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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

M.R. BERESFORD, P.W. ANDREW AND G. SHAMA. 2001.

Aims: To investigate the adhesion of Listeria monocytogenes 10403S to 17 different, food-use approved materials representing metals, rubbers and polymers.

Methods and Results: Adhesion assays were conducted by placing ‘coupons’ of the materials in planktonic cultures at 30°C, and then immediately withdrawing them (‘short contact’) or leaving them submerged in the cultures for 2 h. Adherent cells were recovered by sonication. In the short contact experiments, the logarithm of the mean viable counts ranged from 3·67 ± 0·43 to 4·78 ± 0·38. After 2 h contact time, the numbers of adherent cells had increased significantly for all materials with the exception of polypropylene. The highest count (6·33 ± 0·31) recorded was for stainless steel 405.

Conclusions: Adhesion to a wide range of materials was time-dependent and characterized by reversible and irreversible stages.

Significance and Impact of the Study: Adhesion test protocols must account for cell carry-over and cells which are only weakly bound. Material selection may only have a limited role in reducing food contamination by listeria.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Listeria monocytogenes occurs widely in the environment and has been isolated from a range of sources including vegetables, processed foods, silage and soil (Cox et al. 1989). The bacterium is the causative agent of listeriosis in humans and animals, and food represents a major source of infection (Carpentier and Cerf 1993; Zottola 1994; Hood and Zottola 1995). The threat posed by L. monocytogenes is to some extent a function of its ability to grow over a broad temperature range. This is made possible by the bacterium’s ability to modify its membrane composition in order to maintain membrane fluidity (Jones et al. 1997).

Many different types of food have been implicated in outbreaks of listeriosis. For example, in 1985, consumption of Mexican-style cheese was directly linked to over 142 cases of listeriosis, including 48 deaths (Linnan et al. 1988), and between 1983 and 1987, consumption of contaminated Vacherin Mont d’Or soft-ripened cheese resulted in 31 deaths (Malinverni et al. 1985). In the UK, it was concluded that paté was a contributory cause of the increase in the incidence of listeriosis between 1987 and 1989 (Mclauchlin et al. 1991). Bacteria have been shown to enter foods as a result of contact with a contaminated surface (Eginton et al. 1995). However, the cleaning of surfaces in food-processing facilities is rendered difficult because of the ability of L. monocytogenes to form biofilms (Zottola 1994). Cells in a biofilm have been shown to be significantly more resistant to disinfectants and sanitisers than planktonic cells (Ronner and Wong 1993; Arizcun et al. 1997). Much of the previous work on the attachment of L. monocytogenes to surfaces has tended to focus on only a small number of materials, typically stainless steels and only one type of rubber (Buna-N), and no large comparative studies of its adhesion to a wider range of materials present in food-processing environments have been reported.

In this paper, listerial adhesion to a broad range of materials used to manufacture vessels, pipe-work, cutting surfaces, gaskets and conveyer belts, all of which are commonly found in food-processing facilities, is reported. The materials included stainless steels, rubbers and polymers. Adhesion to these materials was assessed after a short period and also after two hours. As part of the investigation, potential sources of error in previously published methods for determining microbial adhesion were explored.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

Preparation of coupons

The materials tested are listed in Table 1. All test materials were cut into flat coupons (0·8 × 0·8 cm) from sheets, the thickness of which is also shown in Table 1. Coupons were placed in a sterile universal tube (eight coupons per tube) containing 10 ml 1% (v/v) Teepol solution. These tubes were then sonicated for 15 min in a bath sonicator (Kerry Ultrasonics Ltd, Hitchen, UK). After sonication, the coupons were washed in 20 ml sterile distilled water, transferred to a glass universal tube and autoclaved at 121°C for 15 min.

Table 1.   Test materials Thumbnail image of

Growth conditions and media

Listeria monocytogenes strain 10403S (Portnoy et al. 1988) was grown on Luria agar (LA; 1% (w/v) tryptone (Lab M), 0·5% (w/v) yeast extract (Oxoid), 1% (w/v) sodium chloride, agar (Lab M), 2·1% (w/v)) at 30°C. Tryptone Soya Broth (TSB, Lab M) was prepared according to the manufacturer’s directions.

Listeria monocytogenes was grown statically in 20% (w/v) TSB at 30°C overnight. The overnight culture was then centrifuged at 1400 g at 4°C for 15 min. The cell pellet was resuspended 1% (v/v) in fresh sterile 20% (w/v) TSB. For use, cell suspensions were prepared to an O.D.600nm of between 0·15 and 0·2, which corresponded to cell counts of 5·2 × 108–8·6 × 108 cfu ml–1. Aliquots (10 ml) of cell suspension were then dispensed into universal tubes. For short contact experiments, coupons were completely immersed in culture previously raised to a temperature of 30°C, and then immediately withdrawn using forceps. For all other experiments, the coupons were incubated statically at 30°C for 2 h.

Detachment of L. monocytogenes from coupons

Following incubation, the coupons were removed from the universal tubes using sterile forceps to hold the coupon by two edges. Each coupon was touched lightly against the side of the tube to remove as much of the film of liquid adhering to the coupon as possible, before transferral to a Petri dish containing 10 ml sterile phosphate-buffered saline (PBS). The dish was swirled twice gently and the coupon removed with forceps. The coupon was then gently placed in 10 ml sterile 20% (w/v) TSB in a sterile plastic universal tube for sonication in a bath sonicator for 1 min. For enumeration of bacteria, 20 μl cell suspension were serially-diluted in PBS and plated on LA. The Miles and Misra (1938) plating technique was employed, with six 20 μl aliquots of sample being deposited onto the surface of a single agar plate using a micropipette. All samples were plated in replicates of four. Plates prepared in this way were incubated overnight at 30°C. Only dilutions which resulted in counts of between 30 and 150 colonies were enumerated.

A sample was removed before sonication to determine the numbers of viable planktonic bacteria, and after sonication to determine the sum of planktonic and adherent bacteria. Coupon thickness varied according to the material and consequently, counts were normalized by assuming that each coupon had an area of 128 mm2 (i.e. the equivalent surface area of a 0·8 × 0·8 cm coupon of infinitesimal thickness).

Estimation of mass of liquid adhering to coupons

Sterile coupons were immersed in the planktonic culture and removed immediately. The coupons were then placed onto pre-weighed 90 mm Whatman filter paper discs (‘School Grade’, Fisher Scientific, Loughborough, UK), allowing any adherent medium to soak into the paper. The papers were then weighed to determine the mass of liquid.

Statistical analysis

The data were analysed by analysis of variance and the Tukey–Kramer multiple comparison test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

The total number of listeria apparently associated with each material after a short contact time is shown in Table 2 (column 2). No significant difference was observed (P > 0·05). These numbers are made up of cells strongly adherent, those that readily detach into a planktonic population and those carried over in liquid adhering to the coupon. To limit liquid carry-over, washing coupons with PBS and drainage on filter paper were compared, because both methods are widely used in quantitative analysis of bacterial adherence to coupons (e.g. Mafu et al. 1991; Lindsay and Von Holy 1997). There was no significant difference (P > 0·05) in the carry-over between the two methods so washing in PBS was selected. It was found that, on average, only 4·9 μl of liquid were transferred by this method of handling the coupons (about three times the volume is transferred if the coupon is held by the corners). The coupon material did not significantly affect the volume transferred. Thus, carried-over listeria make up only a small fraction of the cells seen in the planktonic phase (in the worst case, only about 690 cells). As Table 2 shows, the number of cells re-entering the planktonic phase can represent a substantial fraction of the total population, in some cases as much as 51%. There was no significant difference between the number of listeria weakly or strongly adhering to the different materials after a short contact time (Table 2, columns 3 and 4, respectively).

Table 2.   Number of Listeria monocytogenes cells associated with coupons after a short contact time Thumbnail image of

Table 3 shows the number of cells adhering to all materials following a contact time of 2 h. There was no significant difference (P > 0·05) in the total number of listeria adhering to each material but for each, the number adhering had increased over 2 h (P < 0·05). Also, the percentage of the cells that were weakly adherent decreased over 2 h. When the number of weakly-adhering cells (those shed into the PBS) was subtracted from the ‘total’ number, the numbers adhering to polypropylene was found to be significantly lower (P < 0·05) than for the other materials. Indeed, the number of cells strongly adhering to this material after a 2 h contact period did not differ significantly from those obtained following only short contact.

Table 3.   Number of Listeria monocytogenes cells associated with coupons after 2 h incubation Thumbnail image of

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References

It has been shown before that L. monocyogenes will adhere to a wide variety of surfaces (Mafu et al. 1990; Helke and Wong 1994; Smoot and Pierson 1998). Some of these studies have been quantitative whereas others have been primarily descriptive. It is thought that this present study is the most comprehensive, quantitative analysis of the adherence of L. monocytogenes to materials encountered in the food-processing industry. Mafu et al. (1990) measured contact angles and surface energies for a restricted range of materials that coincided with those examined here. Given the range of surface properties of the coupons used in the present study, it was surprising that there were no differences in the degree of attachment that occurred either instantaneously or after 2 h contact time. Mafu et al. (1990) had reported the ability of L. monocytogenes to adhere to materials of widely differing hydrophobicity, but without quantification. These findings are confirmed and extended. The adherence ability reinforces the view of listeria as a micro-organism able to populate widespread niches.

Marshall et al. (1971) proposed a two-step, time-dependent model for adhesion of marine bacteria to a surface (glass). First, there was an instantaneous reversible adhesion, with only weak interactions occurring between the bacteria and the surface; this was followed by irreversible adhesion mediated by the formation of extracellular material. The data obtained in the present study show that this model is generally true for L. monocytogenes binding to a wide variety of materials. The data also support the notion of reversibly- and irreversibly-bound populations. However, the data apparently are at variance with the model of Marshall et al. in that reversibly- and irreversibly-bound populations are present instantaneously, and reversibly-bound cells continued to be found after 2 h contact time. However, in accordance with Marshall et al., the proportion of irreversibly-bound cells increases with time. Another view of the comparison of these data and those of Marshall et al. is that while they both support the concept of different populations of bacteria with different adherence strengths, it may be erroneous to conclude that there are just two populations. Thus, in our method where the washing was as gentle as possible, some bacteria were removed, but the irreversibly-bound bacteria were over-estimated. Marshall used a more vigorous washing procedure and therefore may have over-estimated the reversibly-bound fraction. Smoot and Pierson (1998) also examined short contact time adhesion and like Marshal et al., employed a relatively harsh washing procedure with possibly the same outcome. An alternative conclusion is that there are multiple populations with differing adhesive strengths, or that even a continuum may exist. This might occur if, following the initial cell–surface interactions, the strength of the bond formed between any cell and the surface increased with time in such a way that the force required for detachment was also time dependent. In this analysis, no attempt was made to quantify the adhesive forces that define each population. Descriptors such as ‘reversible’, ‘irreversible’, ‘strong’ and ‘weak’ are only operational terms within the limits of the assay used. For exact determination of the forces involved, test materials would need to be placed in well characterized shear fields. Bos et al. (1999) go further, and claim that it is essential to eliminate any manipulations, such as drawing coupons through a liquid–air interface, that might subject the biofilms to unquantifiable shears.

Although only semi-quantitative in terms of analysis of adhesive forces, tests in which samples or coupons of material are immersed in liquid culture and then withdrawn after a specified period of time in order to estimate the number of adherent cells, are valid and widely used for comparative testing of adhesion. However, although apparently simple in concept, care has to be exercised in the undertaking and interpretation of these tests. For example, failure to account for planktonic cells present in medium adherent to the coupon, or very weakly adherent bacteria, may lead to erroneous conclusions. Norwood and Gilmour (1999) reported on the adhesion of over 100 strains of L. monocytogenes to stainless steel. As in the present study, they discounted from their total counts cells present in the carried-over liquid. However, in their account of carry-over they also may have counted some weakly attached cells. They assumed that all bacteria present instantaneously were carried over in adherent liquid. However, when we measured the amount of adherent liquid, it was insufficient to account for the numbers of listeria associated with the coupons. Thus, it was concluded that some listeria become instantaneously bound.

At this stage there is no obvious explanation for the observation that numbers of listeria strongly adhering to polypropylene do not increase with time. Mafu et al. (1990) described the physical surface properties of polypropylene as lying between those of rubber and stainless steel, two materials to which L. monocytogenes binds equally well (this study; Helke and Wong 1994; Smoot and Pierson 1998). An explanation may come with a greater understanding of the mechanism of binding of L. monocytogenes to these various materials. The molecular basis of the capacity of L. monocytogenes to adhere to many materials is unknown, but it argues against a single mechanism. The identity of the listerial adhesins for the material discussed here is the subject of current research.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. References
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