis or alleviation of gastrointestinal disorders.[22–25] Probiotics are recommended as food to provide for the balance of intestinal flora.
Antibiotic resistance refers to the change of the mechanisms toward antibiotics. Due to antibiotic resistance yearly more than 1.2 million fatalities are occurring and this triggers researchers to find alternative approaches for antimicrobial agents. It has been told that 10 million people can be killed per year by 2050.[26] Therefore, the present study was undertaken to reveal the antimicrobial activities of some commercially available probiotic tablets on drug-resistant enteric pathogens isolated from clinical specimens.
METHODS
Probiotics, as an antibacterial agent
Samples: Six (6) types of commercially available probiotics such as Brand 1: Luvena, Brand 2: Acteria, Brand 3: Good-gut, Brand 4: Probio, Brand 5: Prolacto, and Brand 6: Enterogermina were used in this study within a time frame of November 2020 to December 2020. Samples were randomly collected from different shops in Dhaka city. Dates of manufacturing and expiry were checked before evaluate the antimicrobial activity of samples (see the Supplementary).
Test microorganisms
The bacterial isolates used in this study for the antimicrobial assay were previously isolated and biochemically identified from clinical samples in the laboratory of the Department of Microbiology, Stamford University Bangladesh. The selected clinical bacterial isolates include Salmonella spp., Klebsiella spp., Pseudomonas spp., Vibrio spp., and Shigella spp. Bacteria were preserved at −20°C and subcultures were made on Nutrient agar (HiMedia Laboratories, Mumbai, India) before tested in the current study.
Antibiotic susceptibility test of the isolates
Agar-disc-diffusion method (Kirby-Bauer technique) was used to examine the antibiotic assay of the isolates (either sensitive or resistance) on Mueller-Hinton agar (MHA) according to Kirby Bauer disc diffusion method. The antibiotic discs used in this experiment were Streptomycin (10 μg), Gentamycin (10 μg), Ampicillin (10 μg), Methicillin (30 μg), Azithromycin (30 μg), Erythromycin (15 μg), and Ceftriaxone (30 μg). The plates were then inverted and incubated at 37°C for 24 h. After incubation, the plates were examined, and the zone of inhibition was measured in mm according to the standard guidelines.[27]
Determination of anti-bacterial activity of the probiotics
In this experiment, agar well-difusion method on Muller Hington Agar is used to examine the efficacy of probiotics against different laboratory isolates. A single touch of an isolated colony was picked from the bacterial colony and prepared a bacterial suspension with normal saline as 0.5 McFarland standard. By using a sterile one-time-use swab stick, the suspension was lawn into the Muller-Hinton agar media.[28] Varieties of isolates such as Klebsiella spp., Pseudomonas spp., Salmonella spp., Shigella spp., and Vibrio spp. were inoculated into the appropriately labeled sterile tubes containing Mueller Hinton (MH) broth (Oxoid Ltd., England) and the bacterial lawn was prepared onto the surface of the MHA media. Then wells (8 mm) were made with cork bore on the inoculated MHA media and granules of probiotics were added into the wells. After incubation at 37°C for 24 h the presence of clear zone around the sample solution (if any) was analyzed for the existence of the antibacterial activity of the tested samples. The zone of inhibition was measured according to the Clinical and Laboratory Standard Institute (CLSI) guideline, 2020.
Determination of minimal inhibitory concentration
The minimum inhibitory concentration (MIC) assay was performed to determine the lowest concentration of probiotics which can trim down the extent of the viability of the test bacteria.[29] According to the suggested method by CLSI, the two-fold serial broth dilution method was used to determine the MIC (CLSI2006). An overnight culture of each test organism 100 μl was inoculated into the sterile tube containing MH broth (Oxoid Ltd, England) at the turbidity adjusted with 0.5 McFarland standard and the different concentrations of probiotics (32 μL, 64 μL, 128 μL, 256 μL, 512 μL, 1024 μL, and 2048 μL) were subjected to introduce into the inoculum containing broth. All the tested tubes were incubated at 37°C for 24 h. The smallest number of samples which could retard the multiplication of the tested bacteria as indicated by measuring the zero density was regarded as MIC.
Ethical statement
In this study, neither humans/samples or animals/samples were used.
RESULTS
Antibiotic susceptibility patterns of the enteric clinical isolates
In this study, 7 types of selected antibiotics were used to determine drug sensitivity patterns against the pathogens. The degree of susceptibility of these test organisms to each antibiotic was determined and interpreted as either sensitive (S) or resistant (R) by calculating zones of inhibition around the antibiotic discs.
The experiments were conducted three times independently, and the results were found to be reproducible. One representative data has been shown.
Effects of different commercial probiotics samples against the bacteria
All the probiotic samples were found to be effective against the bacteria such as Klebsiella spp., Pseudomonas spp., Salmonella spp., Shigella spp., and Vibrio spp. Several research groups previously found a significant amount of antibacterial activity imparted by Lactobacillus spp. isolated from different dairy and vegetable samples.[30] The inhibitory action of Lactobacillus spp. can be due to the production of main primary metabolites such as lactic and acetic acids, ethanol, and carbon dioxide.[31] In addition, they are also capable of producing antimicrobial compounds such as formic and benzoic acids, hydrogen peroxide, diacetyl, acetoin, and bacteriocins such as Nicin.[32]
The experiments were conducted three times independently, and the results were found to be reproducible. One representative data has been shown.
Minimum inhibitory concentration of the sample
The MIC of probiotic samples against the drug-resistant enteric pathogens was ranged between 512 and 1024 μL. 6 different probiotics were tested against Klebsiella spp., Pseudomonas spp., Salmonella spp., Shigella spp., and Vibrio spp.
The experiments were conducted three times independently, and the results were found to be reproducible. One representative data has been shown.
DISCUSSION
Antibiogram method has to be checked when an alternative method is used as an antimicrobial agent. In this study antibiogram with a few common antibiotics showed that all bacterial isolates were 100% sensitive against Gentamycin, Streptomycin, Azithromycin, and Ceftriaxone. In contrast, all the isolates were found to be resistant against Ampicillin, Methicillin, and Erythromycin [Table 1]. Bacterial resistance is an alarming issue and awareness should be made. The search for alternative of antibiotics is ongoing. Probiotics from natural sources such as from Lactobacillus spp. have some antimicrobial properties thus can be an option to kill pathogenic bacteria. In our study design enteric pathogenic bacteria were targeted against six commercial probiotics. As a result, it was found that in the case of Klebsiella spp., 22 mm zone diameter was estimated by Luvena while 21 mm zone of inhibition was observed against Pseudomonas spp. In addition, for Shigella spp. the zone of inhibition was recorded for Luvena and the minimum zone of inhibition was found for Vibrio spp. Moreover, good bactericidal effects were observed by Good-gut and Probio against Klebsiella spp. and Salmonella spp. whereas Prolacto and Enterogermina did not show good efficacy against all Gram-negative pathogens. Although the composition is the same or similar in these commercial probiotic capsules but different brands showed different results for the pathogens. Prolacto did not show a significant zone of inhibition even though it has the same composition like Good-gut and Probio [Table 2]. It can be assumed that the effectivity is not the same with all the brands found in the market. Interestingly Prolacto and Enterogermina were found poor in performance for bactericidal activity in comparison with other brand probiotics. Regarding the MIC, it was observed that lowest MIC was 256 μL by Acteria, Luvena, and Good Gut against the Pseudomonas and Salmonella spp. while the highest MIC was found at 1024 μL by Prolacto and Enterogermina against most of the tested enteric pathogens [Table 3].
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Table 1: Antibiotic susceptibilities of tested organisms
Table 2: Antimicrobial activity of probiotics against gram negative bacteria
Table 3: Minimum inhibitory concentration of the sample
CONCLUSION
The current exploration established that the probiotic samples have antimicrobial activity against different clinical isolates. Not only these facts, but also these commercially available probiotics were found to be effective against those bacteria with satisfactory levels of antibacterial traits. Therefore, the study findings suggested that probiotic samples can be a potential source or can be a replacement of antibiotics.
Limitations of the study
There are some limitations in this study. Molecular studies are required to understand the mechanism of antimicrobial activity by the probiotics were not done. The sample size was small and it was a laboratory-based experiment. Different types of microorganisms such as fungi and yeast were not checked for the antibacterial effect of these commercial probiotics.
Future studies
Mostly Lactobacillus strains are responsible to inhibit the growth of other bacteria. The metabolites and the mode of action of the metabolites or toxins needed to identify with high throughput machines and sophisticated data analysis are required. Bigger sample size and screening with wide range of microorganisms are required to get clear data about the pattern of antimicrobial activity of various probiotics. Our research, on the other hand, provides an overview on the alternative approach of antibiotic resistance in Bangladesh and needs more attention in this sector in future studies.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
The authors would like to thank Stamford University Bangladesh for providing laboratory facilities, technical assistance, and financial aid.
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