This is an open access article under the CC BY 4.0 license 69
Research Article
BASRAH JOURNAL OF VETERINARY RESEARCH, 2023, 22(1):69-82
https://bjvr.uobasrah.edu.iq/
Inhibitory effect of Probiotics on some Gram positive and negative Bacteria
Aamer Y.H. Alchalaby1, Semaa F.H.AL-Abedi2
1Department of Microbiology, College of Veterinary Medicine, University of Mosul, Mosul, Iraq.
2Department of Mass Media and Public Relations, Mass Media Division, University of Al-Hamdaniya, Mosul, Iraq.
Corresponding Author Email: draameryeh@uomosul.edu.iq
ORCID ID: https://orcid.org/0000-0003-4654-6749
DOI:
Received: 7 March 2023 Accepted: 4 April 2023.
Abstract
The present study's objective was to evaluate the inhibitory activity of the Probiotics Lactobacillus acidophilus and Bifidobacterium (obtained from the Agriculture Research Directorate, Ministry of Science and Technology, Iraq) and a suspension of a mixture between the two mentioned probiotics with two types of Gram-negative bacteria (Pseudomonas spp and Proteus spp) and one type of Gram-positive bacteria (Streptococcus spp) in vitro. The required tests were completed to verify the probiotics' purity, and the bacterial isolates used in the current investigation were assessed using biochemical assays and selected culture medium (culture and microscopic features). In addition, the inhibitory efficacy of the investigated Probiotics in different Gram positive and negative bacteria was evaluated by drug susceptibility testing (disc diffusion test as well as agar well diffusion test). Our data of the current study confirmed an excellent inhibitory activity of each Bifidobacterium (B) and the mixture of the two probiotics (MLB) via measuring the inhibition area, they had 25, 22mm, 28,-30 mm inhibition zone for Pseudomonas spp, 23, 25 mm, 26-27mm inhibition zone for Proteus species spp, and 22,20 mm, 33,29 mm inhibition quarter for Streptococcus species, by way of the usage of disc and agar well diffusion methods respectively. Where it was once weak inhibition activity of Lactobacillus acidophilus (L)on Pseudomonas spp, 0-3 mm and Streptococcus species 1-7 mm by the usage ofthe disc and agar well diffusion respectively. On the other, hand, Probiotic(Lactobacillus acidophilus) had available zone of inhibition on the Proteus spp
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bacteria, which were 24, 24 mm through the disc and agar well diffusion respectively. In conclusion: the Probiotics were found to have good and active inhibitory action on Gram-positive microorganism (Streptococcus) and gram-negative microorganism (Pseudomonas and Proteus) in vitro by way of using disc and agar well diffusion test, and the combination of the two probiotics MBL of present study, had more potent inhibitory action than each one of the studied separate probiotics.
Key words: Probiotics, Lactobacillus, Bifidobacterium, Inhibitory effect, Antibiotics Sensitivity test.
Introduction
Many years ago, there was a revolution in the food industry in the field of functional foods production as a result of increasing awareness of the consumers on the food positive role in wellbeing and health. (1). Development of probiotic foods received more focus due to its potential to prevent disease. Probiotics are numerous types of live microorganisms that have been shown to have positive impacts on human health. (2). According to reports from the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), probiotics are live microorganisms that, when given to a host in sufficient amounts, have a positive effect on their health in ways like improving the balance of their intestinal microbiota, modulating their immune system, lowering their serum cholesterol levels, inhibiting the growth of harmful bacteria, and reducing or preventing intestinal disorders. (3, 4). Present indicator suggests that some probiotics like Lactobacillus and Bifidobacterium can modulate gut microflora homeostasis, and may have protective effects against diarrhea (5). The development of multiple antibiotic-resistant Gram-positive and Gram-negative bacteria in many areas such as new ways for quick healing in hospital and community settings and accurate diagnosis and treatment (6).
Pseudomonas aeruginosa can be defined as a common Gram-negative opportunisticmultidrug-resistant pathogen which causes acute and chronic infections particularly in immunocompromised and diseased animals. (7).
Proteus is considered as one of the common pathogens among gram-negative bloodstream isolates causing the secondary infections of the urinary tract (UTIs) and usually associated with urinary catheters community-acquired. It may result in infections of the biliary tract, surgical wounds, and nosocomial infections. Due to immune evasion and the protective reservoir that urinary stones provide, Proteus has a remarkable ability to remain in the urinary tract despite the use of antibiotics and catheter exchange. It can
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cause opportunistic infections and is a natural component of the gut flora (8).
Streptococcus is a group of gram-positive bacteria that has the ability to infect many animal species, leading to suppurative conditions like mastitis, metritis, polyarthritis and meningitis. It is distributed worldwide, most species live in the form of commensals on the mucosae of the upper respiratory tract and lower urogenital tract. Streptococcus is catalase-negative, facultative anaerobe, which is non-motile, fastidious and requires the addition of blood or serum to be cultured. (9).
The current study aimed to evaluate the inhibitory effect of probiotics against certain gram-positive and negative bacteria via using disc diffusion and agar well diffusion methods in vitro and to compare between the antimicrobial activities of these probiotics.
Material and Methods
Bacterial Isolates:
In the current study, Pseudomonas spp and Proteus spp as gram-negative and one species of gram-positive Streptococcus spp bacteria were used . All isolates have been taken from laboratory of Microbiology, College of Veterinary Medicine, University of Mosul, Iraq , during the period December 2021 to March 2022.
The isolates were similarly purified via subculture on brain heart infusion broth, cultured for two days at 37°C, and then transported to selective medium that included Pseudomonas agar, Edward agar, and regular media such Nutrient broth, Nutrient agar, and. To verify the purity of all isolates, specific biochemical tests were carried out (10).
Probiotics:-
The two isolates of Lactobacillus acidophilus and Bifidobacterium spp. were obtained from the agriculture Research Directorate, Ministry of Science and Technology, Iraq.
The probiotic isolates were further purified using a subculture of brain-heart infusion broth, after which they were incubated for one day at 37°C to achieve further differentiation. Finally, the isolates were cultured on specific media (De-Man, Rogosa, and Sharpe broth (M.R.S. broth and Agar) with 5% Co2 for 48 hours at 37°C. In addition, we examined the combination of these two probiotics (Lactobacillus and Bifidobacterium spp) (MLB) for the estimation of the antagonism effects of these Probiotics on the bacterial study.
Preparations of Bacterial Suspension:
These procedures involved transferring 4-5 colonies of each isolate of bacterialspecies from the selective media intonutrient broth-filled tubes, which werethen incubated for 14–16 hours at 37°C(11). Physiological salts solution wasused for dilution, and comparisons weremade with the standard control tubes(McFarland tubes), which were used inthis learning experience (10).
Estimation of the inhibitory effect:
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Technique and the Agar well diffusion method had been performed by means of taking 0.1 ml from each and every bacterial suspension (1.5x108 cfu) which used to be inoculated to the Muller Hinton agar and spread via the usage of an L. shape glass spreader and left for 30 minutes to confirm suspension diffusion.
To evaluate the influence of these probiotics on the isolates of bacterial study, a disc (6 mm) from filter paper (Whitman No 1) was prepared and later autoclaved, 0.1 ml of “each probiotics” cultured into each 10 discs to be saturated with it (12, 13). Later the discs were cultured on the Muller Hinton agar,(use 0.1 ml from each probiotics used to be used to prepare identical 10 discs to measure the antagonism of these probiotics together) and was once incubated for about 14-16 hours at 37°C, the effects were revealed by size of the inhibition zone which was obtained at the areas around the discs which have been saturated by Probiotics of current study in the culture media (Muller Hinton agar) (14). Similar approach was also applied concerning agar well diffusion (The 6‐mm or 7‐mm wells were bored in each plate) but without the use of discs, 0.1 ml of the isolates were directly inoculated in the well (15).
Results
The effect of the current study revealed that probiotic Bifidobacterium and probiotic which are a combination of Bifidobacterium and lactobacillus acidophilus (MBL) were able to distinguish inhibition activity on the bacterial study (Pseudomonas spp and Proteus spp and Streptococcus spp, they had 25, 22mm, 28,30 mm inhibition zone for Pseudomonas spp, 23,25 mm, 26-27mm inhibition zone for Proteusspp and 22,20 mm, 33,29 mm inhibition zone for Streptococcus species by using the methods of disc and agar well diffusion respectively.
The results related to probiotic Lactobacillus acidophilus showed that it had susceptible inhibitory action on Pseudomonas spp 0-3 mm and Streptococcus species 1-7 mm by using the method of disc and agar well diffusion respectively. On the other hand, probiotic Lactobacillus acidophilus was found to have available zone of inhibition on the Proteus spp bacteria, 24 mm, 24 mm through the disc and agar well diffusion respectively. Shows, Tables 1 and Figure (1), Pictures (1, 2, 3, 4, 5, 6).
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Table (1) : The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on bacterial isolates is indicated by Inhibition Zone (mm).
Bacterial Species
Disc Diffusion Test (mm)
Agar well diffusion test (mm)
L
B
M
L
B
MLB
Pseudomonas spp
0
25
28
3
22
30
Proteus spp
24
23
26
24
25
27
Streptococcus spp
1
22
33
7
20
29
L – Lactobacillus acidophilus.
B – Bifidobacterium spp.
MLB – Mixture of Lactobacillus and Bifidobacterium.
Figure (1): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on bacterial study isolates
Disc diffusion test (mm)
Agar well diffusion test
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Picture (1): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on Pseudomonas spp culture by using agar well diffusion test.
Picture (2): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on Proteus spp culture by using agar well diffusion test.
M
L
B
B
M
L
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Picture (3): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on Streptococcus spp culture by using agar.
Picture (4): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on Pseudomonas spp culture by using agar Disc diffusion test.
L
B
M
L
B
M
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Picture (5): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on Proteus spp culture by using agar Disc diffusion test.
Picture (6): The effect of Probiotics Bifidobacterium, Lactobacillus, and the mixture (MBL) on Streptococcus spp culture by using agar Disc diffusion test
Discussion
The Probiotic approach aims for repair of the deficiencies in the microflora and restoration of the animals' resistance to disease and it is now replacing the chemical growth promoters for farm animals (16, 17 ). Several in vitro studies suggest that probiotic lactic acid bacteria for many enteric bacterial infections may inhibit the growth of food-borne pathogenic microbes (18).
L
B
M
L
B
M
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In the present study, it was noticed that Probiotic Lactobacillus possess weak antimicrobial action on the Sreptococcus 1-7 mm (Gram positive bacteria) and Pseudomonas bacteria 0-3 mm (Gram negative bacteria) by disc and agar well diffusion test respectively, and these outcomes are different with the study done by (18). in general, and also differ with the study done by (3). in relation with Pseudomonas spp. It was also observed that lactobacillus revealed the strongest inhibitory action on Proteus spp bacteria (24-24 mm) by using disc and agar well diffusion test respectively, these results agree with the study done by (18, 8).
The small intestine represents an important site of infection in humans as well as animals (19), Probiotics are thought to modulate the indigenous intestinal microbiota and improve health through different mechanisms of action, including the direct preventing of the growth of enteric pathogens via decreasing luminal pH, and the secretion of bactericidal proteins (20). The selection of Lactobacilli as a potential probiotic which promotes health in food as well as pharmaceutical Preparations necessitate to be screened in vitro to confirm certain criteria, that involve antibiotic tolerance, bile tolerance, growth inhibition of other microorganisms as well as gastric juice that permit them to be established in the intestine (21 and 22).
Zone of inhibition of probiotic Bifidobacterium (B) has good inhibitory efficacy on bacterial study, Streptococcus 22-20 mm, Pseudomonas 25-22 mm andProteus spp, 23-25 mm by using disc anddisc and agar well diffusion testrespectively, the proportion results of theBifidobacterium in the present study was inagreement with those reported in differentstudies done such as (17), who studied Gramnegative bacteria. Also is agree with a studyabout pseudomonas spp done by (21) byusing agar well diffusion.
The results were interpreted by measure thezone around the discs and compared with thebreak points of CLSI (clinical laboratoryinstitute 2015) (23).
Lactobacillus are important group of probiotic organisms that play an important role in human health by inhibiting pathogenic bacteria growth, induce immune response (24). The strains which are presently used as a type of probiotics, belong to both Bifidobacterium and Lactobacillus, are naturally exist as a form of microbiota of human intestine and has the ability to produce metabolites of antimicrobials like organic acids, hydrogen
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peroxide, ethanol, diacetyl, acetaldehyde, saturated or unsaturated form of free fatty acids and compounds like peptides and bacteriocins (25 and 26). On the other hand, the outcomes of mixture MBL which had inhibitory action on bacterial study Streptococcus 33-29 mm , Pseudomonas 28-30 mm and Proteus spp,26-27 mm byusing disc and disc and agar well diffusiontest respectively, these results agreed withthe study performed by (25). Previousstudies confirmed the antimicrobial activityof probiotic strains cultures on pathogenicbacteria but few studies reported influencesas it was gained from co-cultured strains ofprobiotic (25).
Conclusions
In conclusion, the results of the present study showed that the Probiotics had good and active inhibitory action on Gram-positive microorganisms (Streptococcus) and gram-negative microorganisms (Pseudomonas and Proteus) in vitro by using disc and agar well diffusion tests. Furthermore, the present study proved that the combination of two Probiotics MBL, were of more potent inhibitory action than each separate one of probiotics.
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التأثیر التثبیطی للبروبیوتیک على بعض أنواع البکتیریا الموجبة والسالبة الجرام
عامر یحیى الجلبی , جامعة الموصل , العرا ق
سیماء فیصل حسب لله , جامعة الحمدانیة , العرا ق
الخلاصة
Bifidobacterium و Lactobacillus acidophilus کان الھدف من الدراسة الحالیة ھو تقییم النشاط المثبط للبروبیوتیک
(تم الحصول علیھ من مدیریة البحوث الزراعیة ، وزارة العلوم والتکنولوجیا ، العراق) وعمل خلیط من مزیج بین البروبیوتیک
ونوع واحد من البکتیریا موجبة Proteus spp) و (Pseudomonas spp المذکورین مع نوعین من البکتیریا السالبة الجرام
(Streptococcus spp) الجرام فی المختبر. تم الانتھاء من الاختبارات المطلوبة للتحقق من نقاء البروبیوتیک ، وتم تقییم
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العزلات البکتیریة المستخدمة فی الاستقصاء الحالی باستخدام المقایس البیوکیمیائیة ووسط الاستزراع الانتقائی (الخصائص
الزرعیة و المجھریة). بالإضافة إلى ذلک ، تم تقییم الفعالیة التثبیطیة للبروبیوتیک التی تم فحصھا فی أنواع مختلفة من البکتیریا
موجبة وسالبة الجرام عن طریق اختبار حساسیة الدواء (اختبار انتشار القرص وکذلک اختبار الانتشار فی حفر الآجار). أکدت
عن (MLB) وخلیط اثنین من البروبیوتیک (Bifidobacterium (B بیانات الدراسة الحالیة نشاطًا مثبطًا ممتازًا لکل بکتیریا
منطقة تثبیط ، Pseudomonas spp 30 ملم منطقة تثبیط ل - ، 22 ملم ، 28 ، طریق قیاس منطقة التثبیط ، وکان لدیھم 25
عن ، Streptococcus و 22،20 ملم ، 33،29 مل م ربع تثبیط لأنواع ، spp Proteus 27 ملم لأنواع - 25 ملم ، 26 ، 23
طریق استخدام طرق الانتشار بالقرص و الانتشار فی حفر الاکار على التوالی. حیث کان نشاط التثبیط الضعیف ل
ملم عن Streptococcus 1- 3-0 ملم وأنواع 7 ، Pseudomonas spp على Lactobacillus acidophilus (L)
(Lactobacillus طریق استخدام القرص والانتشار فی حفر الاجار على التوالی. من ناحیة أخرى ، کان لدى البروبیوتیک
24 ملم من خلال القرص والانتشار فی ، والتی کانت 24 ، Proteus spp منطقة تثبیط متاحة على بکتیریا acidophilus)
حفر الأجار على التوالی. فی الختام: وجد أن البروبیوتیک لھا تأثیر مثبط جید وفعال على الکائنات الحیة الدقیقة إیجابیة الجرا م
فی المختبر عن طریق استخدام (Pseudomonas and Proteus) والکائنات الحیة الدقیقة سالبة الجرام (Streptococcus)
فی الدراسة الحالیة ، کان لھا تأثیر مثبط أقوى MBL اختبار الانتشار بالقرص و حفر الأجار ، والجمع بین الاثنین البروبیوتیک
من کل واحدة من البروبیوتیک المنفصلة المدروسة.
الکلمات المفتاحیة: بروبایوتک , لاکتوباسیللس , التأثیر المثبط , اختبار الحساسیة الدوائی ة
