Pesq. Vet. Bras. 35(4):353-359, abril 2015 DOI: 10.1590/S0100-736X2015000400007

Effect of sp. isolates supernatant on Escherichia coli O157:H7 enhances the role of organic acids production as a factor for pathogen control1

Larissa B. Poppi2, Javier D. Rivaldi2,4, Thais S. Coutinho2, Claudete S. 3 3* and Ismael M. Mancilha2,5

ABSTRACT.-Astolfi-Ferreira , Antonio J. Piantino Ferreira Mancilha I.M. 2015. Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157:H7 enhancesPoppi L.B.,the role Rivaldi of organic J.D., Coutinho acids production T.S., Astolfi-Ferreira as a factor C.S., for Ferreira pathogen A.J.P. con &- trol. Pesquisa Veterinária Brasileira 35(4):353-359. de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Mar- ques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: Departamento [email protected] de Patologia, Faculdade Many attempts have been made to establish the control of foodborne pathogens through Lactobacillus isolates and their metabolism products with success being obtained in sev- eral situations. The aim of this study was to investigate the antagonistic effect of eight Lac- tobacillus isolates, including L. casei subsp. pseudoplantarum, L. plantarum, L. reuteri and L. delbrueckii subsp. delbrueckii, on the pathogenic Escherichia coli strain O157:H7. The inhibitory effect of pure cultures and two pooled cultures supernatants of Lactobacillus on the growth of pathogenic was evaluated by the spot agar method and by monitor- L. reuteri and L. delbrueckii subsp. delbrueckii and for a pool of bacteria. The neutralized supernatant of the pool ex- ing turbidity. Antimicrobial activity was confirmed for - tic acid and acetic acid were produced during growth of the Lactobacillus isolates studied. INDEXerted a TERMS: higher Lactobacillusantimicrobial, antagonism, activity than Escherichia that of the coli individual O157:H7, organic strains. acid. Furthermore, D-lac

RESUMO.- [Efeito do sobrenadante de isolados de Lac- isolados de Lactobacillus, incluindo L. casei subsp. pseudo- tobacillus sp. sobre Escherichia coli O157:H7 reforça o plantarum, L. plantarum L. reuteri e L. delbrueckii subsp. del- papel da produção de ácidos orgânicos como fator de brueckii, sobre Escherichia coli amostra O157:H7. Os efeitos controle patogênico.] Muitas tentativas têm sido feitas inibidores de culturas puras e de dois “pools” de cultura de para se estabelecer o controle de patógenos de origem ali- Lactobacillus sobre o crescimento da bactéria foram avalia- mentar através do uso de estirpes de Lactobacillus e dos dos através do método de inibição em ágar e através do mo- seus produtos de metabolismo, com sucesso sendo sucedi- nitoramento da turbidez da cultura bacteriana. A atividade do em várias situações. O objetivo deste trabalho foi investi- Lactobacillus reuteri e gar o efeito antagônico do sobrenadante de culturas de oito Lactobacillus delbrueckii subsp. delbrueckii e para o “pool” deantimicrobiana bactérias acido-láctica. foi confirmada O sobrenadante para neutralizado do 1 Received on January 13, 2015. “pool” de Lactobacillus exerceu uma atividade antimicro- Accepted for publication on April 21, 2015. biana mais elevada do que aquela das estirpes individuais. 2 - Além disso, ácido D-láctico e ácido acético foram produzi- de de São Paulo (USP), Área I, Estrada Municipal do Campinho s/n, Lorena, dos durante o crescimento dos Lactobacillus estudados. SP 12.602-810,Departamento Brazil de Biotecnologia, Faculdade de Engenharia, Universida 3 - TERMOS DE INDEXAÇÃO: Lactobacillus, antagonismo, Escherichia ques de Paiva 87, São Paulo, SP 05508-270, Brazil. *Corresponding author: coli O157:H7, ácido orgânico. [email protected] Departamento de Patologia, FMVZ-USP, Avenida Prof. Dr. Orlando Mar 4 INTRODUCTION Universitaria, San Lorenzo, Paraguay. 5 Facultad de Quimica, Universidad National de Asunción, Barrio Villa Escherichia coli O157:H7 represents one of the most impor- de Viçosa, Av. Peter Henry Rolfs s/n, Campus Universitário, Viçosa, MG tant enteropathogenic bacteria; it is responsible for nume- 36570-000, Departamento Brazil. de Tecnologia de Alimentos, Universidade Federal rous reports of is transmitted through food, water

353 354 Larissa B. Poppi et al. and the environment, affecting mainly infants and immu- of toxic anions. The decrease in the cell’s internal pH affects nosuppressed adults, all over the world (Monteiro-Neto et - E. coli sipates the proton-motive force, reducing cellular energy O157:H7 is attributed to the production of potent entero- the influx of protons through the cell membrane, which dis- toxins,al. 1997, the Fábrega Shiga-like et al. toxins 2002). (Stx1 The and pathogenicity Stx2, also called of ve- rocytotoxins), that directly affect the activity of cells in the (ATP)Several and affectingin vitro and substrate in vivo studiesuptake indemonstrated the cell (Axe the & Bai an- intestinal wall, resulting in hemorrhage and thousands of tagonismley 1995, Diez-Gonzalezof numerous strains & Russell of Lactobacillus 1997). , including L. deaths annually (Kudva et al. 1996, Cantarelli et al. 2000, delbrueckii var delbrueckii, L. plantarum, L. acidophilus, L. Garcia et al. 2010). Many different types of food were iden- reuteri and L. casei, against a variety of pathogens (Poppi et Escheri- chia coli (STEC), including raw and undercooked foodstuffs Jin et al. 1996). In spite of many detailed studies concer- (Garciatified as etpotential al. 2010). sources The ofnatural Shiga-toxin-producing hosts of Escherichia coli ningal. 2008, the Servinantagonistic & Coconnier effects 2003,of these Kalantzopoulos bacteria on patho 1997,- O157:H7 are wildlife and farm ruminants, mainly cattle and gens, there is still a need for new bacterial strains with an- swine. In the state of Rio de Janeiro (Brazil), studies con- ducted by Cerqueira et al (1999) reported the presence of In vitro screening STEC in 71 % of the fecal samples of healthy cattle from dai- methodstimicrobial such properties as agar forspotting clinical and and the commercial monitoring benefits of tur- bidity(Chaucheyras-Durand represent a fast &and Durand effective 2010). tool for this purpose. report concerning the isolation of STEC from the intestines Therefore, it is desirable to use these methods to select pro- ofry farms,dairy and beef beef farms cattle and inslaughterhouses. Brazil, although This several was thestudies first mising strains of Lactobacillus for the development of new had already reported the presence of other enteropathoge- preparations at the industrial scale. nic strains in the food industry (Cerqueira et al. 1999). The main objective of this work was to evaluate the in One approach that led to the reduction and, in a number vitro performance of eight strains of Lactobacillus isolated of cases, the elimination of intestinal pathogenic bacteria from poultry litter with respect to their inhibitory effect on in humans and animals includes the ingestion of the growth of Escherichia coli O157:H7.

2001). Probiotics are live microorganisms that, when ad- MATERIALS AND METHODS in the diary diet (Guarner & Schaafsma 1998, Gopal et al. Lactobacillus isolates and Escherichia coli strain O157:H7 on the host by altering indigenous microbiota and pre- Eight isolates of Lactobacillus, previously isolated from poul- ministered in adequate amounts, confer beneficial effects try litter (Paço et al. 2003), were selected to study their antago- (LAB) with probiotic properties, such as nistic effects on an Escherichia coli O157:H7 strain that was kin- spp.venting and infectionsLactobacillus (FAO/WHO spp. were 2001). used to Lactic prevent acid some bacteria in- dly provided by Dr. Isabel Scaletsky (Department of Microbiology, testinal pathogenic infections and to stimulate the host’s São Paulo, Brazil). The bacterial isolates subsp. immune system in both humans and animals (Perdigón et L. casei pseudo- plantarumImmunology (isolates and Parasitology, 30b and 30c), Federal L. plantarum University of São Paulo, 22c and 41b), L. reuteri (isolates 18fa and 19fa) and L. delbrueckii documented that Lactobacillus spp. with probiotic proper- subsp. delbrueckii (isolates 11fb,- tiesal. 1999, prevent Fang the et growthal. 2000, and Nakazato toxin production et al. 2011). of Itbacteria is well tory of Avian Diseases at the School of Veterinary Medicine and such as Campylobacter jejuni, Listeria monocytogenes, He- Animal Science of (isolatethe University 17fb) were of São obtained Paulo, Brazil. from the The Labora Lacto- licobacter pylori, Salmonella, Shigella and Escherichia coli bacillus isolates were maintained at 4°C in MRS (Manosa-Rogosa and Sharpe) agar slant. The pathogenic strain of Escherichia coli 2003, Poppi et al. 2008, Scapin et al. 2013). 0157:H7 was grown in BHI broth (Difco, Sparks, MD, USA) under (JinThe et al. antagonist 1996, Kalantzopoulos activity of probiotics 1997, Servin on pathogenic & Coconnier bac- aerobic conditions at 37°C for 18h. teria could be associated with the competition for nutrients The physiological characteristics and purity of the Lactoba- and sites of adhesion in the mucosa of the small intestine cillus isolates were analyzed by observation of their morphological characteristics using a light microscope, Gram staining, a catalase and the production of carbon dioxide, hydrogen peroxide test, a motility test and their ability to ferment various substra- tes. Sugar and sugar- alcohol assimilation tests were performed effect on the growth of several enteropathogenic bacteria is in test tubes by inoculation of the isolates into sterilized media, as likelyand diacetyl associated (Gopal with et al.the 2001). antimicrobial Furthermore, compounds the inhibitory produ- described. The following substrates were used: peptone (10 g.L-1), ced by , such as and lactic, ace- yeast extract (5 g.L-1) , phosphate (5 g.L-1), ammonium tic and other short-chain organic acids, which are responsi- citrate (2 g.L-1) acetate (5 g.L-1), sulfate (0.5 g.L-1), sulfate (0.01 g.L-1), Tween 80 (0.05 g.L-1), phe- 2003, Cheng et al. 2003, Varalakshmi et al. 2013). Lactic acid nol red (0.5 g.L-1). All products have been purchased from Merck, representsble for a reduction the main in antimicrobial the intestinal compound pH (Servin present & Coconnier in cul- Darmstadt, Germany. The carbohydrate sources used: arabinose, tures of lactic acid bacteria (Earnshaw 1992, Navarro et al. cellobiose, galactose, glucose, lactose, maltose, mannitol, manno- trehalose and xylose (Sigma, St. Louis, MO, USA) at concentration - 10g.L-1se, melezitose, for each melibiose, sugar type. raffinose, The stock rhamnose, solutions salicin, were prepared sucrose, ty2000, than Todorov strong acids, & Dicks which 2005, ionize Rossland completely et al. in2005, an aqueous Moraes 0.45 µm et al. 2013). Weak acids possess higher antimicrobial activi membrane filter (Millipore Corp., Billerica, MA, USA). The tubes organic acids are able to function as protonophores, indu- werefrom ainoculated 20% (w/v) and solution incubated previously at 37oC filtered for 7 days through to observe a red solution (Axe & Bailey 1995). The non-dissociated forms of cing the acidification of the cytoplasm and the accumulation phenol reduction by the sugar test (Kandler & Weiss 1986). Pesq. Vet. Bras. 35(4):353-359, abril 2015 Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157:H7 enhances the role of organic acids production as a factor for pathogen control 355

Growth conditions Effect of pH on Escherichia coli O157:H7 The strains of Lactobacillus were activated by transferring a To investigate the survival of Escherichia coli O157:H7 in the - culture media at different pH values. Afterwards, pH was adjusted taining 45mL of MRS broth, followed by incubation at 37°C for to 3.6 and 4.2, respectively, with 1.0 N HCl. The bacterial cultures 18full h. loop After from three the successive stock culture propagations to a 125mL in Erlenmeyer the same conditions, flask con were inoculated with an aliquot of Escherichia coli O157:H7 cell strains were grown independently in test tubes containing 5.0mL suspension (108 of MRS broth at 37°C for 16h. growth was monitored following the procedures described above. Two different pools of Lactobacillus were prepared, which CFU/mL) and incubated at 37°C for 7h. The cell were named pool A (PA) and pool B (PB). PA was prepared by mi- Lactic acid and acetic acid assays xing 4.0-mL of each pre-activated culture (32-mL total) in a 500- The D-lactic acid and acetic acid concentrations were determi- the mixture at 37°C for 18 h. PB consisted of the same mixture with a refractive index (IR) detector and Bio-Rad HPX-87-H (300 asmL PA, flask but containingwithout the 268-mL subsequent sterile incubation. MRS broth Cells and of incubatingindividual xned 7.8mm) by HPLC column Waters at 45°C 786 using(Spectralab 5 mM sulfuricScientific, acid Ontario, as the eluent,Canada) a and pooled cultures were collected by centrifugation (10,000xg -1 and a sample volume of 20 µl. All samples at 4°C for 10 min), washed twice with 0.1 M PBS (phosphate sa- - line buffer, pH 7.4), suspended in 20 % sterile sucrose solution mnflow (Millipore rate of 0.6ml.min Corp., Billerica, MA, USA). and frozen at -80°C for later use. The supernatants from these were conveniently diluted and filtered using a Sep Pak C18 colu cultures were used to study their inhibitory capability against Es- RESULTS AND DISCUSSION cherichia coli O157:H7. All of the experiments were carried out at least in duplicate. Replicates differ by less than 10%, and typically The physiological characteristics of Lactobacillus strains previously isolated from poultry litter (Paço et al. 2003) Student’s t test. were analyzed with regard to their morphology, physiology by less than 5%. The statistical significance was evaluated by the Antibacterial activity against Escherichia coli O157:H7 - agar as Lactobacillus using characteristics and properties expec- spot test tedand tobiochemical be on this characteristics. bacterium, such All as strains a rod were shape, confirmed positive The antimicrobial activity of Lactobacillus strains against Es- Gram staining, lack of motility, catalase-negativity, absence O157:H7 was analyzed by a spot agar test as descri- cherichia coli of endospores, fermentation of most of the sugars and su- cell suspensions of pure cultures and pool A of Lactobacillus were gar alcohols tested and resistance to the phenol compound. spottedbed by Schillinger on the surface & Lüke of MRS (1989). agar Aliquots plates and of 2μLMRS of agar concentrated plates bu- ffered with 35 mM sodium bicarbonate. The plates were dried out similar to L. plantarum, L. reuteri, L. delbruecki subsp. del- at room temperature for 30 min and incubated microaerobically brueckiThe strains and exhibitedL. casei subsp. a carbohydrate pseudoplantarum fermentation profile at 37°C for 24 h. Afterward, the plates were covered with 10mL of soft BHI (Brain Heart Infusion) agar containing 108 (Kandler & overnight culture of Escherichia coli O157:H7 suspension. Then, aceticWeiss 1986).acid concentrations of cell-free supernatants obtai- the plates were incubated under anaerobic condition CFU/mL at 37°C of foran nedTable from 1 pure shows and the pooled total viable cultures cells, of finalLactobacillus pH, lactic. Theand an additional 24h. The formation of clear zones of growth inhibi- tion around Lactobacillus colonies and their diameters were re- 7 9 -1 corded. Inhibition was considered positive if the diameter of the were generally high, ranging from 1.10 to 1.10 . clear zone that formed around colonies was 5 mm or larger. total number of colony formation units (CFU) of the strains De Vuyst (2001) using seven commercial strains of CFU lactoba mL - Inhibitory effect of Lactobacillus supernatants on Escherichia cilliThese grown results in MRS were broth similar under to those the same obtained conditions, by Avonts also as& coli O157:H7 shown in Table 2. The supernatants from each culture and pool of Lactobacil- lus were separated into two groups, a neutralized fraction and an Table 1. Results of colony-forming unit, pH values and acidic fraction. One fraction was adjusted to pH 7.0 with sodium organic acid concentrations from Lactobacillus isolates after bicarbonate, while the pH of the other fraction remained unal- 24 h of cultivation Lactobacillus -1 pH Organic acid production (x107) after cultivation on MRS furthertered. Both use. fractions were filtered using a 0.45 μm membrane isolates CFU.mL Lactic acid (g.L-1)* Acetic acid (g.L-1)* filterThe (Millipore direct antagonism Corp., Billerica, of compounds MA, USA) contained and stored in at Lactobacil -80°C for- lus cell-free supernatants against E. coli O157:H7 was monitored L. plantarumisolate L. delbruecki the acidic fraction of supernatants from each pure culture and PA 11fb 62 4.10 15.0±0.2 4.4±0.1 by turbidimetry. Aliquots (300 μL) of the neutralized fraction of and an L. reuteri subsp. 100 3.86 17.6±0.6 4.1±0.2 E. coli O157:H7 cell suspension (108 delbrueckiisolateL. reuteri 17fb 37°Cand PB for were 7h. Thetransferred blank used to sterile for standardization tubes containing consisted 300μL of a L. plantarum isolate 18fa 22 4.29 16.1±0.5 4.9±0.1 CFU/mL) and incubated at L. casei isolate 19fa 1 3.96 17.8±0.7 4.3±0.4 - Pseudoplantarum isolate isolate 22c 30b 90 3.83 18.5±0.3 4.2±0.5 genicmixture cell of suspension, 300μL each followed of sterile by MRS incubation and BHI asbroth. indicated As a control above. L. casei subsp. 330 3.95 8.6±0.1 2.1±0.2 Samplesrun, 300μL were of sterile taken MRSat intervals broth was of 1added hour, to and 300 their μL of OD the was patho de- Pseudoplantarum isolate 30c L. plantarum subsp. 790 3.98 16.7±0.2 2.7±0.1

isolate 41b 39 3.88 18.4±0.4 3.7±0.3 termined using a spectrophotometer Biomate 3 (Thermo Fisher Pool A 83 3.93 19.1±0.1 2.8±0.5 incubatedScientific, Waltham,in the same MA, conditions. USA) at 600 nm. After each absorbance *These values are means of triplicate experiments. Pool B 90 3.90 12.4±0.2 2.7±0.1 measurement, cell viability was confirmed using a BHI agar plate

Pesq. Vet. Bras. 35(4):353-359, abril 2015 356 Larissa B. Poppi et al.

All strains, except L. casei subsp. pseudoplantaum iso- (Table 2). The strains L. delbrueckii isolate L. casei late 30b, were able to produce lactic acid at concentra- subsp. pseudoplantarum tions higher than 12.4g. L-1. The highest concentration greater (p<0.05) inhibitory effects on the pathogen17fb and than the was attained with pool A (19.1g.L-1), followed by L. plan- other studied strains, with isolate zones 30c of displayedinhibition significantly larger than tarum isolate 41b (18.4g.L-1) and L. plantarum isolate 22c L. plantarum isolate 41b exhi- (18.5g.L-1). In addition, all lactobacilli produced acetic acid bited a similar pattern (19mm of inhibition halo). Among at concentrations higher than 2.0g.L-1. These short-chain the20mm strains (Fig.1). studied, Pool differences A and between 10 and 77% of the organic acids are known to exhibit high antimicrobial ac- diameter of clear zone formation were observed. The total tivity against microorganisms due to the easy diffusion of the non-dissociated form through the cell membranes of culture on an MRS plate ranged from 1.105 to 1.108 - - mLcolony-1, while count the in inoculum2 μL of cell concentration suspension obtained of E. coli by O157:H7 parallel was 108 CFU propionicpathogens acid (Axe and & Baileylactic acid1995). on Chengthe growth et al. and(2003) survival stu of inhibition by L. reuteri isolate 18fa, L. reuteri isolate 19fa ofdied E. thecoli influenceO157:H7. ofThe organic authors acids, reported including that acetic lactic acid,acid and L. plantarum CFU in 10 isolate mL of liquefied22c in MRS BHI media agar. Thewas lowerlikely levelrela- exerts greater inhibitory effects on the growth of the pa- ted to their slower growth capacity (Table 2). thogenic bacteria than acetic acid and propionic acid; ho- Lac- wever, the combination of the organic acids exhibited an tobacillus were tested on neutralized MRS medium (with important synergic effect in the inhibition of this pathogen sodiumFurthermore, bicarbonate, when 3g.L the-1 ),same the strainsmixed andstrains pools (Pool of A) (Cheng et al. 2003, Varalakshmi et al. 2013). According to exhibited higher inhibitory activity (19mm halo diameter) than the pure cultures (0 to 15mm). Differences in the halo acetate (1.6g. L-1) can inhibit the growth of E. coli O157:H7. diameter of between 30 and 100% were observed for cul- ThisDiez-Gonzalez level of acetate & Russell is similar (1997) to smallthe levels amounts observed of sodium in the - present work. milar results have been described in a study that examined The antagonistic effects of pure and pooled cultures of thetures inhibitory in MRS and effects MRS-neutralized of different strains agar medium of Bifidobacterium (Fig.1). Si Lactobacillus strains on E. coli O157:H7 were studied using against E. coli; halo diameters in the range of 13 to 30mm - were reported for the un-neutralized agar media associa- -neutralized media, all of the strains and the pool of Lacto- ted with a considerable reduction in halo size during gro- bacillusthe agar strainsspot method. were antagonisticBased on the towards data in Figure E.coli 1,O157:H7 in non wth in media containing sodium bicarbonate (Gagnon et al. 2004). Table 2. Results of optical density of Escherichia coli O157:H7 In our study, the E. coli O157:H7 inhibition by the Lacto- after 7 h of incubation in acidified BHI and MRS media bacillus isolates in the presence of sodium bicarbonate may Broth Optical density* (600 nm) be associated with non-acidic substances, such as hydro-

L. plantarum isolate 22c and L. Initial OD Final OD reuterigen peroxide isolate 19fa and .to grow in the Furthermore, media containing it has sodium been MRS, pH 3.6 0.063±0.005 0.076±0.004 bicarbonateconfirmed that was the due failure to an ofunfavorable pH for the growth of MRS, pH 4,2 0.103±0.008 0.117±0.003 these microorganism. MRS, pH 6,3 0.083±0.003 0.212±0.010 The combination of strains in pool A displayed greater BHI, pH 3,6 0.090±0.004 0.113±0.005 BHI, pH 4,2 0.094±0.002 0.120±0.008 inhibitory activity toward E. coli O157:H7 than the indivi- MRS =Manosa, Rogosa, Sharpe media, BHI = Brain Heart Infusion, *Avera- BHI, pH 7,4 0.111±0.010 1.015±0.016 dual cultures in almost all cases, regardless of the presen- ge of three experiments, OD = Optical density. demonstrate variation in the inhibitory strength of pure culturesce or absence of Lactobacillus of sodium, bicarbonateas well as in (Fig.1).the synergistic These results effect of co-cultures against E. coli O157:H7, emphasizing the im- portance of the precise selection of the strains for probiotic applications. E. coli O157:H7 grown in media containing the supernatants from different culturesFigure of 2A Lactobacillus. shows the optical The absorbance density of values demons- trate that E. coli was able to grow in media containing the neutralized fraction of supernatants from pure and pooled cultures of Lactobacillus. However, the pathogenic strain

OD values in neutralized media compared to the control exhibited slower growth rate and reached lower final The results suggest that pH adjustment results in the Lactobacillus isolates supernatant on loss(Fig.2A). of the antibacterial properties of the supernatants. Ho- Escherichia coli O157:H7 by the spot agar method using MRS wever, inhibitory activity was observed in all of the media Fig.1.agar Inhibitory and MRS effects after treatment of with sodium bicarbonate. containing the acidic fractions of the supernatants, as con-

Pesq. Vet. Bras. 35(4):353-359, abril 2015 Effect of Lactobacillus sp. isolates supernatant on Escherichia coli O157:H7 enhances the role of organic acids production as a factor for pathogen control 357

res, pHs and after treatment with proteolytic enzymes. The results revealed that the supernatant from L. plantarum lost its antimicrobial activity when the pH was adjusted to values higher than 5.0 and lower that 4.0, suggesting that the compound responsible for the inhibition was active only in this pH range. In the present work, a similar effect was detected with the supernatants from Lactobacillus iso- - bial activity toward E. coli O157:H7 except at pH 7.0, where thelates pathogenic 17fb and 19fa, bacteria and grewpool B,normally. which exhibited antimicro Ogawa et al. (2001) reported that L. casei strain Shirota and L. acidophilus strain YIT 0070 exerted inhibitory effects on E. coli growth. Their antimicrobial activity was assayed in co-culture, suggesting that the antimicrobial effect of Lactobacillus depends on the pH, due to accumulation of lactic acid in the culture medium. The authors also tested the antagonistic effect of L. brevis; however, this organism was unable to affect E. coli growth. The lack of antagonis- tic effects indicates that, as observed in the present work, the antimicrobial activity is dependent on the Lactobacillus strain. On the other hand, Aricia et al. (2004) observed a decrease in the antibacterial activity of supernatants of Lactobacillus that were adjusted to pH 6.5 and contained catalase, suggesting that hydrogen peroxide and organic acids promoted the inhibition of the growth of pathogens, including E. coli. The pH exerts an important negative effect on bacterial metabolism and growth. In this study, the E. coli O157:H7 was subjected to a moderately acidic environment at 37°C for 7 h. Table 2 shows the optical density (OD 600 nm) of the pathogenic E. coli inoculated in BHI and MRS broth whi- ch had been adjusted to different pH values. The results de- monstrate that E. coli O157:H7 was unable to grow in both Growth of Escherichia coli O157:H7 in cultural media con- media at pH 3.6 and 4.2; the effect on pathogen growth was taining the neutralized fraction of supernatants of Lactoba- Fig.2.cillus cultures (A) and the acidic fraction of supernatants (B) of L. plantarum- £), L. delbruecki subsp. delbruecki- bactericidal effect on E. coli; after 7h of exposure, the patho- (), L. reuteri -18fa (r), L. reuteri-19fa (s), L. plantarum genicbacteriostatic. strains were However, able to the grow acidified on BHI media agar plates did not (data exert not a -22c (v), L. casei11fb subsp. ( pseudoplantarum -30b (w), L. casei17fb shown). Previous studies showed that E. coli and other pa- subsp. pseudoplantarum-30c (¯), L. plantarum -41b( ), pool thogenic bacteria are sensitive to pH 3.5 and that exposure A ( ), pool B ( ) and control(). to low pH can result in adaptive resistance to acidic media E.coli and - others pathogens bacteria can survive under conditions of ed, it is well known that most of the antagonistic effects of (Koutsoumanisextreme acids stress & Sofos thanks 2004; toCheng an acidic-resistance et al. 2003). (AR) Lactobacillusfirmed by optical on E. density coli growth values could that be were due near to the zero. presence Inde systems, which is a group of amino acid decarboxylases of organic acids that are produced during cell growth (Axe and antiporters amino-acid dependent. Cells that have gro-

- succumb at pH 2.5 given the lack of glutamate or arginine ces& Bailey present 1995, in Diez-Gonzalezthe supernatants & Russell exerted 1997). bacteriostatic or wn in amino-acid deficient medium (minimal medium) can bactericidal According effects to theon resultsthe pathogenic shown in Escherichia Figure 2B, substancoli. The Cheng et al. (2003) reported that the percentage of E. supernatants from L. delbruecki subsp. Delbrueckii isolate coli(Foster O157:H7 2004). cells that survived in MRS broth at pH 3.0 L. reuteri isolate 19fa and pool B cultures exhibited were 4.2%. However, the authors mentioned that the bac- bactericidal activity on E. coli; after 7 h of exposure to these tericidal effect was not observed after 120 min of exposure supernatants,17fb, no colony formation was observed on spe- to these conditions and that by increasing the pH of the cul- L. ture broth to 4.0, the percentage of survival grew to 70% of reuteri, can produce reuterin, a non-proteic substance that the control growth (pH 7.0). Previous researches showed iscific soluble agar mediain water (data and not possesses shown). antibacterial,Some strains, antifungalsuch as that many Lactobacillus isolates may prevent the binding and antiprotozoan properties (Sung et al. 2003). of pathogens bacteria in the human and animal intestinal Lash et al. (2005) studied the inhibitory effects and sta- cells. After the banning of growth promoters antibiotics bility of L. plantarum supernatants at different temperatu- (GPA) in worldwide, many alternatives were launched, but

Pesq. Vet. Bras. 35(4):353-359, abril 2015 358 Larissa B. Poppi et al. still the Lactobacillus strains are the best choice for many situations, like the use as probiotics for chicken, cattle, swi- 2002. Cell-detaching Escherichia coli (CDEC) strains from children with Fábrega V.L.A., Ferreira A.J.P., Patrício F.R.S., Brinkley C. & Scaletsky I.C.A.- ne and others production animals, such as meat-type or biol. Lett. 217:191-197. egg-type. Nevertheless, we must consider the metabolism diarrhea: identification of a protein with toxigenic activity. FEMS Micro- product of these bacteria for use in animal production fee- ding. The Lactobacillus studied could prove useful as dieta- Fang29:47-52. H., Elina T., Heikki A. & Seppo S. 2000. Modulation of humoral immu ry supplements as well as antimicrobial agents in food and ne response through probiotic intake. FEMS Immunol. Med. Microbiol. packaging applications. health and nutritional properties of probiotics in food including powder FAO/WHOmilk with 2001. live lactic Joint acid FAO/WHO bacteria. Expert Available consultation at Accessed CONCLUSIONS Almost all strains of Lactobacillus investigated in this Escherichia coli acid resistance: tales of an amateur aci- Febr. 12, 2014. study exhibited bactericidal effects on Escherichia coli dophile. Nature Rev. Microbiol. 2:898-907. Foster J.W. 2004. O157:H7. Es- The highest inhibitory activity corresponded to Lac- cherichia coli Gagnon M., Kheadr E.E., Blay G.L. & Fliss I. 2004. In vitro inhibition of tobacillus delbrueckii subsp. delbrueckii L. casei O157:H7 by bifidobacterial strains of human origin. Int. J. Zoonotic enterohemorrhagic Escher- subsp. pseudoplantarum 30c and, suggesting that antimi- Food Microbiol. 92:69-78. ichia coli: a one health perspective. ILAR J. 51:221-232. crobial compounds production, such as hydrogen 17fb and peroxide Garcia A., Fox J.G. & Besser T.E. 2010. or bacteriocins may be responsible by bacterial inhibition. - ties of Lactobacillus rhamnosus DR20 and Bifidobacterium lactis DR10 All strains tested produced lactic and acetic acids. Gopalstrains P.K., and Prasad their J., antagonistic Smart J. & Gill activity H.S. 2001. against In enterotoxigenicvitro adherence Escheriproper- The amount of lactic acid produced has no demonstra- chia coli with the highest lactic acid concentrations were unable to 237-238.. Int. J. Food Microbiol. 67:207-216. exertble influence a more onpronounced the inhibition; bactericidal in pool effectA, the onsupernatants the patho- Guarner F. & Schaafsma G.J. 1998. Probiotics. Int. J. Food Microbiol. 39: genic bacteria than the other strains. Lactobacillus spp. on in vitro adherence of salmonellae to the intestinal Jin L.Z., Ho Y.W., Ali M.A., Abdullah N. & Jalaludin S. 1996. 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