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Activity of natural antimicrobial  compounds against  Escherichia coli and Salmonella enterica serovar Typhimurium

ABSTRACT
 

Aims: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium.

Methods and Results: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1·0 and 1·2 mmol 1 ¹ against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity.

Conclusions: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity.

Significance and Impact of the Study: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.

INTRODUCTION
 

Many food preservation systems, such as heat treatments and addition of chemical preservatives, are used to reduce the risk of outbreaks of bacterial food poisoning and food spoilage (Periago and Moezelaar 2001). However, some of these systems can have undesired effects, which are against the food industry's and consumer's demands, who desire fresher, additive-free and more natural tasting food products while maintaining microbiological safety and stability (Dillon and Board 1994; Gould 1996). There has been an increasing interest in the development of effective natural antimicrobials as food preservatives. Nisin, a bacteriocin produced by strains of Lactococcus lactis has found practical application in the food industry (Delves-Broughton et al. 1996), whereas other natural compounds such as carvacrol, cinnamic acid, diacetyl, eugenol and thymol have been shown to exhibit inhibitory activity against numerous food related bacteria (Kim et al. 1995; Kang and Fung 1999; Friedman et al. 2002; Roller and Seedhar 2002; Walsh et al. 2003). However, their potential as a novel source of food preservatives has yet to be fully exploited.

Although, nisin is effective against Gram-positive bacteria such as Listeria monocytogenes and is known to inhibit bacterial sporulation, it is inactive against Gram-negative bacteria (Boziaris and Adams 1999). The nisin resistance of Gram-negatives is caused by the protective outer membrane that forms the outermost layer of the cell envelope, functioning as an efficient barrier against certain hydrophobic solutes and macromolecules (Hurst 1981; Hauben et al. 1996).

Both carvacrol and thymol are known to have prominent outer membrane disintegrating properties (Helander et al. 1998). This was indicated by the enhanced uptake of 1-N-phenylnaphthylamine and the release of lipopolysaccharide constituents into the external medium. Previous reports (Boziaris and Adams 1999; Terebiznik et al. 2002) have shown that combination of nisin with treatments such as addition of chelators (e.g. EDTA), pulsed electric fields or reduction in water activity can make Gram-negative bacteria sensitive to nisin inhibition. In the same manner, it could be envisaged that combining potentially membrane active organic compounds with nisin may enhance the overall inhibitory effect against Gram-negative bacteria by allowing nisin to pass through the outer membrane.

The objective of the present study was to evaluate the inhibitory activity of such organic compounds alone and in combination with nisin against Escherichia coli and Salmonella Typhimurium.

MATERIALS AND METHODS

 

Bacterial strains and culture conditions

Escherichia coli and Salm. Typhimurium LT2 were grown in L-broth containing (l ¹): bacteriological tryptone (Becton Dikinson, Oxford, UK), 10 g; yeast extract, 5 g; NaCl, 5 g and glucose, 1 g (pH 6·0). Cells were incubated with continuous agitation (150 rev min ¹) at 37°C. Both strains were obtained from the Institute of Food Research (IFR) culture collection.

Chemical preparation

The inhibitory compounds used in the assay included nisin and five natural organic compounds, namely diacetyl and the essential oils components, thymol, trans-cinnamic acid, eugenol and carvacrol (Sigma, UK). Nisin A (HPLC purified) was supplied by Aplin & Barrett (Beaminster, UK). In all cases, chemicals were of the highest grade available (98-100% pure). Stock solution (1 mmol 1 ¹) of nisin was prepared in 0·02 mol 1 ¹HCl and further dilutions were carried out in the same acidic solution. Stock solutions (1 mol 1 ¹) of the natural organic compounds were prepared in 95% (v/v) ethanol, with the exception of diacetyl that was prepared in water; further dilutions were made in the same liquids. Where necessary sterilization was achieved by filtration through a 0·22  m membrane filter (Millipore, Watford, UK).

MIC determination and combination assays

The minimum inhibitory concentrations (MICs) were determined using a Bioscreen C (Labsystems, Helsinki, Finland), which measures kinetically, the development of turbidity (i.e. growth) by vertical photometry. The test organisms were grown for 16 h in L-broth and incubated with continuous agitation at 37°C. The optical density (O.D.₆₀₀ nm) of the cultures was then adjusted (0·1) by dilution in L-broth. In the combination assays, the 'checkerboard' procedure described by Davidson and Parish (1989) was followed. Briefly, the method allows varying concentrations of each antimicrobial along the different axis of a 10  10 matrix thus ensuring that each well of the assay plate represents a different combination.

Antimicrobial assays were performed in Bioscreen honeycomb 100-well plates containing L-broth with test compounds whose final concentrations were: cinnamic acid, 0·5-20·0 mmol 1 ¹; diacetyl, 0·5-20·0 mmol 1 ¹; carvacrol, 0·1-5·0 mmol 1 ¹; eugenol, 0·5-10·0 mmol 1 ¹; thymol, 0·1-3·0 mmol 1 ¹ alone or in combination with nisin which was added to final concentrations of 0·1-5·0  mol 1 ¹. Diluted cell cultures were then added (5% v/v) to a final volume of 200  l per well. In all assays, controls grown in the presence of maximum ethanol and/or 0·02 mol 1 ¹HCl (both 1·25% v/v) alone, were set up in parallel.

The plates were incubated at 37°C for 16 h and the optical density (600 nm) was measured at 30-min intervals. The MIC of each natural organic compound alone or in combination with nisin was taken as the lowest concentration that completely inhibited bacterial growth after 16 h. In all cases, the assays were performed in duplicate.

RESULTS AND DISCUSSION
 

The MICs of the natural antimicrobials against E. coli and Salm. Typhimurium are presented in Table 1. Results of the negative controls containing 95% (v/v) ethanol and/or 0·02 mol 1 ¹ HCl (both 1·25% v/v) indicated complete absence of inhibition of both bacterial species (data not shown). As expected, nisin had no inhibitory activity against either of the Gram-negative bacteria tested in this study. Among the natural organic compounds, thymol was found to be the most effective, with the lowest MIC values of 1·0 and 1·2 mmol 1 ¹ against Salm. Typhimurium and E. coli, respectively. After thymol, antimicrobial order of the compounds tested was carvacrol > eugenol > cinnamic acid > diacetyl. Inhibition profiles (growth curves) of all the antimicrobial compounds were obtained and two such profiles for different levels of diacetyl against E. coli and Salm. Typhimurium are shown in Figs 1 and 2, respectively. Generally, at the sub-MIC levels, the lag phase of growth was extended and both the growth rate and final cell density were reduced with increasing concentrations of diacetyl. Complete inhibition of E. coli and Salm. Typhimurium was achieved with concentrations of 7·5 and 12·5 mmol 1 ¹ diacetyl, respectively.

Our observations of the antimicrobial activities of the natural organic compounds alone against E. coli and Salm. Typhimurium were consistent with several earlier studies. MIC values of 1·5 and 1·2 mmol 1 ¹ for carvacrol and thymol were observed here against E. coli. In comparison, slightly higher MIC values of 3·0 mmol 1 ¹ for both compounds were reported by Helander et al. (1998). Identical MIC values (1·0 mmol 1 ¹) were obtained for both carvacrol and thymol against Salm. Typhimurium in both studies. In an extensive study of antimicrobial activity of plant essential oils, Friedman et al. (2002) measured the effectiveness of eugenol against five strains of E. coli and four strains of Salm. enterica and their results indicated that on average, the E. coli strains were more sensitive to eugenol. The MIC values of 2·5 and 3·0 mmol 1 ¹ for eugenol against E. coli and Salm. Typhimurium observed here further support this finding. Kang and Fung (1999) reported that diacetyl concentrations of 0·57 and 3·41 mmol 1 ¹ were sufficient to inhibit the growth of E. coli O157:H7 and Salm. Typhimurium (ATCC 6994), respectively, in laboratory media and during meat fermentation. The strains used by Kang and Fung (1999) appear to be more sensitive to diacetyl compared with those used in this study (MICs of 7·5 and 12·5). These studies also support the view that Salm. Typhimurium has a greater resistance to diacetyl than E. coli (Jay 1982). The cinnamic acid was active against both strains and the MIC values indicated in Table 1 are the first such data reported for this compound, although its antimicrobial effectiveness was indicated previously in both E. coli and Salm. Typhimurium (Salmond et al. 1984; Dorantes et al. 2000).

Combination of nisin with the organic compounds failed to enhance the antimicrobial activity and the MIC values of the natural organic compounds remained unchanged in comparison with the values obtained when tested alone (data not shown). On the contrary, the combination of nisin with diacetyl against Salm. Typhimurium revealed an antagonistic interaction. The MIC of diacetyl was 12·5 mmol 1 ¹ when used alone, but this value increased with higher levels of nisin so that, at 5·0  mol 1 ¹ nisin the MIC of diacetyl doubled to 25 mmol 1 ¹(Table 2). Although the antimicrobial activity of diacetyl against E. coli and Salm. Typhimurium has been reported (Archer et al. 1996; Kang and Fung 1999), no information is available on its effectiveness in combination with nisin. One possible explanation for the observed antagonism is that nisin may prevent diacetyl from being taken up by the cells. Helander and Mattila-Sandholm (2000) studied the permeability barrier of Gram-negative bacterial outer membrane (OM) and showed that nisin at low levels stabilized the OM and protected the cells of both E. coli and Salm. enterica from the membrane damage caused by citric and lactic acid. In the present study, the antagonistic interaction between nisin and diacetyl was not observed against E. coli and this may reflect the structural differences in the outer membranes of the two test organisms. The mechanism of action of diacetyl is not yet understood although its interaction with arginine residues of enzymes has been suggested (Jay and Rivers 1984).

Previously Helander et al. (1998) reported that natural organic compounds including carvacrol and thymol degraded the outer membranes of E. coli and Salm. Typhimurium. In this study the addition of these organic compounds did not make the bacterial cells amenable to nisin inhibition as would be expected if the outer membrane was made nisin permeable. This would therefore suggest that if the organic compounds do actually damage the outer membrane, it is probably of an insufficient magnitude to permit the entry of nisin through it. Additional studies are clearly required to understand the mode of action of these compounds on the outer membranes of Gram-negative bacteria.

FIGURES

Fig. 1 The growth of Escherichia coli in the absence () or presence of 1 mm (), 3 mm (), 5 mm () or 7·5 mm... ...

Fig. 2 The growth of Salmonella Typhimurium in the absence () or presence of 2·5 mm (), 5 mm (), 7·5 mm (),...

 
Table 1 Minimum inhibitory concentration (MIC) of the natural antimicrobials against Escherichia coli... ...

 
Table 2 Effect of increasing nisin concentrations on the MIC of diacetyl against Salm. Typhimurium

ACKNOWLEDGEMENTS

The award of a fellowship by the Royal Society in London to Dr N. A. Olasupo is gratefully acknowledged.

REFERENCES

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