Adherence of Listeria monocytogenes strains to stainless steel coupons

Authors


D. E. Norwood, Department of Food Science (Food Microbiology), The Queens University of Belfast, Newforge Lane, Belfast BT9 5PX, UK (e-mail: dnorwood@alpha1.dani.gov.uk).

Abstract

An assay was developed to measure the number of Listeria monocytogenes cells adhering to stainless steel, and was used to investigate the adherence of 111 strains of the organism, which included representatives with respect to serotype, carriage of plasmids, source and persistence in the food processing environment. Growth and adherence curves of four L. monocytogenes strains over 48 h were obtained. While the growth curves of all four micro-organisms were seen to reach similar levels at stationary phase, there was still substantial variation among the adherence curves. In addition, a scatter-graph of growth vs adherence counts at 24 h showed poor correlation. These factors indicated that interstrain variation in adherence at stationary phase is due to factor(s) intrinsic to each strain of L. monocytogenes. Persistent strains were found to adhere in significantly greater numbers than sporadic strains, and variation was also found among serotypes, with serotype 1/2c showing significantly greater adherence than serotypes 1/2a and 4b; 4b strains were significantly higher than those of 1/2a strains. No significant difference was found between strains according to source or plasmid carriage.

The food-borne pathogen Listeria monocytogenes is of particular concern to the food processing industry because of its ability to grow at refrigeration temperatures (Gray & Killinger 1966) and its tolerance of preservation agents ( Shahamat et al. 1980a; 1980b). Food has been shown to be the primary mode of the transmission of L. monocytogenes, being implicated in numerous food-borne disease outbreaks (Schlech et al. 1983; Linnan et al. 1988; Farber & Peterkin 1991). This pathogen has been isolated from an extensive range of food products including soft cheeses (Pini & Gilbert 1988), fish products (Eklund et al. 1995), vegetables (Breer & Baumgartner 1992), milk (Harvey & Gilmour 1992), paté and other cooked meat products (Gilbert et al. 1993). It is believed that contamination of these food products by L. monocytogenes occurs during post-process procedures rather than being due to survival during the processing itself (McLauchlin 1987). In almost all cases of listeria-contaminated products, L. monocytogenes can be isolated from the environment of the food plants from which the products originated (Ryser & Marth 1991; Jacquet et al. 1993). It has been found in drains, standing water, residues, food-contact surfaces and floors (Cox et al. 1989).

The capacity of pathogens to adhere to surfaces has been well documented (Frank & Koffi 1990; Hood & Zottola 1997). Food processors have always relied on physical and chemical methods to eliminate micro-organisms from these surfaces. However, research in this area has indicated that adherent micro-organisms may be much more resistant to sanitizing compounds than planktonic cells. Frank & Koffi (1990) showed that L. monocytogenes adhering to glass survived more than 10 times longer than free-living cells when exposed to benzalkonium chloride, anionic acid sanitizer or heat. Also, Lee & Frank (1991) found that L. monocytogenes adhering for 8 d were 100 times more resistant to hypochlorite than those adhering for only 4 d. Of concern to the food industry is the fact that these adhering pathogens may detach and contaminate food present on that surface or, if a non-processing surface is colonized, will have the potential indirectly to contaminate foods being processed.

The present study was designed to investigate the adherence capabilities of a broad range of L. monocytogenes strains. Strains were selected to represent a variety of characteristics, i.e. serotype, plasmid carriage, source, and evidence of persistence in the food-processing environment.

Materials and methods

Micro-organisms

A total of 111 L. monocytogenes strains were used in this adherence survey. Of these, 38 were obtained from the strain collection related to a recent international comparative study of L. monocytogenes typing methods (Caugant et al. 1996), 30 were obtained from a survey of milk and milk products (Harvey & Gilmour 1992), 33 were obtained from a survey of various non-dairy foods (Harvey & Gilmour 1993), and the remaining 10 were miscellaneous strains from various sources. During the raw milk and food surveys referred to above, a number of strains were seen to recur in sequential samples from the same food processing establishments and farm environments over a period of months, and these were referred to as persistent strains (Harvey & Gilmour 1994). The other strains from this source were isolated only once throughout the sampling period and were termed sporadic. In all, 35 persistent and 24 sporadic strains were used in this survey. Plasmid carriage was exhibited in 49 strains and 45 were clinical and veterinary strains. Of the 111 strains, 89 had been fully serotyped giving the following numbers: 1/2a, 39, 1/2b, 6,1/2c,11, 4b, 28, 3b, 2 and one each of 4c, 4d and 4e.

Test surface

Stainless steel was the surface chosen as it is used extensively throughout the food processing industry. Flat, stainless steel coupons (grade 304, 0·9 gauge, 2 × 2 cm) were used as the test surface. The coupons were initially soaked in acetone overnight to remove any grease. There then followed a cleaning procedure which was repeated each time the coupons were required to be re-used. After soaking the steel coupons in 2% stericol solution overnight (to destroy attached L. monocytogenes cells), they were placed in a Decon FS200 sonication bath (Decon Ultrasonics Ltd, Hove, UK) containing 5% Decon 90 detergent, and sonicated for 1 h to remove any bacterial and/or extracellular debris. The coupons were then thoroughly washed in tap water followed by three washes in distilled water. The steel coupons were finally autoclaved at 121 °C for 15 min.

Assay

The bead vortexing method of Oh & Marshall (1995) and Lindsay & von Holy (1997) for the removal and quantitative analysis of adherent micro-organisms was used after modification. Stainless steel coupons (2 × 2 cm) fitted neatly into 30 ml sterile plastic universals (Sterilin) so that both sides were available for bacterial adherence. Each L. monocytogenes strain was grown up from cryo-beads in full strength Tryptone Soya Broth (TSB; Oxoid) at 37 °C for 18 h, at which time late exponential phase was attained. A 10–3 dilution was then made and 100 μl inoculated into a sterile plastic universal containing 20 ml of 1/15 strength TSB (2 g l−1). A stainless steel coupon prepared as described above was then inserted and planktonic growth, coupled with adherence to the coupon, was allowed to take place for a 24 h period at 25 °C. Following incubation, the broth was carefully decanted from the plastic universal leaving only the steel coupon within the vessel. Using sterile forceps the coupon was then carefully removed, gently tapping it against the side of the vessel to remove excess liquid droplets, and transferred to a new sterile universal. Phosphate-buffered saline (PBS, 20 ml) was then slowly poured into this universal, the rising liquid level gently washing the coupon. Plate counts of the PBS were obtained at this stage and termed Wash Numbers (W) as shown in Fig. 1. Sterile glass beads (0·7 g, Sigma, 106 μm) were then added to the universal (containing coupon and PBS) and the universal vortexed for 2 min at maximum intensity on a Genie II (Scientific Industries, Bohemia, NY, USA) vortexer. Plate counts of the post-vortexed PBS were then made and termed the Vortex Numbers (V)(Fig. 1).

Figure 1.

Relative counts of reported assay using Listeria monocytogenes Scott A. TC, Transfer Constant; W, after 20 ml PBS wash; V, after bead vortexing for 2 min; X, number of adherent cells removed by 20 ml wash. For each value, n = 6

Transfer Constant

Ten strains were randomly selected and used to obtain a measure of the number of organisms transferred across to the new universal in small droplets still attached to the steel coupons, i.e. non-adherent micro-organisms. Each strain was incubated as before in the dilute TSB at 25 °C for 24 h, but this time the steel coupon was inserted into the TSB at the end of the 24 h incubation period and immediately removed to prevent bacterial adherence. The coupon was then placed into a sterile plastic universal containing 20 ml PBS as before, and a count obtained. The mean of the 10 transfer values, the so-called Transfer Constant (TC), could then be subtracted from V for each of the 111 strains to obtain the adherence value for each strain.

Growth and adherence curves

Growth and adherence curves of four L. monocytogenes strains, LP 2068, Scott A, TS 54 and MQ 2708, were obtained over 48 h at 25 °C. The assay described above was used to obtain the adherence levels in cfu cm–2 for each of the strains, while total viable counts were performed to obtain complimentary planktonic counts. In calculating cfu cm–2 of steel coupon, the coupon edges (1 mm) were deemed insignificant.

Bead vortexing efficiency

In order to obtain the cfu cm–2 steel coupon for the surveyed organisms accurately, the above assay assumed that all the attached organisms had been removed by the 2 min bead vortexing. The nucleotide specific DAPI (4′,6-diamidino-2-phenylindole) stain (Ross et al. 1996) was used on two of the strains of L. monocytogenes, LP2068 and Scott A, to measure the percentage of organisms removed by the bead vortexing method. The assay was performed on four coupons per organism, with the exception that half of the coupons did not undergo the 2 min bead vortexing. Before staining, the cells were fixed by quickly passing the steel coupon through a Bunsen flame three times. The coupons were then stained with 20 μl DAPI stain (0·95 mg l−1) and left for 30 min on ice in darkness. Subsequently, the coupons were gently washed by passing them slowly through a container of distilled water three times. Cell numbers were obtained by counting the number of cells in 20 randomly selected fields of view per coupon using an Olympus BX-50 fluorescent microscope with ultraviolet filter (Olympus Optical Co., UK).

Cell injury

The proportion of cells damaged by the 2 min bead vortexing was measured by plating out five bead-vortexed strains (each from a different serotype) onto Tryptone Soya Agar (TSA; Oxoid) and Listeria Selective Medium (Oxford formulation; Oxoid CM856 plus SR140E); these are complex and selective agars, respectively. The selective agar inhibits the growth of damaged cells using the selective agent, acriflavine, while TSA, a rich complex medium, allows cell membrane repair to occur. The proportion of cells injured can thus be calculated.

Data analysis

Unless stated otherwise, each experiment was replicated twice with triplicate analysis in each replication, giving six observations for each value. Numbers of cfu cm–2 were transformed to log10 for statistical analysis. The Students t-test (two samples assuming equal variance), and the anova, a single factor test, were performed on the results as explained below. Evaluations were based on a 5% significance level unless otherwise stated.

Results

Assay development

The assay methodology developed for this survey entailed the transfer of the steel coupon from the 24 h monoculture broth to a second universal, to which was added 20 ml PBS, with subsequent vortexing with glass beads to detach adhering cells. Figure 1 shows how the numbers of adherent and non-adherent organisms were distinguished for one of the 111 strains, L. monocytogenes Scott A. The total number of adherent cells (N) of the Scott A strains using the results displayed in Fig. 1 is

= V – TC
where V is the total number of organisms within the universal and TC is the Transfer Constant, which was calculated at 4·93 log10 cfu cm–2. This equation was used to measure the number of adherent cells for all 111 strains. W is the count in the 20 ml PBS after transfer from the 24 h broth and before bead vortexing. W was measured in all 111 strains to investigate the effect on adherent cells of using even a minimal wash. For all the strains V > W > TC.

Growth and adherence curves

The growth and adherence curves for LP 2068, Scott A, TS 54 and MQ 2708 are shown in Fig. 2 and the adherence curves are seen broadly to follow the shape of the growth curves, with the latter being generally 2·5 log cycles greater. At 21–22 h, the growth curves were seen to reach stationary phase at similar levels. The adherence levels, however, still showed substantial variation among the strains throughout stationary phase.

Figure 2.

Growth and Adherence best-fit curves for four Listeria monocytogenes strains incubated at 25 °C over 48 h. (—□—), LP2068; (–-○–-) Scott A; (–-▵–-) ST54; (–-•–-) MQ2708. Adherence counts were obtained every 3 h. Growth counts were obtained every hour for LP2068 and Scott A, and every 3 h for ST54 and MQ2708. Each point was the mean of six observations

A scatter-graph of growth vs adherence counts of all 111 strains at 24 h incubation was drawn to identify any possible correlation between planktonic growth and adherence levels. A low correlation coefficient of 0·334 was obtained.

Bead vortexing efficiency

Direct microscopic analysis was used to measure the percentage of organisms removed by the bead vortexing method using the DAPI stain on two strains of L. monocytogenes, LP 2068 and Scott A. By counting the number of cells per unit area before and after bead vortexing, means of 95·2% of LP 2068 cells and 97·1% of Scott A cells were observed to have been removed by the bead vortexing method.

Cell injury

The counts on the selective agar were seen to be consistently lower than those on the non-selective medium, giving a mean damage rate of 8·2% as shown in Table 1.

Table 1.  The percentage of non-injured cells of five Listeria monocytogenes strains following 2 min bead vortexing
L. monocytogenes strainPercentage of uninjured cells
TS 5490·6
TS 8091·3
TS 2499·0
FV 4490·6
TS 3587·4
Mean91·8

Persistent and sporadic strains

The adherence values of the 34 persistent and 25 sporadic strains of L. monocytogenes are shown in Fig. 3. The persistent strains range from 5·02 to 6·35 log10 cfu cm–2, while sporadic strains range from 4·76 to 6·23 log10 cfu cm–2. It is immediately apparent from these values that the upper and lower ranges of the persistent strains are both higher than the upper and lower ranges, respectively, of the sporadic strains. The mean of the persistent strain values is 5·67 log10 cfu cm–2, while that of the sporadic strains is 5·45 log10 cfu cm–2. Using the Students t-test for two samples, assuming equal variances, the two sets of data were analysed for significance. With a hypothesized mean difference of zero, P(two tail) = 0·041. The mean of the persistent strains is therefore significantly higher than that of the sporadic strains.

Figure 3.

The range of adherence values for persistent and sporadic strains. For each value, n = 6

Serotypes

Figure 4 shows the adherence values for each of the four main serotypes. The 39 1/2a strains ranged from 4·67 to 6·13 log10 cfu cm–2, the six 1/2b strains, from 5·46 to 6·11 log10 cfu cm–2, the 11 1/2c strains, from 5·11 to 6·35 log10 cfu cm–2, and the 28 4b strains, from 4·76 to 6·21 log10 cfu cm–2. The mean adherence values for serotypes 1/2a, 1/2b, 1/2c and 4b were 5·45, 5·68, 5·95 and 5·62 log10 cfu cm–2, respectively. Using the ANOVA, a single factor statistical test with a hypothesized mean difference of zero, the mean 1/2c value (P < 0·05) was significantly higher than that of 1/2a and 4b, and the mean of the 4b strains significantly higher than that of the 1/2a strains. The mean 1/2c value was also higher than that of 1/2b, although it fell just outside the 5% significance level.

Figure 4.

The range of adherence values for strains according to serotype. For each value, n = 6

Source and plasmid carriage

The 111 strains were obtained from three broad areas: meat, dairy and clinical sources. The differences in mean adherence values were shown to be non-significant after statistical analysis. The differences in mean adherence values for strains with plasmids and those without were also shown to be non-significant.

Discussion

The procedure used for ‘removing’ non-adherent bacteria with minimal interference to adherent micro-organisms has been an area of debate for many years, with various methods being applied (Frank & Koffi 1990; Mafu et al. 1990; Kim & Frank 1994; Oh & Marshall 1995; Hood & Zottola 1997). The method reported here has the advantage of containing both adherent and non-adherent micro-organisms within one system and has the means of distinguishing between them. In previous methods used for washing the surface, it had to be assumed that the washing method removed only non-adhering cells. Figure 1 shows that the one simple wash reported above actually does remove x adhering cells along with non-adherent cells, but the method is still able to distinguish between them. It is generally accepted that the adhesion of bacterial cells takes place in two main stages: a reversible followed by an irreversible adhesion, as suggested by Marshall et al. (1971). The reversible stage is dependent on physiochemical forces, while the irreversible stage is time-dependent and involves the production of extracellular material that is involved in the attachment of the bacteria to the surface. The definition of adherence used in this survey encompasses both stages of adherence and makes no attempt to separate them.

In the assay developed for this study, no washing of the steel coupon occurred until it was aseptically transferred from the 24 h incubation universal to the new universal into which the 20 ml PBS was carefully poured. The previously determined Transfer Constant could then be subtracted from the vortex number V, producing an accurate measure of the number of adhering cells for each strain of L. monocytogenes. The Transfer Constant (TC) is a measure of the number of organisms transferred across to the new universal in small droplets still attached to the steel coupons. This value will be dependent on the level of planktonic growth surrounding the steel coupons, which was seen to be analogous for L. monocytogenes strains at stationary phase. Furthermore, the transfer value of non-adherent organisms for the 10 control strains had a variance level four times lower than that of the adherence numbers of all 111 strains in the survey. Therefore, it can safely be concluded that the mean of the 10 transfer values, i.e. the Transfer Constant, was an accurate measure of non-adherent organisms being transferred by each steel coupon.

It is believed that starvation of micro-organisms leads to their increased adherence to a surface (Brown et al. 1977). Consequently, the medium chosen to culture the L. monocytogenes strains was dilute TSB (2 g l−1), being 1/15 normal TSB strength. Jeong & Frank (1994) showed that biofilm formed in 2 g l−1 TSB exibited a 10-fold greater population after 10 d of incubation than a biofilm formed in 10 g l−1 TSB.

An incubation time of 24 h was chosen for the adherence survey as adherence variation was seen to be largely independent of planktonic counts at this point in the growth curve.

In the growth and adherence curves of the four strains of L. monocytogenes, adherence was broadly proportional to numbers of planktonic cells in the surrounding environment. At stationary phase, however, growth curves reached analogous levels while there was still substantial variation among the adherence curves. This observation, combined with the low correlation coefficient of the growth vs adherence counts of the 111 strains, seems to indicate that interstrain adherence variation at stationary phase is due to factor(s) intrinsic to each strain of L. monocytogenes.

This study investigated the adherence levels of a broad range of L. monocytogenes strains to identify any possible adherence patterns with respect to serotype, carriage of plasmids, source, and persistence in the food processing environment. The predilection of L. monocytogenes strains to persist in various food processing environments with subsequent contamination of intermediate and final food products has been shown to occur frequently in the food processing industry (Busse 1990; Boerlin & Piffaretti 1991; Wesley & Ashton 1991; Harvey & Gilmour 1994). Boerlin & Piffaretti (1991) concluded that L. monocytogenes strains isolated from meat originated mainly from the processing environment, rather than directly from animals. Among the 111 strains tested were 34 persistent and 25 sporadic strains obtained from surveys of milk, dairy products and non-dairy foods by Harvey & Gilmour (1992, 1993). The mean of the persistent strains was significantly higher than that of the sporadic strains, and their greater adherence ability could be a factor responsible for the persistence of certain strains of L. monocytogenes in the food processing environment. The higher numbers would presumably be more difficult to remove using cleaning and sanitizing agents, especially if these cleaning procedures were not thorough and/or sub-optimal concentrations of sanitizers were used (Hood & Zottola 1995).

The vast majority of the serotyped strains in this survey belonged to serotypes 1/2a, 4b, 1/2c and 1/2b, in descending order. Only four of the strains were outside these serological groups, i.e. 3b, 4c, 4d and 4e. Although L. monocytogenes contains 13 distinct serological groups, 95% of all human isolates comprise the serotypes 1/2a, 1/2b, 1/2c and 4b (McLauchlin 1987). The statistical evaluations of the work reported here showed that the mean adherence of 1/2c strains is significantly higher than that of 1/2a and 4b strains, while the mean of the 4b strains was significantly higher than that of the 1/2a strains. The 1/2c serotype is found in only 4% of human cases, while serotype 4b is found in 59% of human cases (McLauchlin 1987). Thus, while 4b is the dominant serotype in human cases, serotype 1/2c is a minor player. If it is assumed that the results are representative of the whole L. monocytogenes population, then the higher adherence of 1/2c strains clearly do not manifest themselves in increased 1/2c listeriosis cases. It is interesting to note, however, that the most important serotype with respect to listeriosis, 4b, showed significantly greater adherence than the second most important serotype, 1/2a.

No significant variation in adherence numbers occurred between strains with plasmid carriage and those without, which would appear to suggest that any genetic coding for differential adherence does not occur on a majority of these plasmids. Also, no significant variation in adherence values was found between the three main sources of strains, meat, dairy or clinical; this is not surprising as the strains can be transferable (Schlech et al. 1983; Linnan et al. 1988).

Acknowledgements

This work was supported by a studentship from the Department of Agriculture for Northern Ireland.

Ancillary