DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

DETECTION OF LZSTERZA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

S. SHARMA,2 S. KASATIYA2 and J.M. FARBERlS3

I Microbiology Research Division Food Directorate, Health Protection Branch

Banting Building, Tunney’s Pasture Health Canada

Ottawa, Ontario KIA OL2

20ntario Ministry of Health Lab Services Branch, Ottawa Public Health Laboratory

2380 St. Laurent Blvd. Ottawa, Ontario KIG 5A4

Accepted for Publication September 13, 1994

ABSTRACT

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was dificult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes tophenyl Sepharose at pH 3.5 and 4 MNaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaC1. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. 7his study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk

3Corresponding author, tel: (613) 957-0895, Fax: (613) 941-0280, EMAIL: JFARBERaHPB. HWC.CA.

Journal of Rapid Methods and Automation in Microbiology 3 (1994) 151-162. All Righrs Reserved. 151 0 Copyright 1994 by Food & Nutrition Press, Inc., Tnunbull. Connecticuf.

IS2 S . SHARMA. S . KASATIYA and J.M. FARBER

and may be applicable to other food suspensions. It is a gentle method that makes use of the h.vdrophobic surface properties of Listeria for attachment to hydrophobic gels, as Miell as using mild elution conditions to aipoid inactivation of the organism.

INTRODUCTION

Listeria monocytogenes is a Gram-positive, rod-shaped bacterium which is an intracellular pathogen for both humans and animals (Sun et al. 1990; Tilney et al. 1989; Tilney et al. 1990). The organism is widely distributed in both the environment and foods (McLaucNin 1987; Farber 1991; Farber and Peterkin 1991) and has been the cause of several major listeriosis outbreaks associated with con- taminated foods (Fleming et al. 1985; Griffiths 1989: Farber and Peterkin 1991). The organism can survive and grow at refrigeration temperatures and is thus of major concern to public health (Farber 1991: Farber and Peterkin 1991).

This makes it necessary to develop rapid methods of isolation and confirmation to detect L. monocytogenes in foods. Although isolation by culture methods can be sensitive, it can also be time-consuming (Al-Zoreky et al. 1990). Enzyme immunoassays based on monoclonal antibodies (Farber and Spiers 1987) and nucleic acid hybridization assays (Datta el al. 1990; Kim et al. 1991) have been explored as alternatives for rapid detection and confirmation of Listeria spp. in foods. However, the sensitivity of these methods is still in the range of 105-106 cfu/g of food. Polymerase chain reaction (PCR) methods with improved sensitivity have been developed, but components of food matrices, such as inhibitors and enzymes, may affect the sensitivity of the PCR (Niederhauser et al. 1992).

The psychrotrophic nature of L. monocyfogenes, along with its ability to attach to inert surfaces has been shown to be a major problem, especially in the food industry (Herald and Zottola 1988; M a h et al. 1990, 1991a). The mechanisms of adhesion of L. monoqtogenes to inert surfaces are not clearly defined. Several studies have shown that adhesion of bacteria depends upon the nature of the inert surface as well as the surface properties of bacteria (Rosenberg 1981; Melander et al. 1989; Sorongon et al . 1991).

Bacterial adhesion has been described in physicochemical terms such as hydrophobicity (Dahlback et al. 1981 ; Magnusson 1982; Rosenberg and Kjellerberg 1987). surface energy (Absolom et al. 1983) and electrostatic interactions of cell particles with different matrices (Van Loosdrecht et al. 1987). The cell wall of L. monoqtogenes is composed of lipoteichoic acids (Sen and Dhan- da 1968; Ruhland and Fiedler 1987; Leopold and Fischer 1992), which are amphiphilic polymers. The lipid region anchors the polymers to the outside of the cytoplasmic membrane while the hydrophilic chain penetrates the cell wall and becomes a component of the cell surface (Methor et al. 1983; Fiedler and Ruhland

L. MONOCYTOGENES AND HYDROPHOBIC MATRICES 153

1987; Levy et al. 1990). Mafu et al. (1991a) examined the cell surface charge and hydrophobicity of L. monocytogenes and found the microorganism to be hydrophilic, but to also exhibit hydrophobic interactions and binding to octyl Sepharose at low pH.

The amphipathic properties of L. monocytogenes are therefore potentially useful for separating the microorganism from foods using inert hydrophobic supports. Through hydrophobic interactions, L. monocytogenes could be adsorbed from foods and then be eluted in a pure and viable form using mild conditions. Subse- quently, bacteria, free of contaminants and in an active and viable form, could be detected by PCR, or any other sensitive detection method. Thus, in this study, various conditions that affect the adsorption and elution of L. monocytogenes onto hydrophobic matrices were examined.

MATERIALS AND METHODS

Bacteria and Culture Conditions

Listeria monocytogenes strain Scott A (serotype 4b) was used in this study. Cultures were stored at 4C in a semi-solid medium consisting of meat extract (5.0 g), peptone (10.0 g), NaCl(3.0 g), Na2HP0412H20 (2.0 g) and agar (10.0 g), dissolved in 1 L of distilled water; final medium pH 7.4 (Institut Pasteur, Paris). Prior to each experiment, strains were plated onto Tryptose Agar (TA; Difco Laboratories, Detroit, MI), and incubated at 30C for 24 h.

The L. monocytogenes Scott A strain was grown on TA and then a colony transferred into 5 ml of Tryptic Soy Broth (Difco Lab) containing 0.6% yeast extract (Difco) in test tubes and incubated for 24 h at 30C. Bacteria were centrifuged (IEC-Centra TR International Equipment Company, Needham Height, MA) at 5,500 x g for 10 min, washed twice in salt peptone buffer [0.85% sodium chloride, 0.05% Bacto peptone (Difco)], centrifuged again, and the pellet sus- pended in buffer according to the method tested. Viable counts were done using standard plate count agar (Difco). Serial dilutions were performed by adding 0.1 ml of sample to 9.9 ml of salt peptone buffer, with 0.1 ml of the appropriate dilution being plated onto TA plates. The plates were incubated for 48 h at 30C and colonies counted.

Hydrophobic Interaction Chromatography

For hydrophobic interaction chromatography (HIC), the final pellet was resuspended in 5 ml of salt peptone buffer. In addition, all dilutions were done using salt peptone buffer except for the final dilution where 4 M NaCl (pH 3.5)

I54 S. SHARMA. S. KASATIYA and J .M. FARBER

was used, to give a final concentration of I x lo3 cells/nll. Bacterial numbers were estimated by serial dilution and direct plating onto TA.

HIC was carried out as previously described by Smyth et al. (1978). Octyl. phenyl and butyl gels as well as Sepharose C L 4 B were obtained from Pharmacia, Uppsala, Sweden. Hydrophobic derivatives of Sepharose were washed extensively with buffered 4 M NaCl (pH 3.5) to remove fine particles and preservative. A 2 ml sample of gel was equilibrated overnight at 4C in 3 ml of the same buffered NaCl solution. Solutions of NaCl were prepared by mixing an appropriate quan- tity of 0.1 M citric acid with 0.2 M Na2HP0, to obtain the required pH values. Solutions were adjusted with 1 .O M NaOH or 0.1 M citric acid. A 2.5-ml sample of equilibrated Sepharose gel (octyl, phenyl, butyl, or Sepharose CL4B) was added to a chromatographic column (Pharmacia LKB Biotechnology) (20 cm long and 16 mm wide) to obtain a final gel bed volume of 0.8 ml. The gel bed was washed extensively with 10 bed volumes of NaCl solution to remove any traces of ethanol.

A 1 .O ml sample of washed cells of L. r??onocyogenes (ca. 2 x lo2 cfuiml in 4 M NaCI. pH 3.5). prepared as described above, was introduced onto the gel bed. Bacteria were left in contact with the gel bed for 15 min, then washed three times with4 M NaCl (pH 3.5) to remove any loosely bound bacteria. Release of bacteria that adsorbed strongly to hydrophobic gels was attempted by decreasing the ionic strength. Therefore, desorption was performed by washing the gel bed with 1 ml of 10 mM sodium phosphate buffer, pH 6.8. Desorption was also tried with 10 ml of 10 mM sodium phosphate buffer, pH 6.8, then subsequently with 10 ml of l o%, 30% and 50% ethylene glycol. The unadsorbed, washed and eluted fractions as well as the gel, were collected, and viable counts of each frac- tion performed on TA as described above. The plates were incubated at 30C for 48 h and colonies counted with the aid of Henry’s illumination (Henry 1933).

Cell suspensions of L. monoqtogenes were also chromatographed under the conditions described above on Sepharose C L 4 B with no ligand attached, to control for possible nonspecific adsorption effects on the Sepharose gel beds.

Adsorption and Elution of Bacteria from Milk

A 100 ml sample of boiled 2% milk was adjusted to pH 3.5 and 4 M NaC1. A 0.9-ml sample of this milk was artificially inoculated with L. monocytogenes (final concentration; 1 x lo3 cellsiml) and then HIC was performed with the equilibrated phenyl Sepharose column as described above. Ten 1-ml fractions were collected and the adsorbed and eluted bacteria were plated onto a TA plate in order to perform viable counts. All experiments were repeated at least twice.


RESULTS

Adsorption of L. Monocytogenes to Hydrophobic Gels

L. monocytogenes bound to octyl Sepharose at 4 M NaCl, pH 3.5. In contrast, nearly all the bacteria came out in the first wash with underivatized Sepharose CL-4B, which does not contain any hydrophobic ligands (Table 1). Adsorption of bacteria onto octyl gels was also done at 1, 2, 3 and 4 M NaCl, pH 3.5 (Table 2). Maximum adsorption occurred at the highest salt level (4 M) tested. In initial experiments, phenyl Sepharose also exhibited good binding and retention of bacteria at 4 M NaCI, pH 3.5. With adsorption at 1 M (NHJ2S04, Listeria remained bound to the gel. Butyl Sepharose adsorbed bacteria, but the retention was very poor, with most of the bacteria coming out during washing (results not shown).

TABLE 1. ADSORPTION OF L. MONOCYTOGENES ONTO DIFFERENT SEPHAROSE GELS AT

4 M NaCI, pH 3.5

No. of No. of Gel type bacteria bacteria Wash3 Elution4 Gel'

applied' unadsorbed'

Octyl 298.5 75.5 24, 7.5. 2.5 37.5. 27.5, 1 1 83.5 Sepharose

Sepharose

Phenyl' 268.5 40 13, 9.5, 3 07, 44.5. 29.5, 12.5 3.5 Sepharose

' Data represent number of bacteria applied to the different Sepharose gels

Represents the number of bacteria which did not anach to Sepharose

The gel was washed three times with 1 ml of 4 M NaCI, pH 3.5. Each number represents total L. m o n o c v t m i m l of wash fluid.

Sepharose was washed three times wi th 1 ml of 10 mM sodium phosphate, pH 6.8. number represents total I;. monocvtoaeneslml of eluent.

Underivatized 278.5 34 186.5, 33, 15 1, 0, 0 0

Each

' Represents the number of bacteria remaining bound to the gel.

For phenyl Sepharose, one extra elution was done. Each number represents total L. monocvtooeneslgel.

156 S . SHARMA. S. KASATIYA and J.M. FARBER

TABLE 2 . EFFECT OF DIFFERENT SALT CONCENTRATIONS ON THE NUMBER OF L MONOCYTOGENES CELLS ADHERING TO OCTYL SEPHAROSE, pH 3.5

~ ~ u t i o n ~ Gel' NaCl No. of bacteria Wash3 concentration unadsorbed'

4 M 76 24, 11, 2.5 37.5. 29.5, 12.5 85

3 M 91 30, 13, 2.5 30, 28, 12.5 68

2 M 105.5 31, 14, 2 30.5, 20.5, 11.5 50

1 M 120.5 25.5. 16, 1 22.5, 18.5, 7 .5 35

' Ndmber of bacteria applied to the Sepharose pel was ca. 280.

Represents number of bacteria which did not bind to the Sepharose gel.

Three washes were performed with 1 ml each of 4 M NaCI, pH 3.5. Each number represents total .. monocvt0aenes:ml of wash fluid.

Bacteria were eluted out three times each with 1 ml of 10 mM sodium phosphate, pH 6.8. Each number represents total C. monocvtoaenes/ml of eluent.

Represents the number of bacteria remaining bound to the gel. *

Elution of L. Monocytogenes from Hydrophobic Gels

L. monocytogenes adsorbed onto octyl Sepharose at 4 M NaCl (pH 3.5) was washed three times with 1 ml each of 4 M NaCl (pH 3 . 3 , and eluted with 1 ml of 10 mh4 sodium phosphate, pH 6.8. However, substantial amounts of bacteria remained bound to the gels and did not elute. With underivatized Sepharose, most of the bacteria came out in the first wash (Table 1). With butyl Sepharose, some bacteria were removed during washing, some were eluted out with 1 ml of 10 mM sodium phosphate, whde others remained bound to the gel (results not shown).

With phenyl gels, the binding of bacteria in the presence of 1 M (NH4)*S04 was very tight, so that insignificant amounts of bacteria were eluted out when using 10 mM sodium phosphate, pH 6.8. Some bacteria were released from phenyl gels with adsorption at 4 M NaCl (pH 3.5) and elution with 1 ml of sodium phosphate (pH 6.8). Elution was also attempted at pH 8.0, 8.5 and 9.0. but pH 6.8 gave the best recovery (results not shown). When the volume of sodium phosphate (10 mM) was increased from 1 to 10 ml, most bacteria were eluted. A further increase in the volume to 30 ml did not result in a marked increase in the number of cells eluted and some bacteria remained adhered to the gel. To try and further detach Lisferia from the gel, a polar reducing agent was used (10 mi of 10, 30 and 50% ethylene glycol). All bacteria eluted from the phenyl gel using a sequential combination of sodium phosphate and increasing levels of up to 50% ethylene glycol (Table 3). Subsequent experiments demonstrated no added


TABLE 3. EFFECT OF ETHYLENE GLYCOL AND SODIUM PHOSPHATE ON THE NUMBER OF L. MONOCYTOGENES CELLS ELUTED FROM A PHENYL

SEPHAROSE COLUMN'.'

Ethylene Glycol4 10 mM Na Phosohate3 10% 30% 50%

5 9 4 3

4 5 6 2

13 2 1 3

15 6 3 2

21 4 3 1

22 2 2 0

23 2 1 0

13 2 3 1

5 1 1 1

9 1 2 0

34 26 13 Total 203 Total no. of 130 !&c&a eluted

' Milk was adjusted to a pH value of 3.5, NaCl 14 M) added and then the milk (2% milkfat) was artificially inoculated with 4. rnonocvtooenes.

Flow rate, 1 mllmin. Number of bacteria initially adsorbed to the gel was 208. Only 1 bacterium was found in the unadsorbed and wash fractions, and no bacteria remained on the gel.

Ten 1 ml fractions were collected.

Ten 1 ml fractions were collected for each ethylene glycol concentration.

benefit of using 70% ethylene glycol. As with 50% ethylene glycol, all bacteria could be detached and eluted in a viable form. A flow rate of either 1 or 2 ml/min gave good elution. To economize on time and reagents, a flow rate of 2 ml/min and elution conditions consisting of 10 ml of 10 mh4 sodium phosphate pH 6.8, and 10 ml each of lo%, 30% and 50% ethylene glycol, were selected for further study.

Removal of L. Monocytogenes from Milk

Milk was artificially inoculated with L. rnonocytogenes to a final concentration of approximately 2 x lo2 cells/ml. All the bacteria were adsorbed from 1 .O ml milk onto phenyl Sepharose at 4 M NaC1, pH 3.5. The elution of bacteria was done initially by using 10 ml of 10 mM sodium phosphate, pH 6.8, followed

158 S . SHARMA. S . KASATIYA and J . M . FARBER

by 10 ml each of l o % , 30% and 50% ethylene glycol at a flow rate of 2 ml/min. No bacteria remained on the gel after this elution treatment (Table 3). In addition, the method was very rapid. taking about 45 min to complete (15 min for adsorption and 25-30 min for elution).

DISCUSSION

Hydrophobic interactions play a role in the adherence of microorganisms to a wide variety of surfaces (Magnusson et al. 1982). The hydrophobic nature of the outermost bacterial surface has been shown to be a factor in the partitioning of microorganisms at interfaces (Rosenberg and Kjellerberg 1987). in the adherence of bacteria to nonwettable plastic surfaces, and in the attachment of bacteria to phagocytes (Tilney and Portnoy 1989). Adherence of a number of bacterial species to polystyrene has been investigated by several authors and at- tributed to hydrophobic interactions between cells and the plastic surface (Herald and Zottola 1988). Mafu et al . (1990) have previously reported on the attachment of L. tnonocytogenes to surfaces like glass. polypropylene, rubber and stainless steel after short contact times.

In our study, the ability of L. motiocytogenes to bind tightly to hydrophobic matrices was demonstrated. Mafu et al. (1991a) also showed that when using HIC, L. moriocyogenes can adhere to octyl Sepharose at low pH. Although L. monocytogenes appears to be hydrophilic, when the pH is lowered, hydrophobic groups present on the bacterial cell surface can orient themselves and participate in binding. In a low-pH environment, proteins on the bacterial cell surface may undergo a conformational change. causing exposure of hydrophobic groups, which thus promote binding of Listeria to the hydrophobic matrices. Alternatively, pro- tonation of carboxyl groups may be taking place on the cell surface causing charge neutralization, therefore making the cells more hydrophobic. This was very evident with the tight binding observed of L. monocytogenes to octyl Sepharose (see Table 1). Some elution occurred, but the binding was so tight that when using only mild conditions, such as a change of pH or a lowering of the salt concentration, all bacteria could not be removed from the gel matrix. The chain length of octyl Sepharose is long so that even hidden hydrophobic groups on the bacterial cell surface could possibly participate in binding. Harsher conditions can be used for elution during HIC, such as chaotropic agents like detergents; however we wanted to use mild conditions so as not to inactivate the bacteria. The control or underivatized gel without any attached hydrophobic groups adsorbed Listeria well, but all bacteria came out in the initial wash. These experiments performed with both octyl Sepharose and underivatized Sepharose indicate that alkyl or aromatic substituents are involved in binding (see Table 1).


Adhesion of Listeria to phenyl Sepharose was higher in higher ionic strength environments like 4 M NaCl and 1 M (NH&SO4. This finding was also reported by Piette and Idziak (1992), who found that adhesion of P. jluorescens to meat tendon was higher in a 85 mM solution of NaCl than in deionized H20, and that it was ionic strength per se and not sodium ions that aided adherence. The strength of adhesion is influenced by cell surface charge and hydrophobicity. In fact, it has been proposed that cell surface characteristics have a greater influence on adhesion strength than on the number of adherent bacteria (Piette and Idziak 1992). These results are in agreement with previous attempts to elucidate the mechanism of adhesion by experiments in which adherent cells were detached with various eluents. For example, Thomas and McMeekin (1981) reported that rinses with physiological saline, but not deionized water, effectively detached a large number of Salmonella typhimurium and S. singapore cells from collagen fibers.

When the bed volume of the phenyl Sepharose gel was increased from 0.8 to 2 ml, nearly all the bacteria in solution adsorbed to the phenyl gels. When the volume of elution buffer was increased from 1 to 10 ml of 10 mM sodium phosphate (pH 6.8), a considerable amount of bacteria eluted out. However, there were still some cells remaining on the gel. From the elution patterns observed, it was evident that some bacteria were eluted out by lowering the salt concentration and changing the pH, but others remained tightly bound to the gel. These were finally desorbed by using an increasing gradient of ethylene glycol. Orstavik et al. (1977) found that Enterococcus faeciurn could be desorbed from glass by a 2% Tween 80 solution, but not by a 2 M NaCl solution. These results were interpreted as an indication that hydrophobic rather than ionic interactions are involved in the adhesion process (Orstavik et al. 1977). Jones et al. (1981) also found that S. typhimurium cells could not be washed off HeLa cells by low ionic strength solutions, even though decreasing the ionic strength in the suspension medium substantially reduced adhesion. Piette and Idziak (1992) found that removal of bacteria by rinsing with solutions compatible with meat integrity (dilute salt/or organic acid solution) was ineffective. This has also been observed by other research groups (Appl and Marshall 1984; Delaquis and McCurdy 1990).

To test the potential application of the HIC procedure, milk was inoculated with L. monocytogenes and passed through a phenyl Sepharose column. L. monocytogenes adsorbed well from the milk onto the phenyl gel. Upon changing the pH and lowering the salt concentration, a large number of bacteria were eluted out. The remaining more tightly-bound bacteria were then eluted out by using 10 ml each of lo%, 30% and 50% ethylene glycol. The eluted bacteria were pure and viable.

This study has shown the potential of using the amphiphilic nature of L. monocytogenes to adsorb bacteria onto hydrophobic matrices, and then to elute them out in a form that can be detected by PCR and/or any other rapid, sensitive

160 S . SHARMA. S . KASATIYA and J . M . FARBER

method. This could eliminate problems associated with food particle contamina- tion and time-consuming enrichment techniques in detection methods, as bacteria can be adsorbed directly from food samples onto hydrophobic matrices. The method is cost effective. rapid and amenable to automation. Future studies will concentrate on the attachment and desorption of Listeria from pasteurized milk and other more complicated food matrices, such as muscle foods.

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DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES