Antibacterial Activity Lactic Acid Bacteria (LAB) Isolated Native Yogurt against ESBL Producing E. coli Causing Urinary Tract Infection (UTI)

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1 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 heshmatipoor@toniau.ac.ir and zheshmat@gmail.com Aslikousha H Msc. Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran haniyeasli@ymail.com Mohamadiebli F Department of Microbiology, Infection Control Comimittee, Pars Hospital Laboratory, Tehran, Iran farzad.ebli@gmail.com Ashrafi Eslami A Bsc. Genetic, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran alireza_ashrafi_eslami@yahoo.com 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 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 2015 Int. J. Life Sci. Biotech. Pharm. Res. 117

2 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. A. Collection of Samples MATERIALS AND METHODS 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 ( ) 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 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. 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) Int. J. Life Sci. Biotech. Pharm. Res. 118

3 TABLE I. ANTIBIOTIC RESISTANCE PATTERN OF ESBL E. COLI ISOLATESIN URINARY TRACT Antibiotic Concentration(μg) Resistant Sensitive Cefazoline Ceftazidime Ceftriaxone Ciprofloxacin Gentamicin Meropenem Tobramycin Cefotaxime Terimetoperim&Sulfomethoxazole 1.25/ Ceftazidime+cluvalonic acid Cefotaxime+cluvalonic acid Cefpodoxime Piperacillin+Tazobactam Ampicillin Amikacin Nitrofurantion 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 Int. J. Life Sci. Biotech. Pharm. Res. 119

4 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) ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES ES 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. REFERENCES [1] P. A. Bradford, Extended-spectrum β-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat, Clin. Microbiol. Rev., vol. 14, pp , [2] A. Andremont, Commensal flora may play key role inspreading antibiotic resistance, ASM News, vol. 69, pp , [3] E. J. Vollaard and H. A. Clasener, Colonization resistance, Antimicrobial Agents and Chemotherapy, vol. 38, pp , [4] A. Sullivan and C. E. Nord, The place of probiotics inhuman intestinal infections, International Journal of Antimicrobial. Agents, vol. 20, pp , [5] E. A. Zottola, T. L. Yessi, D. B. Ajao, and R. F. Roberts, Utilization of cheddar cheese containing Nisinas an antimicrobial agents in other foods, Int. Food Microbial., vol. 24, pp , [6] P. I. Alade and O. N. Irobi, Antimicrobial activity of extracts of Acalyphawikesina, J. Ethnopharmacol., vol. 39, pp , [7] M. F. Bernet-Commard, F. Leivin, D. Brassanrt, A. L. Sewin, and S. Hundnait, The human Lactobacillus acidophilus strain LAU secrets and non-bacterium antibacterial substance activity in vitro and in vivo, Applied Environmental Microbiology, vol. 63, pp , Int. J. Life Sci. Biotech. Pharm. Res. 120

5 [8] P. A. Gibbs, Novel uses for lactic acid fermentation in food preservation, Journal of Applied Bacteriology, vol. 63, no. 16, pp , [9] O. B. Olorunfemi, T. T. Adebolu, and F. C. Adetuyi, Antibacterial activities of Micrococcus lactis strains isolated from nigerian fermented cheese whey against diarrhea causing organisms, Nigeria J. Microbiol., vol. 24, no. 1, pp , [10] L. Morelli, In vitro selection of probiotic lactobacilli: A critical appraisal, Current Issues in Intestinal Microbiology, vol. 1, pp , [11] S. Hudault, V. Lievin, M. F. Bernet-Camard, and A. L. Servin, Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection, Appl. Environ. Microbiol., vol. 63, pp , [12] M. Cheesbrough, District Laboratory Practice in Tropical Countries, 2nd ed. New York: Cambridge University Press, [13] Clinical and Laboratory Standards Institute, Performance standards for antimicrobial susceptibility testing, Seventeenth informational supplement, CLSI document M100-S16, Clinical and Laboratory Standards Institute, Wayne, PA, [14] J. C. M. De Man, Rogosa, and M. E. Sharpe, A medium for the cultivation of Lactobacilli, Journal of Applied Microbiology, vol. 23, no. 1, pp [15] G. M. Garrity, J. A. Bell, and T. G. Lilbum, Taxonomic Outline of the Prokaryotes Bergey s Manual of Systematic Bacteriology, 2nd ed. New York, Berlin, Heidelberg: Springer, [16] U. Schillinger and F. Lucke, Antibacterial activity of Lactobacillus sake isolated from meat, Appl. Environ. Microbiol., vol. 55, pp , [17] V. Miteva, T. Z. Stefanova, I. Budakov, I. Ivanova, V. Mitev, A. Gancheva, et al., Characterization of bacteriocins, produced by strains from traditional Bulagarian dairy products, Syst. Appl. Microbiol., vol. 21, no. 1, pp , [18] I. Ivanova, V. Miteva, T. Stefanova, A. Pantev, I. Budakov, S. Danova, et al., Characterization of a bacteriocin produced by Streptococcus thermophiles 81, Int. J. of Food Microbiology, vol. 42, pp , [19] I. Ivanova, P. Kabadjova, A. Pantev, S. Danova, and X. Dousset, Detection, purification and partial characterization of a novel bacteriocins substances produced by Lactococcus lactis subsp. Lactis B14 isolated from boza - Bulgarian traditional cereal beverage, Biocatalysis: Fundamentals & Applications, vol. 41, pp , [20] S. D. Todorov and L. M. Dicks, Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria, Enzyme and Microbial Technology, vol. 36, pp , 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 Int. J. Life Sci. Biotech. Pharm. Res. 121