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International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
Antibacterial Activity Lactic Acid Bacteria (LAB)
Isolated Native Yogurt against ESBL Producing
E. coli Causing Urinary Tract Infection (UTI)
Heshmatipour Z
Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
Email: [email protected] and [email protected]
Aslikousha H
Msc. Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
Email: [email protected]
Mohamadiebli F
Department of Microbiology, Infection Control Comimittee, Pars Hospital Laboratory, Tehran, Iran
Email: [email protected]
Ashrafi Eslami A
Bsc. Genetic, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran
Email: [email protected]
Index Terms—Lactic Acid Bacteria (LAB), Urinary Tract
Infection (UTI), Extended Spectrum β-lactamases (ESBLs),
CFCS.
Abstract—The Lactic Acid Bacteria (LAB) is well known
probiotics with beneficial effects to human health. Their
antimicrobial activity is one of the most important probiotic
characteristics. Urinary tract infections (UTIs) are the most
frequent bacterial infections encountered in community
settings. Extended spectrum β-lactamases (ESBLs) are
enzymes produced by pathogenic bacteria that are capable
of hydrolyzing oxyimino-cephalosporins, and are inhibited
by β-lactamase inhibitors. Microbial products have used the
best source for therapeutic agents worldwide.
The lactic acid bacteria were isolated from native yogurt in
North IRAN. Theantimicrobial activity of cell-free
supernatant and partially purified bacteriocin was
determined by well diffusion method. All isolated ESBL
were processed and identified as per the standard
bacteriological division of microbiology lab. The bacteria
were primarily identified by colony morphology,
microscopy of Gram`s stain and routine biochemical tests
and antibiotic disk sensitivity tests.
The current study showed that ESBL E. coli resistant to
Cefazoline (96.6%), Ceftazidime (43.3%), Ceftriaxone
(3.3%), Ciprofloxacin (76.6%), Gentamicin (26.6%),
Meropenem (0%), Tobramycin (43.3%), Cefotaxime
(93.3%), Terimetoperim & Sulfomethoxazole (83.3%),
Ceftazidime + cluvalonic acid (3.3%), Cefotaxime +
cluvalonic acid (3.3%), Cefpodoxime (96.6%), Piperacillin +
Tazobactam (0%), Ampicillin (100%), Amikacin (0%),
Nitrofurantion (6.6%).
The CFCS and PP exhibited an antibacterial effect on a
narrow range of ESBL strains. However the effect is isolates
the invitro is better than large number broad-spectrum
antibiotics such: third-generation cephalosporins (eg,
cefotaxime, ceftriaxone, ceftazidime).

I.
One group of β lactamases, extended-spectrum β
lactamases (ESBLs), have the ability to hydrolyse and
cause resistance to various types of the newer β-lactam
antibiotics, including the expanded-spectrum (or thirdgeneration) cephalosporins (eg, cefotaxime, ceftriaxone,
ceftazidime) and monobactams (eg, aztreonam), but not
the cephamycins (eg, cefoxitin and cefotetan) and
carbapenems (eg, imipenem, meropenem, and ertapenem)
[1].
The gastrointestinal microbial ecosystem is relatively
stable but quantitative and qualitative disturbances are
seen after oral administration of antibiotics [2]. The
normal flora limits the concentration of potentially
pathogenic microorganisms, which can reach high
numbers in connection with intake of antimicrobial
agents [3]. The use of probiotics for prevention of
gastrointestinal diseases is well established [4].
Lactic acid bacteria and their metabolites have been
shown to play an important role in improving
microbiological quality and shelf life of many fermented
food products and provide a good example of
biopreservation [5]. Their antimicrobial activity is one of
the most important probiotic characteristics.
The application of antagonistic compounds by
lactobacilli are not limited to food preservation
antimicrobials of LAB have been employed successfully
to prevent the formation of biogenic amines [6], to inhibit
Manuscript received December 26, 2014; revised March 18, 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
INTRODUCTION
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International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
Entero pathogens in the small intestines of animals [7].
Some of the inhibitory components produced by lactic
acid bacteria have been intensively studied by application
in food preservation [8]. The use of bio-therapeutic
agents is presently one of the avenues being exploited for
the possible treatment of diarrhea [9].
One of the most significant criteria for a probiotic
selection is the capability to enhance innate host defences
by production of antimicrobial substances, and the
growth inhibition and/or competitive exclusion of the
enteric pathogens [10]. Probiotics control intestinal
pathogens by production of antibacterial compounds,
including lactic and acetic acid and antibioticlike
substances, competition for nutrients and adhesion sites,
increased and decreased enzyme activity, increased
antibody levels and increased macrophage activity [11].
The aim of current study was to determine the
antibacterial activity lactic acid bacteria isolated Native
Yogurt north IRAN against ESBL Producing E. coli
Causing Urinary Tract Infection (UTI).
II.
plated onto MRS(De Man Rogosa and Sharpe) and M17
agar(Merck, Germany) to isolate the Lactobacillus spp
and incubated at 37°C for 48 - 72 h at condationanerobic
jar [14]. The strains were subcultured onto MRS agar
incu-bated at 30°C for 24 h and preserved in DMSO at 80°C. One of the isolates was selected for further
studies.It was identified on the basis of growth, cell
morphology, gram staining and catalase activity. Further,
identification was performed according to carbohydrate
fermentation patterns and growth at 15°C and 45°C in the
MRS broth based on the characteristics of the lactobacilli
as described in Bergey’s Manual of Determinative
Bacteriology [15] and 16s rRNA sequencing.
E. Antimicrobial Activity Determination
The antimicrobial activity of cell-free supernatant and
partially purified protein was determined by well
diffusion method [16]. Antibacterial activity of LAB
isolated from Native Yogurt was screened by Agar well
diffusion method. Antibacterial activity of LAB isolated
from Native Yogurt was tested against target bacterial
pathogens of health significance like ESBL isolated by in
vitro techniques using Muller-Hinton agar plates at 37 °C
for 24 h. ESBL suspensions of 10 8 colony forming units
(CFU)/ml were grown to log phase, and the well
diffusion were treated with the antibacterial compounds.
The plates were incubated at 37°C for 24 h, and the size
of the inhibition halos diameter was evaluated (mm). The
antibacterial effect was determined by measuring the size
of inhibited halos formed around clinical samplese wells.
MATERIALS AND METHODS
A. Collection of Samples
One hundred diarrheic fecal samples were collected
from different hospital Tehran (IRAN), over a period of 3
months. Samples were aseptically collected onto sterile
bottles and were immediately taken to the laboratory.
B. Culture and Identification E. coli
Fecal samples were suspended in saline (0.85%) and
100μl portion of the suspended fecal samples were plated
on EMB agar and MacConkey agar plates by spread
method. The plates were incubated at 37°C for 24 hours.
Pure cultures of the isolates were subjected to Gram
staining and biochemical tests [12].
III.
Eighty Escherichia coli isolated from different Urinary
Tract Infection (UTI). The80 E. coli isolated 30 (37.5%)
ESBL E. coli isolated and 50 (62.5%) non-ESBL
producing E. coli isolated. The current study showed that
ESBL E. coli resistant to Cefazoline (96.6%),
Ceftazidime (43.3%), Ceftriaxone (3.3%), Ciprofloxacin
(76.6%), Gentamicin (26.6%), Meropenem (0%),
Tobramycin
(43.3%),
Cefotaxime
(93.3%),
Terimetoperim
&
Sulfomethoxazole
(83.3%),
Ceftazidime + cluvalonic acid (3.3%), Cefotaxime +
cluvalonic acid (3.3%), Cefpodoxime (96.6%),
Piperacillin + Tazobactam (0%), Ampicillin (100%),
Amikacin (0%), Nitrofurantion (6.6%) (Table I).
C. Confirmation of ESBL
A total of 80 E. coli isolated from clinical specimens of
urine tract. All samples were processed and identified as
per the standard bacteriological division of microbiology
lab. The antibiotics disk used were Amikacin (30μg),
Ceftriaxone
(30μg),
Ceftazidime
(30μg),
Ceftazidime/Clavulanicacid,
Terimetoperim
&
Sulfomethoxazole, Gentamicin (10μg), Ciprofloxacin
(5μg), Meropenem (10μg), Pipercillin/Tazobactum,
Nitrofurantoin, Cefazoline (30μg), Tobramycin (10μg),
Cefotaxime/Clavulanic acid, Cefotaxime (30 μg),
Cefpodoxime (30), Ampicillin (10μg) was used to
detected and confirm ESBL producers. The test was
carried out in Mueller-Hinton agar and interpreted
according to the standards established by the CLSI third
generation Cephalosporins with and without Clavulanic
acid. An increase of more than 5mm in the diameter of
the inhibition halos around disks containing Clavulanic
acid as compared to the diameter around disks free of
Clavulanic acid inhibitor indicated ESBL activity. [13]
A. Antimicrobial Activity Cell Free Supernatant and
Purified Protein
The antimicrobial activity of CFCS and purified
protein (PP) were tested against ESBL by well diffusion
assay (Fig. 1). The CFCS and PP exhibited an
antibacterial effect on a narrow range of ESBL strains.
In this study, it showed that CFCS and PP
Lactobacillus sp. isolated from Native Yogurt narrow
antibacterial spectrum against ESBL isolated urinary tract
infection (UTI). All isolates have little sensitivity to
CFCS Lactobacillus sp. But did not show sensitivity to
PP (purified protein) (Table II).
D. Isolation and Identification LAB from Native Yogurt
Native yogurt was serially diluted in saline (0.85%)
and 100 μl of each dilutions (10-1 - 10-6) were spread
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
RESULTS
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International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
TABLE I.
ANTIBIOTIC RESISTANCE PATTERN OF ESBL E. COLI ISOLATESIN URINARY TRACT
Antibiotic
Concentration(μg)
Resistant
Sensitive
Cefazoline
30
29
1
Ceftazidime
30
13
17
Ceftriaxone
30
1
29
Ciprofloxacin
5
23
7
Gentamicin
10
8
22
Meropenem
10
0
30
Tobramycin
10
13
17
Cefotaxime
30
28
2
Terimetoperim&Sulfomethoxazole
1.25/23.75
25
5
Ceftazidime+cluvalonic acid
-
1
29
Cefotaxime+cluvalonic acid
-
1
29
Cefpodoxime
10
29
1
Piperacillin+Tazobactam
100
0
30
Ampicillin
10
30
0
Amikacin
30
0
30
Nitrofurantion
300
2
28
Figure 1. Antimicrobial activity of Cell-Free Culture Supernatant (CFCS) Pseudomonas sp..
IV.
Only a few bacteriocins of LAB with activity against
Gram-negative bacteria have been reported, viz.
thermophilin 81 (4.5 kDa), produced by Streptococcus
thermophiles [18]; a bacteriocin produced by
Lactococcus lactis B14 [19], plantaricin 35d (4.5 kDa),
produced by Lactobacillu splantarum etc. [20].
According to other research, the strain isolated in this
study is a potent antibacterial against Gram-negative
bacteria that could be used in future research in the food
preservation and probiotics.
This study has shown that native yogurt isolates such:
Lactobacillus sp. has inhibitory effects against the ESBL
isolated from UTI. Because, this kind of bacteria is very
resistant to antibiotics therefore, strains selected should
be optimized for the production of antibacterial
metabolites.
DISCUSSION
According to the results obtained in this study, CFCS
and PP Lactobacillus sp. isolated from Native Yogurt
narrow antibacterial spectrum against ESBL isolated
Urinary Tract Infection (UTI). The CFCS and PP
exhibited an antibacterial effect on a narrow range of
ESBL strains. However the effect is isolates the in vitro is
better than large number broad-spectrum antibiotics such:
third-generation
cephalosporins
(eg,
cefotaxime,
ceftriaxone, ceftazidime).
Miteva et al. (1998) reported 36 L. delbrueckii strains
from the ELBY Bulgaricum collection with a broad
spectrum of activity, including closely related LAB
species, pathogenic and food spoilage bacteria [17].
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
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International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
TABLE II. ANTIMICROBIAL ACTIVITY SPECTRUM OF THE CELL-FREE CULTURE SUPERNATANT AND PARTIALLY PURIFIED PROTEIN OF
LACTOBACILLUS SP.
Isolate code
Diameter of Zones Inhibition (mm)
Cell free Supernatant(100μl/well)
Purified Protein(100μl/well)
ES1
14
15
ES2
15
17
ES3
16
16
ES4
15
18
ES5
16
15
ES6
14
15
ES7
15
16
ES8
14
16
ES9
14
16
ES10
14
15
ES11
20
15
ES12
18
18
ES13
18
14
ES14
18
18
ES15
20
19
ES16
17
16
ES17
18
18
ES18
15
18
ES19
15
18
ES20
17
16
ES21
16
16
ES22
15
18
ES23
14
16
ES24
16
16
ES25
17
17
ES26
17
18
ES27
19
18
ES28
18
18
ES29
18
19
ES30
18
18
[3]
LAB, have potential to inhibit the growth of pathogens,
including multidrug resistant such: ESBL, MRSA, etc.
The spectrum of antibacterial activity LAB is highly
specific for each strain. Therefore, the in vitro
antimicrobial testing should be done on a large number of
antibiotic resistant bacteria. Also, a large number of LAB
isolated strains need to selection a suitable candidate for
probiotics.
[4]
[5]
[6]
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Zoheir Heshmatipour received the B.S. in microbiology from Azad
Universiy Tonekabon Branch (2000), and the M.S. (2002) in
microbiology from the Islamic azad University Lahijan Branch and the
Ph.D. (2012) in microbiology from the Islamic Azad University of
Science and Research Tehran Branch. Zoheir Heshmatipour teaches
courses in Basic microbiology 1 and 2, microbial physiology, microbial
ecology and microbiology standards in foods in Azad University
Tonekabon Branch (from 2002 to present). The activity and interest of
Zoheir Heshmatipour are in environmental microbiology and food
microbiology, especially in extremophiles and probiotics.
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