Abstracts. U. Schillinger

Use of bacteriocinogenic lactic acid bacteria to inhibit spontaneous nisin-resistant mutants of Listeria monocytogenes Scott A

U. Schillinger, H. -S. Chung, K. Keppler and W. H. Holzapfel

ABSTRACT

Nisin is a bacteriocin with a broad antibacterial spectrum including strains of Listeria monocytogenes. Populations of L. monocytogenes, however, frequently contain spontaneous nisin-resistant mutants. When a culture of L. monocytogenes Scott A was exposed to nisin concentrations between 10 and 500 IU ml¹, the initial decrease in viable numbers was followed by regrowth of survivors to nisin. Nisin-resistant mutants of L. monocytogenes Scott A were isolated after a single exposure to nisin at 100 IU ml¹ and were shown to be sensitive to the non-nisin bacteriocins, sakacin A and enterocin B, produced by Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900, respectively. The regrowth of L. monocytogenes Scott A following the initial decrease due to exposure to nisin was prevented by nisin-resistant Lact. sake Lb 706-1a and to a somewhat lesser extent, by Ent. faecium BFE 900-6a. Listerial cells surviving nisin action were thus inhibited by the bacteriocin-producing strains that might be used as starter or protective cultures in foods. Growth of a nisin-resistant mutant of L. monocytogenes Scott A (Li3) was also suppressed by the bacteriocinogenic cultures. Use of nisin in combination with a starter culture producing a non-nisin antilisterial bacteriocin may therefore prevent the emergence of nisin-resistant mutants of L. monocytogenes.

INTRODUCTION

Nisin is widely used as a preservative in several food products such as processed cheese, dairy desserts and canned foods. It shows inhibitory activity against spore-forming bacteria and other Gram-positive spoilage and pathogenic bacteria including Listeria monocytogenes. Among the sensitive bacteria, however, strains differ greatly in their relative sensitivity to nisin. Some strains of L. monocytogenes may be inhibited by low concentrations of nisin (below 200 IU ml¹) whereas others are relatively resistant and require much higher amounts of nisin to be inhibited (Ferreira & Lund 1996; Ukuku & Shelef 1997). Even within a population of sensitive bacteria, some cells show elevated resistance to nisin. Spontaneous nisin-resistant variants of L. monocytogenes are reported to occur at a relatively high frequency (Harris et al. 1991; Ming & Daeschel 1993; Davies & Adams 1994). Even a single exposure to nisin may result in the emergence of a nisin-resistant population. Therefore it cannot be excluded that nisin-resistant variants of L. monocytogenes arise from widespread application of nisin in foods. On the other hand, the resistance problem may be minimized by the use of nisin in combination with other food preservatives or preservation techniques. Low pH, increased salt concentration or modified atmosphere packaging are examples of additional 'hurdles' in foods to be overcome by the target bacteria. The application of nisin in combination with such preservation measures may reduce the risk of emergence of nisin-resistant populations of L. monocytogenes.

Another approach could be the use of starter or protective cultures producing antilisterial bacteriocins active against nisin-resistant cells of L. monocytogenes. Numerous studies have shown that bacteriocin-producing lactic acid bacteria are able to suppress growth of L. monocytogenes in meat and other foods (Schillinger et al. 1991; Foegeding et al. 1992; Degnan et al. 1992; Maisnier-Patin et al. 1992; Winkowski et al. 1993; Stiles 1996). There are, however, no studies on the effectiveness of bacteriocinogenic cultures towards nisin-resistant mutants of L. monocytogenes.

The objective of this study was to investigate whether the emergence of nisin-resistant mutants of L. monocytogenes Scott A can be prevented by the addition of a bacterial strain producing a non-nisin antilisterial bacteriocin.

MATERIALS AND METHODS

Nisin

Nisaplin, a commercial product of nisin that contained 10⁶ IU g¹, was provided by Aplin & Barret Ltd (Trowbridge, UK). Stock solutions of nisin were prepared by dissolving 100 mg of Nisaplin in 10 ml of 0·02 N HCl. The pH was adjusted to 2·0 with 1 N NaOH. The solutions were subjected to heating at 100 °C for 10 min and then stored at - 20 °C.

Bacterial strains and culture media

Lactobacillus sake Lb 706 (Schillinger & Lücke 1989) and Enterococcus faecium BFE 900 (Franz et al. 1996) were used as bacteriocinogenic protective cultures and L. monocytogenes Scott A as target organism. In some experiments, a bacteriocin-negative mutant of Lact. sake Lb 706 (Lb 706-B) was included (Schillinger & Lücke 1989). Lactobacillus sake DSM 20017 was used as indicator strain for the determination of bacteriocin activity. Listeria monocytogenes was grown in Standard I Nutrient broth (Merck) and the lactic acid bacteria were cultivated in MRS broth (Merck) at 30 °C. The cultures were maintained as frozen stocks at - 20 °C in 15% glycerol.

Preparation of sakacin A and enterocin B and bacteriocin assay

Partial purification of the bacteriocins sakacin A from Lact. sake Lb 706 and enterocin B from Ent. faecium BFE 900 was performed as described by Keppler et al. (1994) for carnocin 54. In the present purification protocol, Amberlite XAD-7 (Fluka, Switzerland) was used instead of Amberlite XAD-2 and the concentrated bacteriocin solution obtained after rotary evaporation was adjusted to pH 5·0 with ammonia. Bacteriocin activity was tested using the agar spot assay described previously (Schillinger et al. 1993).

Determination of Minimum Inhibitory Concentrations (MIC)

The minimum inhibitory concentration (MIC) of nisin, sakacin A and enterocin B was determined for L. monocytogenes Scott A and mutants derived from this strain. Serial 1:1 dilutions of the respective bacteriocin were prepared using Standard I Nutrient broth in microtitre plates and each well was inoculated with about 10⁶ cells ml¹. The MIC was recorded as the lowest concentration of the bacteriocin which prevented visible growth after incubation for 20 h at 30 °C.

Sensitivity of Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900 to nisin and generation of nisin-resistant derivatives

Sensitivity of Lact. sake Lb 706 and Ent. faecium BFE 900 to nisin was determined by growing them in MRS broth containing various concentrations of nisin for 20 h at 30 °C. In order to generate nisin-resistant mutants, Lact. sake Lb 706 and Ent. faecium BFE 900 were inoculated at 1% in MRS broth. Following incubation for 24 h at 30 °C, cultures were serially transferred to MRS broth containing increasing concentrations of nisin in 100 IU ml¹ steps. Transfers were carried out every 24 or 48 h or as soon as turbidity indicated growth. Turbid growth cultures with 500 IU of nisin ml¹ were streaked onto MRS agar plates with 500 IU of nisin added. Colonies were isolated and checked for nisin resistance by determining the MIC. Bacteriocin production was tested by using culture supernatant fluids in the agar spot assay.

Alternatively, nisin-resistant mutants were obtained by direct streaking of overnight cultures onto MRS agar plates containing 300 IU of nisin. Several colonies isolated from these plates were transferred to MRS broth containing 300-500 IU of nisin ml¹, and after incubation at 30 °C for 24-48 h, they were checked for nisin resistance (300 IU ml¹) and bacteriocin production.

SDS PAGE electrophoresis of whole cell proteins of those isolates was done to confirm the identity of the derivative and parental strains (Pot et al. 1994).

Stability of the nisin-resistance phenotype

The stability of the nisin-resistant derivatives of Lact. sake Lb 706 and Ent. faecium BFE 900 was determined as described by Breidt et al. (1993). Nisin-resistant cultures of Lact. sake Lb 706 and Ent. faecium BFE 900 were transferred daily for 14 d in MRS broth in the absence of nisin, with a 1% inoculum at each transfer. Samples were withdrawn every second day and frozen at - 20 °C in MRS broth containing 15% glycerol. At the end of this 14 day period, growth of each sample in the presence of nisin was tested using an automated turbidometric system, BIOSCREEN C (Labsystems, Helsinki, Finland) according to Holck et al. (1996). Honeycomb wells with MRS broth containing 300 IU of nisin ml¹ were inoculated with the cultures at 1% and were incubated for 24 h at 30 °C. The non-resistant parental cultures and MRS broth without nisin were included as controls.

Bactericidal effect of nisin against Listeria monocytogenes Scott A

Standard I Nutrient broth was supplemented with nisin at a concentration of 10, 50, 100 and 500 IU ml¹ and was inoculated with about 10⁸ viable cells of L. monocytogenes Scott A ml¹. Numbers of survivors were determined after incubation for 2 h and 24 h at 30 °C.

Isolation of nisin-resistant mutants of Listeria monocytogenes Scott A

Listeria monocytogenes Scott A (10⁵ cfu) were inoculated into 1 ml Standard I Nutrient broth containing 100 IU of nisin. After incubation for 3 h at 30 °C, diluted samples were plated on Standard I Nutrient agar plates. Seven colonies were isolated from these plates, transferred into nisin-free broth and re-tested for nisin susceptibility by determining the MIC. To test stability of the nisin resistance phenotype of isolate Li3, the culture was transferred daily for 6 d in nisin-free Standard I Nutrient broth. Samples from every day were tested for nisin resistance by determining the MIC value.

Inhibition of Listeria monocytogenes by nisin in combination with a nisin-resistant derivative of Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900

Standard I Nutrient broth, pH 7·5 and with 100 IU of nisin ml¹ added, was inoculated with L. monocytogenes Scott A or Li3 at an initial level of either 10⁴ or 10⁶ cfu ml¹, and with Lact. sake Lb 706-1a or Ent. faecium BFE 900-6a at about 10⁵-10⁶ cfu ml¹. Incubation was at 30 °C for 48 h. Bacterial viable counts were determined after different time intervals using MRS agar for Lact. sake and Ent. faecium, and PALCAM agar (Merck) for Listeria. In some experiments, a bacteriocin-negative mutant of Lact. sake Lb 706 (Lb 706-B) was included for comparison, and pH and bacteriocin activity of the supernatant fluids were determined after different time intervals by using the critical dilution method (agar spot assay). All experiments were done in duplicate.

RESULTS

Isolation of nisin-resistant mutants of Listeria monocytogenes Scott A

Exposure of L. monocytogenes Scott A to increasing concentrations of nisin resulted in an initial decrease of viable counts followed by a resurgence of growth to high cell numbers (Fig. 1). Even at the highest nisin concentration of 500 IU ml¹, Listeria numbers did not remain at a low level below 10 cfu ml¹ but increased to 6 10⁴ cfu ml¹ within 24 h and to 10⁸ cfu ml¹ after 48 h. Nisin was not inactivated by the listerial cells as the culture supernatant fluid was still active against L. monocytogenes Scott A. Surviving cells of Listeria were obviously able to grow in the presence of nisin, indicating resistance to nisin levels of 100-500 IU ml¹. From a culture of L. monocytogenes Scott A exposed to 100 IU of nisin ml¹, seven survivors were isolated and their resistance to nisin determined. For six of them, a two- to threefold increased MIC of nisin was found. The seventh strain (Li3) was four times more resistant to nisin than the parental strain and was chosen for the further experiments. Several transfers in media without nisin did not reduce the resistance level of this strain.

Sensitivity of Listeria monocytogenes Li3 to sakacin A and enterocin B

Determination of the MIC of sakacin A from Lact. sake Lb 706 and enterocin B from Ent. faecium BFE 900 for L. monocytogenes Li3 showed that this nisin-resistant mutant was still susceptible to both non-nisin bacteriocins (Table 1). Listeria monocytogenes Scott A and its derivative Li3 did not differ in their sensitivity to sakacin A and enterocin B. The finding that L. monocytogenes Li3 had not acquired resistance to sakacin A is also supported by the observation that inhibition zones of about the same size were produced by Lact. sake Lb 706 against L. monocytogenes Scott A and Li3 in the agar spot assay.

Isolation of nisin-resistant mutants of Lactobacillus sake and Enterococcus faecium

Lactobacillus sake Lb 706 and Ent. faecium BFE 900 were both sensitive to low nisin concentrations (below 100 IU ml¹). Lactobacillus sake Lb 706 was inhibited even by 10 IU of nisin ml¹. Nisin-resistant mutants of Lact. sake Lb 706 and Ent. faecium BFE 900 were obtained by step-wise exposure to increasing concentrations of nisin. These mutants were able to grow in the presence of 500 IU of nisin ml¹ and were still able to produce the respective bacteriocin. The stability of the resistance phenotype was determined for three mutants each of Lact. sake Lb 706 and Ent. faecium BFE 900. All of the mutants tested retained the nisin resistance trait for at least 100 generations in the absence of nisin selection. The mutants did not differ in their whole-cell protein pattern obtained by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) from the original cultures of Lact. sake Lb 706 and Ent. faecium BFE 900.

Inhibition of Listeria monocytogenes by a combination of nisin and a nisin-resistant culture of Lactobacillus sake and Enterococcus faecium

When Lact. sake Lb 706-1a, a nisin-resistant mutant of Lact. sake Lb 706, was added to a culture of L. monocytogenes Scott A exposed to nisin at a concentration of 100 IU ml¹, the re-growth of survivors to nisin was inhibited at 30 °C (Fig. 2a). In the presence of Lact. sake Lb 706-1a, Listeria viable counts decreased to a level below 100 cfu ml¹, compared with an increase in Listeria viable numbers to 3 10⁸ cfu ml¹ after 48 h observed in the absence of the bacteriocin-producing Lactobacillus. Similar results were obtained using the nisin-resistant derivative of Ent. faecium BFE 900 as protective culture. Enterococcus faecium BFE 900-6a also prevented restoration of listerial cells surviving nisin amounts of 100 IU ml¹ (Fig. 2b). In the presence of the Enterococcus strain, L. monocytogenes numbers remained below 10⁴ cfu ml¹ throughout the experiment.

The inhibitory effect of Lact. sake Lb 706-1a and Ent. faecium BFE 900 was also investigated in combination with nisin against the nisin-resistant L. monocytogenes Li3. In the presence of 100 IU of nisin ml¹, L. monocytogenes Li3, after a short lag phase, grew rapidly to a high cell density (Fig. 3a,b). This increase in viable numbers was, however, inhibited by both bacteriocinogenic protective cultures inoculated at a level of 10⁶ cfu ml ¹. With Lact. sake Lb 706-1a added, almost no increase in viable counts of L. monocytogenes Li3 was oberved after 24 h and the numbers decreased to a low level after 48 h (Fig. 3a). Enterococcus faecium BFE 900-6a was somewhat less effective than Lact. sake in controlling the growth of L. monocytogenes Li3. It did not prevent an increase of Listeria viable counts by almost 2 log cycles within 24 h (Fig. 3b). Figure 3(a,b) also shows that viable numbers of the nisin-sensitive L. monocytogenes Scott A were kept below 10 cfu ml¹ by Lact. sake Lb 706-1a and Ent. faecium BFE 900-6a.

Thus, the bacteriocinogenic cultures were effective in suppressing growth of both L. monocytogenes Scott A and the nisin-resistant mutant Li3.

In a similar experiment, bacteriocin amounts produced by Lact. sake in a mixed culture with L. monocytogenes Li3 were determined. As a control, a bacteriocin-negative mutant of Lact. sake, Lb 706-B, was included. This derivative of Lact. sake Lb 706 did not produce sakacin A during growth in mixed culture with L. monocytogenes Li3 in Standard I Nutrient broth, whereas Lact. sake Lb 706-1a released large amounts of sakacin A into the medium (Fig. 4b). Growth of L. monocytogenes Li3 was more efficiently suppressed by the sakacin A producing culture than by the bacteriocin-negative variant (Lb 706-B) of Lact. sake, indicating that sakacin A contributed to the inhibition of Listeria (Fig. 4a). The bacteriocin-negative mutant of Lact. sake grew to a lower cell density in Standard I Nutrient broth than Lact. sake Lb 706-1a. Its growth, however, resulted in the same acidification effect. With both strains of Lact. sake, pH dropped to the same level at about the same rate.

Comparison of the growth behaviour of L. monocytogenes Li3 in mixed culture with Lact. sake with and without nisin showed that the inhibitory effect of Lact. sake Lb 706-1a towards Listeria was not more pronounced in the presence of nisin. About the same reduction in viable counts was caused by Lact. sake with nisin added as without nisin (Fig. 5). Exposure to nisin obviously did not render L. monocytogenes Li3 more sensitive to sakacin A produced by Lact. sake Lb 706-1a.

FIGURES

Table 1 MIC of Listeria monocytogenes Scott A and Li3 for nisin (IU ml¹), sakacin A (AU ml¹) and ente...

Fig. 1 Bactericidal effect of various concentrations of nisin against Listeria monocytogenes Scott A a...

Fig. 2 Growth of Listeria monocytogenes Scott A and Lactobacillus sake Lb 706-1a (a) or Enterococcus f...

Fig. 3 Growth of Listeria monocytogenes Scott A and its nisin-resistant derivative Li3 co-cultured wit...

Fig. 4 Growth of Lactobacillus sake Lb 706-1a (bacteriocin-positive), Lb 706-B (bacteriocin-negative) ...

Fig. 5 Influence of nisin (100 IU ml¹) on the growth of Listeria monocytogenes Li3, Lactobacillus sake...

DISCUSSION

Although L. monocytogenes is known to be sensitive to nisin, populations of Listeria frequently contain spontaneous nisin-resistant mutants. Harris et al. (1991) detected mutants of L. monocytogenes resistant to 2000 IU of nisin ml¹ at a frequency of 10⁶-10⁸. Ming & Daeschel (1993) observed resistance frequencies of about 10⁶ for L. monocytogenes Scott A when nisin was added at a level of 400 IU ml¹. The results of our study indicate that mutants resistant to 100 IU of nisin ml¹ occur at a higher frequency. Exposure of 8 10³ viable cells of L. monocytogenes Scott A to 100 IU of nisin did not result in inactivation of all listerial cells as after an initial decrease of viable numbers below 10 cfu ml¹, re-growth was observed (Fig. 1). At least one out of 8 10³ bacteria must have survived nisin action and as a consequence, mutants of L. monocytogenes Scott A resistant to 100 IU of nisin ml¹ can be expected to occur at a frequency of 10³-10⁴.

Different mechanisms may be responsible for the development of resistance. Production of a nisinase seems not to be involved in the resistance of Listeria to nisin (Mazzotta & Montville 1997). Alterations in the cell membrane composition resulting in the inability of nisin to form pores in the membrane may contribute to the resistance of spontaneous mutants of L. monocytogenes (Ming & Daeschel 1993, 1995; Verheul et al. 1997; Mazzotta & Montville 1997). In a mutant of L. monocytogenes Scott A resistant to 2000 IU of nisin ml¹, a higher percentage of straight-chain fatty acids and a lower percentage of branched-chain fatty acids was found (Ming & Daeschel 1993), while the amounts of three different phospholipids in the cytoplasmic membrane were significantly decreased in this mutant (Ming & Daeschel 1995). Similarily, a mutant of L. monocytogenes Scott A which was about 12 times more resistant to nisin than the parental strain contained a reduced amount of diphosphatidylglycerol in the membrane (Verheul et al. 1997). Studies with a nisin-resistant mutant of another strain of L. monocytogenes (F6861) indicated an involvement of the cell wall in the acquisition of nisin resistance (Davies et al. 1996).

Our results indicate that spontaneous mutants of L. monocytogenes surviving certain concentrations of nisin can be inhibited by the addition of bacteriocinogenic protective cultures such as the sakacin A producing strain of Lact. sake or the enterocin B producing Ent. faecium.

A nisin-resistant mutant of L. monocytogenes Scott A which had been obtained following a single exposure of L. monocytogenes Scott A to 100 IU of nisin ml¹ tolerated nisin concentrations of 100-500 IU ml¹. This mutant was able to grow to a high cell density in the presence of 100 IU of nisin ml¹ and was still sensitive to sakacin A and enterocin B. No cross-resistance to these non-nisin bacteriocins was acquired. These results agree with those obtainedby Rekhif et al. (1994) who found that spontaneous nisin-resistant mutants of L. monocytogenes ATCC 15131 were still sensitive to all three different bacteriocins investigated in their study. On the other hand, Song & Richard (1997) reported that exposure of L. innocua Lin11 to 20 IU of nisin ml¹ resulted in survivors that displayed cross-resistance towards nisin, pediocin AcH and enterococcin EFS2. The nisin-resistant mutant L. monocytogenes Li3 was inhibited by both bacteriocinogenic LAB under study when grown in mixed culture in Standard I Nutrient broth. The inhibitory effect of Lact. sake was due to a combination of pH reduction and the release of large amounts of sakacin A detectable in the culture supernatant fluids after different growth periods. A bacteriocin-negative variant of Lact. sake also had some inhibitory effect on growth of L. monocytogenes Li3 that can be attributed to the production of organic acids and the concomitant pH reduction.

Our results obtained with nisin-resistant derivatives of Lact. sake Lb 706 and Ent. faecium BFE 900 added to a culture of L. monocytogenes Scott A in combination with nisin showed that those non-nisin bacteriocin producing cultures are in fact able to prevent re-growth of a nisin-resistant subpopulation of L. monocytogenes. Addition of Lact. sake Lb 706-1a or Ent. faecium BFE 900-6a to L. monocytogenes Scott A exposed to 100 IU of nisin ml¹ resulted in suppression of survivors. The bacteriocinogenic strains thus assured a long-term antilisterial effect.

Inhibition of the nisin-resistant strain of L. monocytogenes (Li3) by Lact. sake Lb 706-1a and Ent. faecium BFE 900-6a was also demonstrated in the presence of nisin. Growth of L. monocytogenes Li3 was hardly affected by the addition of nisin (100 IU ml¹) but was controlled by both bacteriocinogenic strains. However, the combination of nisin with sakacin A produced in situ by Lact. sake Lb 706-1a did not result in an enhanced inhibitory effect against L. monocytogenes Li3 compared with the sakacin A producer alone.

Our results indicate that a combination of nisin and a starter or protective culture producing a non-nisin bacteriocin may be effective in preventing the emergence of spontaneous nisin-resistant subpopulations of L. monocytogenes. The effectiveness of the bacteriocinogenic LAB may be affected by the type of strain, its inoculum size, the medium used as well as pH and temperature. Finally, the behaviour of the protective cultures in food systems has to be investigated. The influence of these factors on the inhibition of nisin-resistant L. monocytogenes is currently being studied.

ACKNOWLEDGEMENTS

This study was supported by funds from the European Commission (FAIR-CT96-1148). The authors would like to thank H. Schäfer for excellent assistance.

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Use of bacteriocinogenic lactic acid bacteria to inhibit spontaneous nisin-resistant mutants of Listeria monocytogenes Scott A