Antimicrobial Activity of Probiotic Lactobacilli against Some Pathogenic Bacteria

Abstract

Probiotics are viable Lactic Acid Bacteria (LAB) that is believed to provide health benefits when administered in appropriate quantities. Lactobacillus is one of the most important genera of LAB that are known to produce substances including bacteriocins which can inhibit the growth of pathogenic bacteria. The current study determined the antibacterial activity of probiotic lactobacilli against some clinical bacterial isolates. A commercially available probiotic lactobacilli product which contains Lactobacillus acidophilus and Lactobacillus plantarum strains were tested for their antibacterial activity against the clinical bacterial isolates. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus, following disk diffusion method. The cell-free supernatant of Lactobacilli strains isolated in the present study exhibited very remarkable and noticeable antimicrobial activity against pathogenic bacteria.

Full text

Journal of Probiotics & Health
1
J Prob Health, Vol. 10 Iss.7 No: 1000284
OPEN ACCESS Freely available online
Research Article
Correspondence to: Radwan R. Mohammed, Department of Medical Laboratory Technology, Erbil Polytechnic University, Erbil, Iraq, Tel: +964 751-
229-4346; E-mail: razwanrushdi@gmail.com
Received: 24-Jun-2022, Manuscript No. JPH-22-18845; Editor assigned: 27-Jun-2022, PreQC No. JPH-22-18845 (PQ); Reviewed: 11-Jul-2022, QC
No. JPH-22-18845; Revised: 18-Jul-2022, Manuscript No. JPH-22-18845 (R); Published: 26-Jul-2022, DOI: 10.35248/2329-8901.22.10.284.
Citation: Mohammed RR, Mohammed MR (2022) Antimicrobial Activity of Probiotic Lactobacilli against Some Pathogenic Bacteria. J Prob Health.
10:284
Copyright: © 2022 Mohammed RR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
J
o
u
r
n
a
l
o
f
P
r
o
b
i
o
t
i
c
s
&
H
e
a
l
t
h
ISSN: 2329-8901
Antimicrobial Activity of Probiotic Lactobacilli against Some Pathogenic
Bacteria
Radwan R. Mohammed*, Maryam R. Mohammed
Department of Medical Laboratory Technology, Erbil Polytechnic University, Erbil, Iraq
ABSTRACT
Probiotics are viable Lactic Acid Bacteria (LAB) that is believed to provide health benefits when administered
in appropriate quantities. Lactobacillus is one of the most important genera of LAB that are known to produce
substances including bacteriocins which can inhibit the growth of pathogenic bacteria. The current study determined
the antibacterial activity of probiotic lactobacilli against some clinical bacterial isolates. A commercially available
probiotic lactobacilli product which contains Lactobacillus acidophilus and Lactobacillus plantarum strains were tested
for their antibacterial activity against the clinical bacterial isolates. Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, and Staphylococcus aureus, following disk diffusion method. The cell-free supernatant of Lactobacilli strains
isolated in the present study exhibited very remarkable and noticeable antimicrobial activity against pathogenic
bacteria.
Keywords: Antimicrobial activity; Lactobacillus; Probiotics; Pathogens; Invitro diagnostics; Clinical strains
INTRODUCTION
Throughout the history of microbiology, most human studies
have been focused on the disease-causing organisms found on or
in people, whilst fewer studies have examined the benefits of the
bacteria. However, we are surrounded by beneficial microorganisms
that live in or on the human body. Probiotics are defined as live
microorganisms when administered in adequate amounts confer
a health benefit on the host [1,2]. Lactic Acid Bacteria (LAB) are
considered as Generally Recognized as Safe (GRAS) organisms
and can be safely used as probiotics for medical applications [3].
Members of the genus Lactobacillus are the most common probiotics
used in commercial fermented and non-fermented products, such
as in some yogurts and fermented milk drinks, as well as dietary
supplements, which are available in the market in the form of
capsules, liquid/gel, and powdered, to combat the diseases due
to their ability to inhibit the pathogenic microorganisms [4].
Lactobacilli, primarily facultative or strict anaerobes generally have
fastidious growth requirements. They prefer an acidic environment
by producing lactic and other acids.
In general, Lactobacilli have not been associated with disease
and have been regarded as non-pathogenic and isolates were
able to tolerate the acidic condition of the environment, Nacl
concentration, and resistance to bile. The reported health benefits
of probiotics include: boosting of the immune system, inhibition
of the growth of pathogenic organisms, prevention of diarrhea
from various causes, improvement of digestion of proteins and fats
and synthesis of vitamins [5]. This study was conducted to detect
the antimicrobial activity of the probiotic lactobacilli isolated from
a commercial probiotic product against some bacterial pathogens.
Probiotics, a word derived from Latin, that means ’for life’, have
been with us for as long as people have eaten fermented milk, but
their association with health benefits dates only from the turn
of the last century. Growing awareness of the health benefits of
consuming microorganisms such as probiotics have encouraged
consumers worldwide. These probiotic bacteria are essential for
their beneficial effect on a particular organism’s health and host
nutrition for healthy gastrointestinal function.
The original modern hypothesis of the positive role played by
certain bacteria was first introduced by Russian scientist Elie
Metchnikoff, who in 1907 suggested that it would be possible to
modify the gut microbiota and replace harmful microbes with
useful microbes [6]. On the other hand, the rise in antibiotic
antibiotic-resistant has awakened the scientific community to the
prophylactic and therapeutic uses of probiotics and to reconsider
them as alternatives to antibiotics [7]. Over the last years, there has

2
Mohammed RR, et al.
OPEN ACCESS Freely available online
J Prob Health, Vol. 10 Iss.7 No: 1000284
been increasing public and scientific interest in the administration
of these live micro-organisms to prevent or treat disease. Twenty-
three publications were retrieved from PubMed for the year 1995
using the search term ‘probiotic’ compared with about 200 in the
year 2000 and more than 600 for the first half of 2012.
Much of the focus of this research has been on the use of probiotics
for the prevention or treatment of gastrointestinal conditions
such as inflammatory bowel disease and inflammatory bowel
syndrome [8]. Antimicrobial activity of Lactobacillus strains against
bacterial pathogens emerges to be multifactorial and includes
the production of hydrogen peroxide, lactic acid, bacteriocin-like
molecules, and unknown heat-stable, non-lactic acid molecules
[9]. Other mechanisms proposed for their activity are competition
for nutrients [10,11], adhesion inhibition of pathogens to the
surface, and stimulation of the immune system [12]. One of the
important features of probiotic lactobacilli to achieve antagonistic
activity against bacterial pathogens because of their capacity to
produce lactic acid and other organic acids that lower the pH in the
human intestine, and to produce H2O2 and bacteriocin, thereby
establishing a hospital environment for the growth and survival of
various human pathogenic bacteria.
There are also other physiological benefits of probiotics that have
been published as it helps in the removal of carcinogens, lowering
of cholesterol, immune-stimulating and allergy lowering effect,
synthesis and enhancing the bioavailability of nutrients, alleviation
of lactose intolerance [13]. One important limitation is that only
one kind of probiotic bacteria may not exert protection against all
harmful strains that cause gastrointestinal pathogenic infections
[14].
MATERIALS AND METHODS
Isolation of probiotic strains
Commercial probiotic product ‘Vitalactic B’ was used to isolate the
probiotic lactobacilli used in the study. According to the product
content information given on the "Vitalactic B" packet, two
probiotic strains, Lactobacillus plantarum and Lactobacillus acidophilus
were present in it. "Vitalactic B" is in form of a capsule so the
powder from the capsule were suspended in Brain Heart Infusion
Broth (BHIB) and kept in aerobic and anaerobic conditions
at 37°C for 24 hrs. The probiotic lactobacilli were subjected to
cultural and morphological characterization. Then, the whole
broth was centrifuged at 3000 rpm for 10 minutes and the was-free
supernatant was collected and tested for its antibacterial activity
against the clinical bacterial isolates.
Bacterial cultures and growth conditions
Clinical isolates of Staphylococcus aureus, Escherichia coli, Klebsiella
pneumoniae and Pseudomonas aeruginosa with their case profiles
were obtained from the laboratory of Nanakali hospital, Erbil, and
maintained in Brain Heart Infusion (BHI) agar and MacConkey
agar [2,3] isolated colonies of each bacterial pathogen were
sub-cultured onto BHI broth under aerobic conditions for 24
hours before testing. These bacteria served as test pathogens for
antibacterial activity assay.
Detection of antimicrobial activity
The antimicrobial activity of probiotic lactobacilli from a commercial
probiotic product was investigated against Staphylococcus aureus,
Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa
using the method. For the purpose of production of paper discs,
Whatman filter paper No.3 was used. Using an ordinary office
hole punching machine, holes of approximately 6 mm diameter
were punched. The discs were then autoclaved at 15 lbs pressure
for 20 minutes [15]. Susceptibility of the pathogens to 2 antibiotics
including Gentamicin (GEN) and Ampicillin (AMP) was also
determined by the diffusion method. The Muller-Hinton agar
plates were examined for the presence of inhibition zones around
the paper disks. The result was considered positive when a clear
zone around the paper disk was present whereas, negative was
defined as the absence of a growth inhibition zone around the disk.
Zones of inhibition were measured after incubation at 37°C for 24
hours and the results were recorded.
RESULTS
Isolation and identification of
Lactobacillus
species
Probiotic lactobacilli were isolated from the commercial probiotic
product "Vitalactic B" and identified by noticing their colony
morphology as shown in Figure 1A, and cultural as welcome
biochemical characteristics. Microscopically they were gram-
positive as shown in Figure 1B, rod rod-shaped-motile, and absence
of endospore (Figure 1).
Antimicrobial activity
Lactobacilli strains were tested for their antimicrobial activity
against some pathogenic bacteria by the disk diffusion method.
The results revealed that the cell-free supernatant of Lactobacillus
Plantarum and Lactobacillus acidophilus, in combination, exhibited
the average inhibition (15-35 mm) on the growth of test pathogens:
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, but
had no activity against Klebsiella pneumoniae. On the other hand, all
the test pathogens were resistant to Ampicillin (AMP) and varieco
Gentamycin (GEN) (Figure 2 and Table 1).
Figure 1: Isolation and identification of Lactobacillus species.
A: Characteristic colonies of lactobacillus species on BHIA; B:
Microscopically appearance of Lactobacilli with gram stains.
Figure 2: Antimicrobial activity. A: Staphylococcus aureus; B:
Pseudomonas aeruginosa; C: Escherichia coli; D: Klebsiella pneumoniae
by the disk diffusion method. Note: (1) Probiotic Lactobacillus
strains supernatant; (2) BHIB (negative control); (3) Ampicillin; (4)
Gentamicin against different test pathogens.

3
Mohammed RR, et al.
OPEN ACCESS Freely available online
J Prob Health, Vol. 10 Iss.7 No: 1000284
DISCUSSION
It is a long time since scientists are trying to formulate alternative
therapeutic protocols with non-antibiotic agents against bacterial
infection. Nowadays, various natural products and methods are
used to prevent or treat diseases. The use of probiotics is one of
these methods. Lactobacilli are normal intestinal flora that plays
an important role in human health. Probiotic Lactobacilli produce
antimicrobial compounds such as bacteriocin, which can be used
to prevent the growth of many bacteria. In this study, it was found
that the cell-free supernatant of these bacteria, was able to inhibit
the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa, the latter was in the highest inhibitory effect, which
comes in compatible with a study by 16 which study the inhibitory
activity of bacteriocin producing Lactic Acid Bacteria (LAB)
against P. aeruginosa and Escherichia coli using free cell supernatant
and disk diffusion method. Jamalifar et.al., [16,17] showed that
Lactobacillus acidophilus exhibit significant inhibitory activity
against clinical isolates of P. aeruginosa by different mechanisms.
Similarly, a bacteriocin from L. plantarum was found to be active
against pathogenic bacteria including Cl. sporogeneses, E. faecalis,
E. coli, and S. aureus [18,19]. Antibacterial activity of bacteriocin
produced by isolated probiotics showed that L. plantarum has the
strong antibacterial effect against enteric bacterial pathogens [20].
In our study, the cell-free supernatant of Lactobacillus plantarum
and Lactobacillus acidophilus, have no antimicrobial effect against
Klebsiella pneumoniae. According to previous studies, a large number
of lactic acid bacteria strains showed different bioactive potentials,
especially in the form of antibacterial properties. The spectrum of
antimicrobial activity for the species suggested that the inhibitory
components were differ [21]. Similarly, Hami [22] and Hassan Ali
Maarof et al., [23] observed varying degrees of inhibition of various
pathogens by the culture filtrate of lactic acid bacteria, although
these inhibitory substances produced by the lactic acid bacteria
strains, they act differently on the pathogenic reference indicator
strains. On the other hand, the inhibitory activity of probiotic
Lactobacillus supernatant was compared with the inhibitory activity
of AMP and GEN against the clinical pathogens. None of the
antimicrobial agents was effective against all the tested pathogens
demonstrating the current problem in the treatment of multi-
drug resistant bacteria. In a previous study, P. aeruginosa isolates
showed intermediate or full resistance to antimicrobial agents
[24].Unfortunately, P. aeruginosa and S. aureus showed complete
resistance against gentamicin and Ampicillin (AMP) and varies in
their susceptibility to Gentamycin (GEN) as shown in Figure 2 and
Table 1.
CONCLUSIONS
The results of the present study revealed the following:
1. The probiotic bacteria isolated in our study possess varying
degrees of inhibition towards tested pathogenic bacteria.
2. Lactobacilli strains are potentially promising because they
generate bactericidal bioactive agents that can control the growth
of pathogens.
3. Results from our present study are expected to encourage people
to consume probiotic products.
4. Given the increasing use of probiotics as health supplements
and therapeutic agents, clinicians need to be aware of the risks
and benefits. Although probiotics have an excellent overall safety
record, they should be used with caution in certain patient groups
particularly neonates born, prematurely or with immune deficiency.
5. Lactobacillus strains isolated in this study from the different
probiotic medicine have in vitro properties that make them
potential against pathogenic bacteria which were susceptible to all
the strains of Lactobacillus. These results collectively suggest that
probiotics particularly commercially available types are important
for combating pathogens.
COMPETING INTERESTS
No competing interests.
FUNDING
The research was self-funded. The authors' independently design
the study, analyze and interpret the data, prepared the manuscript.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.
ACKNOWLEDGEMENT
This paper and the research behind it would not have been possible
without the exceptional support of our supervisor, Sanaria Fawzi
Jarjes. Her enthusiasm, knowledge, and exacting attention to detail
have been an inspiration and kept us fully supported.
REFERENCES
1. World Health Organization (WHO). Health and nutritional properties
of probiotics in food including powder milk with live lactic acid
bacteria, a joint FAO/WHO expert consultation. Cordoba, Argentina,
1-4 October 2001.2001.
2. Joint FAO/WHO working group, Joint FAO/WHO working group.
Guidelines for the evaluation of probiotics in food. London: World
Health Organization (WHO), ON, Canada: Food and Agriculture
Organization. 2002.
Table 1: Antibacterial activity of probiotic Lactobacillus strains supernatant compared with AMP and GEN in terms of zone of inhibition using the agar
disk diffusion method.
Pathogenic bacteria Source
Zone of inhibition
Probiotic
Lactobacillus
strains supernatant AMP GEN Control
Escherichia coli Stool +(15 mm) - +(20 mm) -
Klebsiella pneumoniae Urine - - +(30 mm) -
Pseudomonas aeruginosa Wound +(35 mm) - - -
Staphylococcus aureus Urine +(20 mm) - - -

4
Mohammed RR, et al.
OPEN ACCESS Freely available online
J Prob Health, Vol. 10 Iss.7 No: 1000284
3. Victor SD, Francois ZN, Marie KP, Alberto C, Florence F. Probiotic
properties of lactobacilli strains isolated from raw cow milk in
the western highlands of Cameroon. Innovative Romanian Food
Biotechnology. 2011;9:12-28.
4. Sonomoto K, Yokota A. Lactic acid bacteria and bifidobacteria: Current
progress in advanced research. Caister Academic Press. 2011.
5. Prabhurajeshwar C, Chandrakanth RK. Probiotic potential of
Lactobacilli with antagonistic activity against pathogenic strains: An in
vitro validation for the production of inhibitory substances. Biomed J.
2017;40(5):270-283.
6. Metchnikoff E. Lactic acid as inhibiting intestinal putrefaction. The
prolongation of life: Optimistic studies. 1907:161-183.
7. Ahmed FE. Genetically modified probiotics in foods. Trends
Biotechnol. 2003;21(11):491-497.
8. Fijan S. Antimicrobial effect of probiotics against common pathogens.
Probiotics and prebiotics in human nutrition and health. 2016;10:5772.
9. Servin AL. Antagonistic activities of lactobacilli and bifidobacteria
against microbial pathogens. FEMS Microbiol Rev. 2004;28(4):405-
440.
10. McFarland LV. Normal flora: Diversity and functions. Microb Ecol
Health Dis. 2000;12(4):193-207.
11. Reid G, Burton J. Use of Lactobacillus to prevent infection by pathogenic
bacteria. Microbes Infect. 2002;4(3):319-324.
12. Gill HS, Rutherfurd KJ, Cross ML, Gopal PK. Enhancement of
immunity in the elderly by dietary supplementation with the probiotic
Bifidobacterium lactis HN019. Am J Clin Nutr. 2001;74(6):833-839.
13. Ouwehand AC, Salminen S, Isolauri E. Probiotics: An overview
of beneficial effects. Lactic acid bacteria: genetics, metabolism and
applications. 2002:279-289.
14. Boyle RJ, Robins-Browne RM, Tang ML. Probiotic use in clinical
practice: What are the risks? Am J Clin Nutr. 2006;83(6):1256-1264.
15. Vineetha N, Vignesh RA, Sridhar D. Preparation, standardization of
antibiotic discs and study of resistance pattern for first-line antibiotics
in isolates from clinical samples. Int J Appl Res. 2015;1(11):624-631.
16. Daba H, Saidi S. Detection of bacteriocin-producing lactic acid bacteria
from milk in various farms in north-east Algeria by a new procedure.
Agron Res. 2015;13(4):907-918.
17. Jamalifar H, Rahimi HR, Samadi N, Shahverdi AR, Sharifian Z,
Hosseini F, et al. Antimicrobial activity of different Lactobacillus species
against multi-drug resistant clinical isolates of Pseudomonas aeruginosa.
Iran J Microbiol. 2011;3(1):21-25.
18. Abo-Amer AE. Characterization of a bacteriocin-like inhibitory
substance produced by Lactobacillus plantarum isolated from Egyptian
home-made yogurt. Sci Asia. 2007;33(3):313-319.
19. Atta HM, Refaat BM, El-Waseif AA. Application of biotechnology for
production, purification and characterization of peptide antibiotic
produced by probiotic Lactobacillus plantarum, NRRL B-227. Glob J
Biotechnol Biochem. 2009;4(2):115-125.
20. Tambekar DH, Bhutada SA. An evaluation of probiotic potential
of Lactobacillus sp. from milk of domestic animals and commercial
available probiotic preparations in prevention of enteric bacterial
infections. Recent Res Sci Technol. 2010;2(10):82-88.
21. Raja A, Gajalakshmi P, Raja MM, Imran MM. Effect of Lactobacillus
lactis cremoris isolated from kefir against food spoilage bacteria. Am J
Food Technol. 2009;4(5):201-209.
22. Hami K. Antibacterial effects of probiotics isolated from yoghurts
against some common bacterial pathogens. Afr J Microbiol Res.
2011;5(25):4363-4367.
23. Maarof HA, Abdallah MI, Bazalou MS, Abo-Samra RG. Effect of
probiotics bacteria isolated from yoghurts produced in Damietta city
on some pathogenic bacteria. Proc. of the 6th Scientific Conference
of Animal Wealth Research in the Middle East and North Africa,
Hurghada, Egypt. 2013;27-30.
24. Nicasio AM, Kuti JL, Nicolau DP. The current state of multidrug-
resistant gram-negative bacilli in North America: Insights from the
society of infectious diseases pharmacists. J Human Pharmacol Drug
Ther. 2008;28(2):235-249.