Inhibition of food-related bacteria by antibacterial substances produced by Pseudomonas sp . strains isolated from pasteurized milk

In this work, the production of antimicrobial substances by strains of Pseudomonas sp. isolated from pasteurized milk and their potential action against food-related bacteria were investigated. Samples of pasteurized milk were purchased from arbitrarily chosen commercial establishments in the city of Rio de Janeiro, Brazil. Of the four samples analyzed, three presented several typical colonies of Pseudomonas. About 100 colonies were chosen and subjected to biochemical tests for confirmation of their identity. Eighteen strains of the Pseudomonas genus were identified and submitted to tests for the production of antimicrobial substances. Twelve strains (66.7%) were identified as Pseudomonas fluorescens, four (22.2%) as P. aeruginosa, one (5.5%) as P. mendocina and one (5.5%) as P. pseudoalcaligenes. Only two P. fluorescens strains were unable to produce any antimicrobial substance against any of the indicator strains tested. Most of the strains presented a broad spectrum of action, inhibiting reference and food-related strains such as Proteus vulgaris, Proteus mirabilis, Hafnia alvei, Yersinia enterocolitica, Escherichia coli and Salmonella typhi. Five antimicrobial substance-producing strains, which presented the broadest spectrum of action, were also tested against Staphylococcus aureus reference strains and 26 Staphylococcus sp. strains isolated from foods, some of which were resistant to antibiotics. The producer strains 8.1 and 8.3, both P. aeruginosa, were able to inhibit all the staphylococcal strains tested. The antimicrobial substances produced by strains 8.1 and 8.3 did not seem to be typical bacteriocins, since they were resistant to the three proteolytic enzymes tested. Experiments involving the characterization of these substances are being carried out in order to evaluate their biotechnological application.


Introduction
Although designed to supply complete nutrition for growing calves, bovine milk also provides an appropriate growth medium for a variety of microorganisms.The abundance of carbohydrates, proteins and fats in association with the neutral pH allows for the development of a microbial community which may be highly variable (ALI and ABDELGADIR, 2011).
A m o n g s t t h e m a i n re a s o n s c a u s i n g m i l k contamination are the poor conditions of hygiene during milking, insufficient cleaning procedures of the utensils and equipments and also problems related to the storage and transport (SILVA et al., 2011).
A variety of microorganisms with human pathogenic potential, including Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and Mycobacterium tuberculosis, can be found in raw milk (ARCURI et al., 2006).However the presence of psychrotolerant bacteria belonging to various genera are extremely important in relation to the shelf life of milk and its derivatives, since they may develop even during long periods of cooling.During the storage of raw milk, these microorganisms can produce many proteolytic and lipolytic enzymes responsible for the spoilage of milk and dairy products, reducing both the quality and shelf life of the processed milk and resulting in important economic losses (FRANZETTI and SCARPELLINI, 2007;DE JONGHE et al., 2011).
Although the pseudomonas are also psychrotolerant microorganisms and are generally isolated from milk, they can also produce antimicrobial substances (AMS) able to inhibit other bacteria.Some Pseudomonas aeruginosa strains, for example, produce AMS called pyocins (MICHEL-BRIAND and BAYSSE, 2002;ELFARASH et al., 2012).
Since therapeutic antibiotics are prohibited for use in foods, the interest in these natural antimicrobial substances is increasing exponentially.Additives with antagonistic properties have become a trademark in the search for food safety and preservation.In food and drinks, the addition of antimicrobial compounds to processed products has become a powerful weapon in the arsenal of food preservation.These compounds, especially the bacteriocins, can be interesting strategies against the growth of undesirable microorganism (RILEY and WERTZ, 2002;ELFARASH et al., 2012, NISHIE et al., 2012).
Given the relevance of the research on antagonistic additives, this work aimed to characterize strains of Pseudomonas sp.isolated from pasteurized milk, and investigate the production of antimicrobial substances and their potential for action against the Gram-negative bacteria and staphylococci strains associated with food.

Results and Discussion
Of the four pasteurized milk samples analyzed, three presented several typical colonies suggestive of Pseudomonas sp..About 100 colonies were arbitrarily chosen and subjected to biochemical tests for confirmation of their identity.
Of the eighteen strains analyzed belonging to the Pseudomonas genus, twelve (66.7%) were identified as P. fluorescens, four (22.2%) as P. aeruginosa, one (5.5%)as P. mendocina and one (5.5%)as P. pseudoalcaligenes (Table 2).The significant presence of P. fluorescens was expected, since other studies have also reported this species as the dominant spoilage species in refrigerated raw and pasteurized milk (DOGAN and BOOR, 2003;MUNSCH-ALATOSSAVA and ALATOSSAVA, 2006;2007).
All the 18 strains selected were tested for resistance to different classes of antimicrobials.Five strains were only resistant to aztreonam, another only to cefotaxime and four to aztreonam and cefotaxime (Table 2).According to the (Sigma-Aldrich, São Paulo, Brazil) on the antimicrobial substance activity was determined according to Giambiagi-Demarval et al. (1990).Forty microlitres of the enzymes (1 mg/mL, prepared in 0.05 M Tris (pH 8.0) with 0.01 M CaCl 2 ) were applied around the producer strain after chloroform treatment.The plates were incubated at 37 °C for 4 h and then sprayed with the indicator strain.After treatment with the enzymes, the absence of inhibition zones indicates that the antimicrobial substance presents an active proteinaceous compound.

Susceptibility of the inhibitory substances to NaOH
The antimicrobial substances were also treated with 0.2 M NaOH to rule out the possibility that the inhibition exhibited could have been due to the production of organic acids by the producer strain during its metabolism.This assay was carried out according to Giambiagi-Demarval et al. (1990).The eighteen Pseudomonas sp.strains were also submitted to assays for the production of antimicrobial substances.All were able to produce antimicrobial substances against at least one of the indicator strains tested (Table 3).Differently from some of the antimicrobial substance-producer Pseudomonas, the antimicrobial spectrum of action of the strains isolated in this work was not restricted to pseudomonas (LAVERMICOCCA et al., 1999;RILEY and WERTZ, 2002).
In relation to Gram-negative indicator strains, P. aeruginosa 8.1, 8.2 and 8.3 presented the broadest spectrum of action and also the largest inhibition zones, inhibiting Proteus mirabilis, P. vulgaris, Hafnia alvei, Yersinia enterocolitica, Escherichia coli, Salmonella spp.
Figure 1 shows some examples of the antimicrobial activity exhibited by strains 8.1 ad 8.3.
The inhibition of Salmonella sp. by Pseudomonas strains was also verified by Hubert et al. (1998).A Pseudomonas strain isolated from well water sediment produced the bacteriocin named PsVP-10, which showed a wide range of antibacterial action, inhibiting bacterial species such as S. typhi, S. typhimurium and S. sonnei, besides other microorganisms.
Interestingly, all the Gram-positive reference strains used as indicators were inhibited by P. aeruginosa 8.1, 8.2 and also 8.3, including the three Staphylococcus reference strains (Table 3).So the inhibition ability of these three Pseudomonas producer strains against 26 strains of Staphylococcus spp.isolated from food in previous studies carried out by our research group was evaluated and the results are shown in Table 1.Some differences in the diameters of the inhibition zones were observed in this experiment, but all the staphylococcal strains were inhibited by these Pseudomonas strains.Strain 8.2 was not included in these tests, since its spectrum of action and the plasmidial DNA profile (data not shown) suggested that this strain was identical to strain 8.1.literature, resistance to antibiotics has become common amongst strains isolated from milk and dairy products (ARSLAN et al., 2011;BEENA et al., 2011). Straley et al. (2006) verified that Pseudomonas spp.isolated from bulk tank milk samples were the dominant non-coliform Gram-negative bacteria and showed the highest levels of resistance to antibiotics.In a recent study, Arslan et al. (2011) verified that all 32 Pseudomonas strains isolated from cheese were susceptible to ciprofloxacin, gentamicin and imipenem.Antibiotic-resistant Pseudomonas spp.have significant importance in the dairy industries, since they are able to grow at low temperatures and have the potential to form biofilms (MARCHAND et al., 2012).A few studies have described the inhibition of Staphylococcus sp. by Pseudomonas, generally related to clinical strains.Qin et al. (2009) found that the extracellular products of P. aeruginosa PAO1, mainly polysaccharides, disrupted established S. epidermidis biofilms.Iwalokun et al. (2006) and Saleem et al. (2009) also showed that some pyocins obtained from Pseudomonas sp.associated with patients had antistaphylococcal activity.On the basis of the present results and those of the above mentioned studies, it appears that antimicrobial substances produced by Pseudomonas sp. could have the potential to be used not only in medical situations but also in the food industry.
To evaluate the presence of an active proteinaceous compound, the sensitivity of the antimicrobial substances to the proteolytic enzymes trypsin, proteinase K and pronase XXIII was verified.There was no loss of the inhibitory activity, indicating that the substances were resistant to these enzymes.This fact suggests that they are not typical bacteriocins, although some bacteriocins produced by Pseudomonas sp. are not susceptible to proteolytic enzymes, such as the pyocin SA188 described by Naz and Rasool (2013).This substance is produced by P. aeruginosa and is not digested by proteases, proteinase K, trypsin or papain.According to these authors, the findings are in agreement with the studies on pyocins, in which, of the three types of pyocin, S, R and F, only the S type is sensitive to proteolytic enzymes (NAZ and RASOOL, 2013).
The substances produced by strains 8.1 and 8.3 were also resistant to NaOH, ruling out the possibility that the inhibition of the indicator was the result of acids released by the producer strains.

Conclusions
The consumer demand for food without the addition of chemical preservatives and the spread of antibioticresistant bacteria in food has driven the research for natural inhibitory substances.The results presented in this work highlight the antimicrobial activity of Pseudomonas spp.isolated from pasteurized milk against Gramnegative and Gram-positive food-related pathogens, such as Salmonella typhi and Staphylococccus sp., which play important roles in foodborne diseases around the world, even in developed countries.Since the inhibitory compounds produced by strains 8.1 and 8.3 could have a potential application in reducing the levels of pathogens in foods, further studies are being carried out, including the determination of the best conditions for the production of antimicrobial substances that have proved promising.

Figure 1 .
Figure 1.Agar-spot assay showing antimicrobial activity against (A) Proteus mirabilis, (B) Salmonella Typhi and (C) Staphylococcus aureus ATCC12600.The inhibitory activity is represented by a clear zone around microbial inoculums (spots).Strains 8.1 and 8.3 are highlighted.
(diameter of inhibition zones ≥ 20 mm); -, no inhibition; ±, small inhibition zones (diameter of inhibition zones ≤ 19 mm); ATCC, American Type Culture Collection; LMIFRJ, Laboratory of Microbiology of Instituto Federal do Rio de Janeiro; NCTC, National Type Culture Collection.

Table 1 .
Inhibition of staphylococcal strains isolated from food by the Pseudomonas producer strains 8.1 and 8.3.

Table 2 .
Description of the Pseudomonas spp.strains isolated

Table 3 .
Inhibition of Gram-positive and Gram-negative bacteria by Pseudomonas spp.strains isolated from milk.