Malaysian Isolates of Lactic Acid Bacteria with Antibacterial Activity against Gram-Positive and Gram-Negative Pathogenic Bacteria

Contamination of foodstuff with foodborne and pathogenic bacteria are global issue and it is serious hazard for www.ccsenet.org/jfr Journal of Food Research Vol. 1, No. 1; February 2012 Published by Canadian Center of Science and Education 111 the health of the human. Lactic acid bacteria are well known for their health properties and their antimicrobial activity against spoilage and pathogenic bacteria. In this study, three isolates Lactobacillus fermentum Te007, Pediococcus pentosaceus Te010, L. pentosus G004 isolated from Malaysian fermented foods and fruits such as (tempeh, tempoyak, guava and banana) were evaluated for their antibacterial activity and antibiotic resistant against Gram-positive and Gram-negative bacteria by dual agar overlay method. The three isolates inhibited the growth of indicator bacteria and the activity was varied between weak and strong. All the isolates were resistant to the antibiotic nalidixic acid and vancomycin. The tested bacteria can be added to food as antibacterial agents to prevent the growth of harmful microorganisms.


Introduction
The food safety is important concern in the processed foods industry because of the contamination with food borne pathogenic bacteria.The consumers demand for minimal processed food with less chemical additives (Cleveland et al., 2001).These reasons have led to the increase of interest to introduce the biopreservatives to food to replace chemicals with natural preservation agent (José et al., 2007).The foodborne and pathogenic bacteria, especially anaerobes and facultative anaerobes, even when they are present in low numbers they can grow during storage at low temperatures as in the refrigerators.These pathogens can multiply and cause risk to the safety of raw, processed and bakery products.Biopreservation is the extension of shelf life and food safety by the use of natural or controlled microbiota and/or their antimicrobial compounds (Stiles, 1996).Among the biopreservatives is lactic acid bacteria (LAB).They are known to produce variety of antibacterial substances as reported by several researchers (Moreno et al., 2000;Mallesha et al., 2010;Akpinar et al., 2011;Muhialdin & Hassan, 2011a).LAB are safe and they have the status of general recognized as safe (GRAS), and have an important role in the preservation of foods and fermented products (Cintas et al., 2001).The inhibition activity of LAB to the growth of pathogenic bacteria is most likely due to the production of organic acids and bacteriocin (De Vos, 1993;Klaenhammer, 1993).
There are more than a few reports about the antimicrobial activity of LAB.A total of 16 strains (5 Enterococcus faecium, 5 Enterococcus mundtii, 4 Pediococcus pentosaceus, 1 L. coryniformis and 1 Lactococcus garvieae) were reported to have inhibition activity to the growth of Listeria innocua.Bacteriocin-like inhibitory substances were also reported to maintain the inhibition activity against non-pathogenic and pathogenic food-associated and human pathogenic bacteria (Corsetti et al., 2005).L. pentosus TV35b found to have inhibitory activity against the growth of Clostridium sporogenes, Cl. tyrobutyricum, and Listeria innocua, among others, the active compound produced was produced a bacteriocin-like peptide (pentocin TV35b) (Okkers et al., 1999).Moreno et al., (2002) reported that LAB isolated from fermented food Tempeh produced bacteriocins that inhibited the growth of Gram-positive indicators, including Listeria monocytogenes.Liasi et al., (2009) reported that Malaysia isolates (L.casei LA17, L. plantarum LA22 and L. paracasei LA02) isolated from the fermented fish Budu inhibited the growth of (B.cereus, S. aureus, Salmonella enterica, Listeria monocytogenes, E. coli and Lactococcus lactis).The objective of this study is to evaluate the antimicrobial activity of lactic acid bacteria isolated from Malaysian fermented foods and fruits and their antibiotic susceptibility.

Cultures and isolates
L. fermentum Te007, Pediococcus pentosaceus Te010, L. pentosus G004 were isolated from Malaysian fermented fruits and foods and evaluated for their antibacterial activity against (Bacillus subtilis, Serratia marcescens, Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Shigella sonnei, Klebsiella pneumonia and Salmonella Typhimurium) obtained from the Microbiology Laboratory, Faculty of Food Science, University Putra Malaysia.The pathogenic bacteria were chosen to represent potential food borne bacteria.They were grown on nutrient broth (NB, Oxoid) at 30 °C for 24 h (Table 1).

Growth inhibition of pathogenic bacteria by LAB
The inhibition activities of the isolates were determined by using the dual agar overlay method as described by Magnusson & Schnurer (2001) with some modification.LAB isolates were inoculated in 2-cm lines on MRS agar plates and were grown at 30 °C for 24 h in anaerobic jars.The plates were then overlaid with 10 ml of nutrient agar containing 10 5 CFU/ml of each bacterium separately.The zone of inhibition was measured after 24 h of aerobic incubation at 30 °C; the inhibition was measured by depending on the size of the inhibition zone.The scale was used is: + weak inhibition, ++ moderate inhibition and +++ strong inhibition.The test was done in duplicate.

Antibiotic resistant of lactic acid bacteria
LAB isolates were tested for their resistant against several antibiotics using disk diffusion method (Herreros et al. 2004) with MRS agar.The antibiotics tested were vancomycin (5µm), nalidixic acid (30 µm), gentamycin (10 µm), streptomycin (10 µm), tetracycline (30 µm), penicillin G (10 µm) and chloramphenicol (30 µm) (Sigma).The antibiotic disc were placed on each plate and incubated at 37 O C for 24 hours in an anaerobic jar.The results were taken by detection the inhibition zone around the antibiotic discs.The test was done in replicate.

Growth inhibition of pathogenic bacteria by LAB
The three isolates of LAB (L.fermentum Te007, P. pentosaceus Te010, L. pentosus G004) showed antibacterial activity against tested strains of Gram positive and Gram negative bacteria.The results showed very strong inhibition activity from the isolate L. pentosus G004 and L. fermentum Te007 and moderated activity from the isolate P. pentosaceus Te010 (Table 2).The isolate L. pentosus G004 had very strong activity against all the tested bacteria except (Salmonella Typhimurium and Bacillus subtilis) which the isolate had moderated inhibition activity.
In contrast, with the isolate L. fermentum Te007 that showed very strong activity against (Bacillus subtilis, Serratia marcescens, Escherichia coli and Staphylococcus aureus) and moderated activity against (Enterobacter aerogenes, Staphylococcus epidermidis, Shigella sonnei, Klebsiella pneumonia and Salmonella Typhimurium).The isolate P. pentosaceus Te010 showed fair and good inhibition activity against all the examined bacteria.The three LAB isolates had moderated activity against Salmonella Typhimurium but no isolate showed very strong activity against it.In addition, Bacillus subtilis was sensitive for the isolate Te007 and the sensitivity was less for the other two LAB isolates.

Antibiotic resistant test of LAB isolates
All LAB isolates were resistant to nalidixic acid and vancomycin.Additionally, isolate L. pentosus G004 was resistant to Gentamycin, Streptomycin, Tetracycline and Penicillin G.. Isolate L. fermentum Te007 was sensitive to Gentamycin, Tetracycline and Penicillin G activity to gentamycin.Isolate P. pentosaceus Te010 showed resistance against nalidixic acid and vancomycin but sensitive to the other examined antibiotics.All LAB isolates were sensitive to Chloramphenicol (Table 3).

Discussion
The LAB isolates inhibited the growth of all tested bacteria including the pathogenic strains in different spectrum range of activities and the activity range was between weak to strong depends on the LAB strain.The highest activity was from the isolate L. pentosus G004 which show high activity against the tested microbes, followed by the isolate L. fermentum Te007 and P. pentosaceus Te010, respectively.Moreno et al., (2002) reported that LAB isolated from fermented food Tempeh produced bacteriocins that inhibited the growth of Gram-positive indicators, including Listeria monocytogenes.S. aureus, B. subtilis, S. typhimurium and E. coli are pathogens that have been involved in outbreaks of food-borne disease in the several foodstuffs (Lindqvist et al., 2001;Matarante et al., 2004).K. pneumonia, S. epidermidis Shigella sonnei and Serratia marcescens are human pathogens and involve in many serious infectious diseases (Wang et al., 1998;Ammor et al., 2006).The concern of food safety is on increase and this comes with big responsibility of finding new, natural and inexpensive methods such as the use of the biopreservatives from safe microbes such as LAB.There are many reports described the success of the LAB isolates to inhibit the growth of pathogenic and foodborne bacteria in vivo and in vitro (Herreros et al. 2004;Ammor et al., 2006;Al-Allaf et al., 2009;Akpinar et al., 2011).
In this study the selected LAB isolates showed good inhibition activity against indicator strains of the pathogenic bacteria and this promote the use of these isolates as antimicrobial agent that can be added to food to prevent the growth of spoilage and foodborne bacteria and prevent the production of the different toxins in foodstuff by these bacteria.The inhibition was fast after 24 h; we could observe the inhibition zone around the two lines of the LAB isolates on the MRS agar plates.Liasi et al., (2009) reported Malaysia isolates (L.casei LA17, L. plantarum LA22 and L. paracasei LA02) to have inhibition activity against (B.cereus, S. aureus, Salmonella enterica, Listeria monocytogenes, E. coli and Lactococcus lactis), The LAB was isolated from the fermented fish Budu.
In this experiment, the selected isolates were resistant to antibiotic vancomycin and nalidixic acid.Vancomycin resistance is of major concern because it is one of the antibiotics that broadly efficacious against clinical infections caused by multidrug-resistant pathogens (Johnson et al., 1990;Woodford et al., 1995).It is very important for LAB that added to food to be not carry transmissible antibiotic resistance genes, there are reports which indicate that LAB from fermented products may act as a vehicle of antimicrobial-resistance genes that could be transferred to pathogens, either in the food matrix or in the gastrointestinal tract (Mathur and Singh, 2005).This can lead to the development of new antibiotic-resistant pathogens and that is why those strains of LAB undesirable to be added to foodstuff (Morelli and Wright, 1997;Salminen et al., 1998;Saarela et al., 2000).In this study, all the isolates showed resistance to vancomycin and nalidixic acid, and the isolate L. pentosus G004 was resistant to the seven tested antibiotic except of Chloramphenicol.This study demonstrates that these isolates can be added to food as conservative's agents against the foodborne pathogenic bacteria and to improve the human's health especially the isolate L. pentosus G004.

Conclusion
Lactic acid bacteria isolated from different Malaysian environment inhibited the growth of (Bacillus subtilis, Serratia marcescens, Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Shigella sonnei, Klebsiella pneumoniae, and Salmonella Typhimurium).The three isolates had inhibition activity against tested microorganisms and the spectrum range was varied between fair and strong.The isolates were antibiotic resistant against vancomycin and nalidixic acid.LAB isolates from Malaysian fermented foods and fruits have inhibition activity against foodborne and pathogenic bacteria and it have potential to be use as food preservatives because of their ability to eliminate the growth of spoilage microorganisms.

Figure 1 .
Figure 1.Clear zone of growth inhibition of Staphylococcus epidermidis formed around the streak lines of lactic acid bacteria (LAB) (Te007) incubated at 30 ºC for 24 h by dual agar overlay method A. Te007, B. G004 and C.Te010

Table 1 .
Bacterial strains and media of growth used in this study

Table 2 .
Growth inhibition of Gram positive and Gram negative bacteria after 24 h incubation at 30ºC by dual agar overlay method

Table 3 .
Antibacterial activity of selected antibiotics against lactic acid bacteria measured by diameter of inhibition zone (mm) around the discs