Antibacterial Effects of Lactic Acid Bacteria Cell Free Extract on the Growth of Local Isolates of Listeria moncytogenes from some Food Sources

The present study aimed to determine the antibacterial activity of lactic acid bacteria metabolites against Listeria monocytogenes which was isolated from some foods in local markets of Erbil and Koya cities; 225 food samples were examined for the presence of Listeria species particularly L. monocytogenes from October 2010 to March 2011. The studied samples included 70 samples of raw chicken meat, 55 samples of raw milk, 50 samples of cheese and 50 samples of raw red meat. According to motility test, hemolysin production, sugar fermentation test, 8 isolates of L. monocytogenes were identified, 1 (2 %) isolate from cheese, 2 (4 %) isolates from red meat and 5 (7.3 %) isolates from chicken meat. The percentages of contaminated foods for Listeria species from chicken meat, raw milk, cheese and red meat were 20, 9, 20 and 22% respectively. With respect to the biocontrolling attempt, the effect of cell free extract (CFE) of lactic acid bacteria (LBA) which is considered as safe against isolated L. monocytogene was used as biocontrolling agent. The CFE of lactic acid bacteria which isolated from local dairy products, was tested for its controlling ability by agar diffusion method, the CFE showed inhibition zone from (15-23 mm) in diameter. Also the minimum inhibitory concentration MIC of CFE was determined at three additive percentages (0.5, 1.0 and 1.5ml to 10 ml of nutrient broth), the inhibition percentage ranged from (28.75 48.97) at (0.5 %), (52.13-95.55) at (1 %) and (82.93 -98.15) at (1.5 %) additive percentage of CFE of LAB.


INTRODUCTION
Listeria monocytogenes is widely distributed in the environment and has been isolated from avariety of sources, including soil, vegetation, meat, silage, faecal material, sewage, and water. This bacterium is resistant to various environmental stresses, such as highly salty or acid solutions, which allows it to survive longer under stressful conditions than most other non-sporeforming bacteria of foodborne disease concern (Dimic et al., 2010), and can survive for a long time in foods, processing plants, households, at refrigeration temperatures. Although it commonly exists in raw foods of both plant and animal origin, it is also present in cooked foods due to post-processing contamination, from food processing environments; especially those that are cool and wet (Tompkin, 2002). There are seven species within the genus Listeria (L. monocytogenes, L. ivanovii, L.innocua, L. seeligeri, L. welshimeri, L. grayii, and L. murrayi), only two, L.monocytogenes and L. ivanovii, are pathogenic, causing disease in both humans and animals (Roberts and Greenwood, 2003;Swamina, 2006). Almost 99% of human listeriosis has resulted from the consumption of contaminated foods (Mead et al., 1999). Listeriosis is among the most common bacterial food-borne pathogens worldwide and the disease emerging as an important food -borne pathogen of public health concern (Chukwu et al., 2007) L. monocytogenes causes listeriosis which can be a non-invasive disease, but primarily occurs in an invasive form. It affects those with a severe underlying disease or condition, as immunosuppression and HIV/AIDS, pregnant women, unborn or newly delivered infants, and the elderly. The clinical signs of the invasive form are flu-like illness including fever, headache, nausea, vomiting, and diarrhea appear about 12 hours or more after ingestion. After several days, the more serious symptoms appear, including meningitis, encephalitis, septicemia, and intrauterine/cervical infections that may result in spontaneous abortion in pregnant women (Swamina and Gerner, 2007;Nayak et al., 2010). The term "biocontrol" refers to the use of microorganisms or their metabolites to inhibit or inactivate undesirable microorganisms in food. Biocontrol was first associated with the use of starter cultures, but metabolites and fermentation products also contribute to the safety and microbiological quality of food. Biocontrol of L. monocytogenes in food is achieved by adding bacteriocin-producing microorganism, bacteriocin-containing fermentate, bacteriocin crude extract, or purified bacteriocin ,in fact lactic fermentations are believed to be the oldest preservation known to human kind (Ross et al., 2000). Such an application i.e., the antimicrobial properties of lactic acid bacteria due to their ability to produce substance as a natural competitive means to overcome other microorganisms sharing the same ecological niche (Pattnaik et al., 2005) Bacteria that produce antilisterial bacteriocins include strains of lactic acid bacteria, lactic acid bacteria produce various types of compounds such as lactic acid, acetic acid, formic acid, phenyllactic acid, diacetyl, acetoin, reuterin, reutericyclin, cyclic dipeptides, bacteriocins and other inhibitory proteinaceous compounds, 3-hydroxy fatty acids and hydrogen peroxide (Schnurer and Magnusson, 2005;Francoise, 2010).
The aims of this present study were: (1) to evaluate food in local market of Erbil and Koya cities for the presence of Listeria especially L.monocyogenes (2) to test the antimicrobial activity of LAB isolated from ''local dairy products'' against L. monocytogenes .

MATERIALS AND METHODS
Two hundred and twenty five (225) Samples of fresh white cheese and raw milk and beef meat and poultry were collected from and around different market areas in Erbil City and Koya city for a period of 4 months and examined for the presence of L.monocytogenes and other species. Diagnosis: Colonies suspected to be Listeria were characterized using Gram stain; catalase reaction; umbrella-shaped motility pattern by using motility test medium; haemolysis on sheep blood agar; fermentation of mannitol, rhamnose and xylose and CAMP test performed according to Bergey's Manual of Systematic Bacteriology (Seeliger and Jones, 1987).

Determination of Antibacterial Activity of the Cell Free Extract ( CFE) of LAB:
Cell free extract of all LAB which used in this study was prepared according to (Alsheekh 1999;Hameed, 2004) as follow: Lactic acid bacteria (lactic acid bacteria (LAB) include (3) strains of Lactobacillus lactis (LB), (2) strains of Pediococcus petosaceus (PC) and (3) strains of Lactococcus lactis (LC) was inoculated in MRA (De Man Rogosa Sharp) broth as 2 % of broth volume and incubated in anaerobic condition at 37˚C for 72h. The culture then centrifuge at 5000 rpm for 30 min., The Supernatants were sterilized by filtration through 0.22 mm membranes (Millipore).The antibacterial activity of CFE of LAB was determined by agar well diffusion method as described by (Gupta et al.,1998;Alsheekh,1999), 0.1 ml of approximately 10 8 which corresponded to a 0.5 McFarland turbidity standard solution of L.monocytogenes was transfered into nutrient agar and spread by L-shape spreader after one hour then well (5mm) in diameter and (5mm) in height was made by using sterilized cork porrer in nutrient agar then 0.5 ml of CFE was added into the wells, the plates were incubated at 37˚C for 18h,after then the inhibition zone was measured and compared with control which contains MRS media only.

Determination of Minimum Inhibitory Concentration of CFE of LAB
The MIC of CFE of all LAB was determined, the (2) isolates of L. monocytogenes (1%) of 10 5 cfu/ml of fresh culture was inoculated in10 ml nutrient broth containing (0.5, 1, 1.5) % CFE and incubated at 37˚C for 24 h, after that serial dilution for every treatment was done, then the inhibiting activity of CFE was determined by colony count by plating (0.1 ml) for each treatment into sterilized petri dishes then TSA-YE(Liofilchem) was poured into the plates and incubated at 35˚C for 24 hr., after incubation, results were recorded and the percentage of the death were determined comparing with the control treatment ( 0% of CFE), (Suskovic et al., 1997).

RESULTS AND DISCUSSION
Pre-enrichment for samples in half fraser broth (primary Listeria enrichment broth LEB1). This step helps the recovery of stressed Listeria cell in the food samples (Mansilla, 2008), secondary Listeria enrichment broth LEB11, in this broth the concentration of selective agents are double. Positive fraser broth has darkened and may be black, dark brown or dark green appeared in (Fig. 1). A negative MFB has the straw color of newly made broth, these broth contain selective supplements which inhibit unwanted bacteria in the food samples which usually contain other bacteria as well. On the agar PALCAM (PLA) plates, Listeria colonies appeared grey-green. The colonies had black-sunken centers with a black halo against a cherry red background as shown in (Fig. 2). Listeria monocytogenes breaks down the esculin in the medium to glucose and esculetin, which forms an olive-green to black complex with ferric ions which stains the colonies of Listeria monocytogenes. (Walter, 2000). On Oxford agar Listeria showed the colonies grey-green. The colonies had black-sunken centers with that were surrounded by black halos. Selectivity is provided by the presence of lithium chloride in the formula also it is increased by adding various antimicrobial agents to the base. (Bille et al., 1999;Chukwu, 2007).

Confirmation Tests for identification of Listeria species Isolates
Gram stain: Listeria species cells appeared gram-positive rods, arranged singly, or in short chains, or in pairs at V-form angles and in groups that were parallel to each other. Other conformation include catalase, motility test, Listeria species gave a typical umbrella growth pattern on the motility test medium near the microaerophllic subsurface of the medium at (25C˚) as shown in (Fig. 3)

Biochemical examination of Listeria monocytogenes
All isolated Listeria species strains are subjected to biochemical tests for identification and to show their biochemical characteristics. The identification was confirmed by sugar fermentation. The result showed that L.monocytogenes fermented salcin, lactose, galactose and rhamnose, and not fermented manitol, xylose, sucrose, and melbiose. All isolated strains hydrolyzed esculin to ascultin, which reacts with ferric ammonium citrate producing a black precipitate coloring the media and this result agrees with (Fraser and Sperber, 1988). All isolated L. monocytogenes were positive for catalase and negative for oxidase.
L.monocytogenes has been shown to adhere to several different food contact materials such as stainless steel, polypropylene and glass and the adhered cells show an increased resistance to cleaning agents, disinfectants and heat, all of which are used in the sanitation of the food processing plants (Dimic, 2010), therefore the contamination of cheese during processing from environment is possible. The prevalence of the organism in cheese is hazardous to consumers because cheese is ready to eat.
From a total of (70) of raw Chicken (fresh and frozen) samples 19(27.1 %) were positive for Listeria spp. Subdivided as 5 (7.1 %) L.monocytogenes, 5(7.1 %), L.innocua, L. grayi 2(2.8 %), L.welshimeri 1(1.4 %), 1(1.4 %) L.seeligeri. This result is in agreement with Ennaji et al., (2008) who found that (20.3%) of poultry meat were positive for listeria spp., Among the strains of L. species isolates, (1.3 %) L.monocytogenes, (16.2 %) L. innocua, and L. welshimeri (2.7 %) were identified. Abd El-Malek et al., (2010) in Assuit (Egypt) found that listeria spp. was indicated in (52 %) and (8 %) L.monocytogenes in chicken legs and (54 %) and (0 %) L.monocytogenes in chicken fillet samples. The presence of Listeria spp. in (50) samples of red meat was 11(22 %), subdivided as 5(10 %) L. innocua, 2(4 %) L.monocytogenes, 2(4 %) L.grayi, 1(2 %) L.welshmire, and 1(2 %) L.seelgeri as shown in Table (2) and (Fig. 5). The incidence rate of Listeria spp. in red meat was (32 %) isolated from the examined samples. L. monocytogenes occurred in (4%) and L. innocua in (28%) of tested samples in Issuit city in Egypt (Abd El-Malek et al., 2010), Nayak et al., (2010) found that 10 (6.7 %) samples were positive for Listeria species, of which four (2.7 %) were positive for L monocytogenes, two (1.3 %) for L.innocua, three (2.0 %) for L. seeligeri and one (0.7 %) for L. welshimeri. Contamination of the meat with L.monocytogenes generally occurs after the slaughter and may come from the skin of the animals, the hands of the workers, the equipment and the tools used (Marinsek and Grebenc, 2002).  In Vitro Antagonostic activity of Lactic Acid Bacteria against L. monocytogenes Cell free extract (CFE) metabolites of lactic acid bacteria (LAB) grown in MRS media have an inhibitory effect against (2) strains of L.monocytogenes. By using well diffusion method, the inhibitory effect of LAB cell free extract was variable and the inhibition zone was ranged from ( 15-23) mm. as shown in Table (3) and (Fig. 6), and the effect of LC and LB on the growth of L. monocytogenes was higher than PC as shown in Table (3). The minimum inhibitory concentration of CFE of LAB against L. monocytogenes at (0.5,1and 1.5) v/v of CFE which concentrated for two folds was ranged from (28 -98 %) as shown in Table (4) the inhibition percentage increases as the concentration of CFE increases. LAB produces many antimicrobial substances like organic acids, hydrogen peroxide and bacteriocins that inhibit other bacteria and fungi (Mansilla, 2008). The products of LAB metabolites contribute, not only for preservation, but also to the flavor, aroma and texture, thereby helping to determine unique product characteristics (Pereira, et al., 2008). LAB are used as natural or selected starters in food fermentations, especially for the manufacture of dairy products with functional and probiotic properties. The effect of isolates LAB against L.monocytogenes is very important in the dairy industry, as this pathogen was isolated from cheeses manufactured form raw milk, ripened cheeses, and in chicken 20% milk 9% cheese 20% red meat 22% the wash water employed for cheese production (Glass and Doyle, 2005). Svirakova et al., (2009) reported that Lactococcus lactis caused reduction in listeria growth from the level of 10 3 CFU/ml to 10 0 CFU/ml. Nespolo and Brandelli, (2010), reported that L. plantarum showed antimicrobial activity against Listeria monocytogenes. When Pediococcus acidilactici, was added to frankfurters and cooked ham inoculated with L. monocytogenes showed bacteriostatic activity in cooked ham and bactericidal activity in frankfurters (Kumar, 2011). The use of lactic acid bacteria in fermented sausages as protective culture has a bacteriostatic or bactericidal action on L. monocytogenes growth. Biopreservation is an additional factor in the production of safer food but it alone cannot warrant microbiological safety (Zdolec et al., 2007). Lactic acid bacteria metabolites with activity against L. monocytogenes, have attracted great interest in the past ten years in food science area. Particularly, bacteriocins from lactic acid bacteria (LAB) are very important because they constitute a group of industrial microorganisms that may improve sensorial properties, shelf life and safety of foods (Martinez and Martinis, 2006).