Antagonistic activity of dairy lactobacilli against gram-foodborne pathogens

Thirty-five strains of lactic acid bacteria were isolated from artisanal raw milk cheese, presumptively identified and tested against one dairy Escherichia coli strain. Six lactobacilli, exhibiting antagonistic activity, were identified at the species level and their action was evaluated against four strains of Gram-foodborne pathogens (Escherichia coli O26, Escherichia coli O157:H7, Salmonella spp. 1023, and Salmonella Typhimurium) and the control strain Escherichia coli ATCC 45922. The antagonistic activity was determined by spot method and the inhibition zones were measured by Autodesk AutoCAD 2007. Three strains, all Lactobacillus paracasei, were active against all the pathogens; the other strains, all Lactobacillus plantarum, showed antagonistic activity against some pathogens. This study highlights the intense and different antagonistic activity induced by lactobacilli against various foodborne pathogens thus demonstrating that using selected lactic acid bacteria strains as adjunct cultures could be an effective strategy to prevent the development of foodborne pathogens in artisanal raw milk cheeses, and thus improving their safety.


Introduction
Foodborne infections are, nowadays, one of the most important challenges for public health, causing illness, sometimes fatal, and also resulting in high economic loss (KAFERSTEIN, 2003).Small ruminants are reported as important reservoirs of foodborne pathogens -among which Salmonella and verocytotoxin-producing Escherichia coli (VTEC)resulting in a possible contamination of products from these animals (COLAK et al., 2007;ESSID et al., 2009;OGUNBANWO et al., 2004).Strategies based on the use of antagonistic starter cultures allow the production of foods with improved levels of safety (SPELHAUG;HARLANDER, 1989).Antagonistic activities of lactic acid bacteria (LAB) against microbial pathogens may contribute, among other factors, to their inhibition (SERVIN, 2004).It is actually an important way to expand the range of healthful foods, especially in the dairy industry (SIMOVA et al., 2009).
Artisanal raw milk cheeses -made without the addition of LAB starters -are consumer attractive, with an intense flavour, due to the action of endogenous microflora (CARIDI, 2002(CARIDI, , 2003)).However, they could imply hygiene and health concerns; evidence supports the transmission of foodborne pathogens by contaminated raw milk cheeses (MILLER; PAIGE, 1998).Furthermore, some of the above mentioned foodborne pathogens may survive or multiply during the production process of raw milk cheeses (LITTLE et al., 2008).These cheeses are highly prized and, especially in summer when the food-borne risk increases, may reach a large number of consumers.
The aim of the present work was to study influence and antagonistic action of dairy LAB on several foodborne pathogens and to select pathogeninhibiting strains among the endogenous LAB of the artisanal Calabrian raw milk cheese Pecorino del Poro.

Cheese sampling
Seven Pecorino del Poro ewes' cheeses were sampled from dairy farms in the region of Calabria, southern Italy.Following traditional procedures, cheeses were made using raw milk without the addition of selected LAB.

Sample preparation and strain isolation
Representative cheese samples (10 g) were homogenized in 90 mL of sterile physiological saline for 2 minutes in a blade homogenizer 890-48H (Oster ® /Sunbeam ® , McMinnville, TN, USA).After filtering through sterile gauze, aliquots of 1 mL were diluted 10-fold in physiological sterile saline to enumerate LAB and E. coli and to isolate different microbial groups.Rod-shaped LAB were isolated on Man-Rogosa-Sharpe (MRS) agar (Oxoid, Basingstoke, England, UK) and incubated anaerobically by Gas Pack catalysts AnaeroGen (Oxoid, Basingstoke, England, UK) at 30°C for 3 days.Coccal-shaped LAB were isolated on M17 agar (Oxoid, Basingstoke, England, UK) and incubated anaerobically at 30°C for 3 days.LAB cultures were presumptively identified at the genus level.Dairy E. coli strains were isolated on Petrifilm 3M (Microbiology Products, St Paul, MN, USA), incubated aerobically at 37°C for 24h, and identified at the species level using standard methods.Working cultures were grown on broth media MRS, M17, and lactose broth (Oxoid, Basingstoke, England, UK) for lactobacilli, lactococci, and E. coli strains, respectively.

Food-borne bacterial strains
One strain of each of the following Gramfoodborne pathogens was used for the evaluation of the antagonistic activity of LAB: VTEC O26 (VT1 producer), VTEC O157:H7 (VT1 + VT2 producer), Salmonella spp.1023, and S. Typhimurium.E. coli ATCC 45922, not a pathogenic strain, was used as a control strain.With the exception of the ATCC strain, all the strains used had been previously isolated from dairy products, using standard methods.

Antagonistic activity trials
The isolated LAB were screened on plate against one dairy E. coli strain for antagonistic activity, under conditions that eliminated the inhibitory effects of lactic acid and hydrogen peroxide.The active LAB strains were then identified at the species level using standard methods and tested against the abovementioned foodborne pathogens, using the spot method (SPELHAUG; HARLANDER, 1989), in addition to the control strain E. coli ATCC 45922.A 24h culture of LAB grown in appropriate broth media was diluted 10-fold in 10 mmol L -1 Tris-HCl (pH 7.0) and 1 μl aliquots were spotted in triplicate onto MRS agar.The spot method uses the Tris-HCl (pH 7.0) solution to neutralize the organic acid produced by LAB in the broth media thus excluding possible inhibiting effects either on dairy E. coli or on foodborne pathogens (CARIDI, 2002).Plates were incubated anaerobically for 24h to eliminate inhibition due to hydrogen peroxide production (MESSI et al., 2001), and then overlaid either with Lactose soft agar (0.7% agar) or Triptic Soy soft agar (0.7% agar) inoculated, respectively, with 0.1 mL of overnight cultures of E. coli strains and the other foodborne pathogens.Plates were incubated for additional 18h and then checked for clear inhibition zones around spots of the presumed bacteriocin producers.The inhibition zones, measured using AutoCAD 2007 by Autodesk, Inc. (San Francisco, CA, USA), were expressed in mm and referred to the difference between the outer and the inner circles (Figure 1).

Results and discussion
The evaluation of the amount of LAB and E. coli in the artisanal Calabrian raw milk cheese in current research is quite interesting.In the seven cheeses, LAB ranged from 4 x 10 7 cfu g -1 to 3 x 10 10 cfu g -1 in the cheeses considered, whereas the amount of E. coli varied from zero to 1.4 x 10 6 cfu g -1 .With only one cheese over the limit imposed by the 2005/2073 European Commission Regulation (ECR, 2005), these results could be positively considered from a health and safety point of view.Although low values of E. coli could be due to the good hygienic quality of the milk or the good hygienic conditions during cheese manufacturing, in our opinion they are also due to the presence of antagonistic LAB.
Among the thirty-five LAB isolated, six out the rod-shaped LAB possessed antagonistic activity against the dairy E. coli strain, whereas none of the coccal-shaped LAB did.These six strains were identified as Lactobacillus paracasei (three strains) and Lactobacillus plantarum (three strains); subsequently, the six lactobacilli were tested for their antagonistic activity against foodborne pathogens.
Table 1 highlights the antagonistic activity of the six lactobacilli against the four Gram-pathogens and the control, based on their inhibition zones expressed in mm.Five of the six Lactobacillus strains showed antagonistic activity against E. coli ATCC 45922; means of inhibition zones ranged from 6.25 mm for strain Lb17 to 9.66 mm for strain Lb07.Five of the six Lactobacillus strains showed antagonistic activity against VTEC O26; means of inhibition zones ranged from 4.73 mm for strain Lb06 to 8.08 mm for strain Lb23.Five of the six Lactobacillus strains showed antagonistic activity against VTEC O157:H7; means of inhibition zones ranged from 4.46 mm for strain Lb21 to 7.06 mm for strain Lb23.Four of the six Lactobacillus strains showed antagonistic activity against Salmonella spp.1023; mean of inhibition zones ranged from 6.68 mm for strain Lb23 to 10.77 mm for strain Lb07.All the Lactobacillus strains showed antagonistic activity against S. Typhimurium; means of inhibition zones ranged from 5.22 mm for strain Lb23 to 12.47 mm for strain Lb07.
The three strains of L. plantarum selected in current research exhibit antagonistic activity against some of the tested pathogens; the antagonistic activity of other strains of L. plantarum had been previously studied.
It was reported that L. plantarum NCIM2084 produced a bacteriocin (plantaricin LP84) resistant to heat and catalase and was able to inhibit E. coli D21 (SUMA et al., 1998).Agar spot and well diffusion assay tests were applied to determine the antagonistic activity of 19 strains of L. plantarum against E. coli NRRL B-3704.The vast majority of the strains were able to inhibit the strain in agar spot, conducted by placing 0.5 mL of an overnight LAB culture onto MRS agar containing 0.2% glucose and incubated for 24h at 25°C under anaerobic condition.However, no strain was able to inhibit E. coli NRRL B-3704 by well diffusion assay test, using cell-free supernatants adjusted to pH 6.5 and sterilized by filtering through a 0.22 mm pore size cellulose acetate filter (ÇON; GÖKALP, 2000).
Supernatants of two dairy strains, Z11L and Z10, of L. plantarum produced strong inhibition against E. coli ATCC25922; however, when the supernatants were treated with catalase to eliminate the possible presence of hydrogen peroxide or neutralized to pH 6.5-7.0,only the supernatant of the strain Z10 produced weak inhibition (ASLIM et al., 2005).
L. plantarum ATCC8014 was tested for production of antimicrobial compounds; the study demonstrated that the cell-free supernatant of the strain was effective in inhibiting the growth of E. coli 15-5065 and S. Typhimurium 15-5351A (LASH et al., 2005).

Figure 1 .
Figure1.The inhibition halo size is expressed in mm and is referred to the difference between the outer (r 2 ) and the inner (r 1 ) radius.

Table 1 .
Antagonistic activity of the six Lactobacillus strains and the control strain of L. paracasei subsp.paracasei L356 against the four Gram-pathogens and the control strain of E. coli ATCC 45922.
a Mean of the inhibition zones, expressed in mm, measured around the three spots; b Standard Deviation; c Lactobacillus paracasei; d Lactobacillus plantarum; e Absence of inhibition zone.