antibacterial activity lactic acid bacteria (lab) … activity lactic acid bacteria (lab) isolated...

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


Ashrafi Eslami A Bsc. Genetic, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran


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).

Manuscript received December 26, 2014; revised March 18, 2015

Index Terms—Lactic Acid Bacteria (LAB), Urinary Tract

Infection (UTI), Extended Spectrum β-lactamases (ESBLs),

CFCS.

I. INTRODUCTION

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 third-

generation) 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

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015

©2015 Int. J. Life Sci. Biotech. Pharm. Res. 117

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. 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].

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]

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

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 108 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.

III. RESULTS

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).

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).



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. 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].

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.



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

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.

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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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