Biofilm formation and the survival of Salmonella Typhimurium on parsley

International Journal of Food Microbiology 109 (2006) 229 – 233 www.elsevier.com/locate/ijfoodmicro

Short communication

Biofilm formation and the survival of Salmonella Typhimurium on parsley Anat Lapidot a , Ute Romling b , Sima Yaron a,⁎ a

b

Department of Biotechnology and Food Engineering, Technion, Haifa, Israel Microbiology and Tumorbiology Center (MTC), Karolinska Institutet, Stockholm, Sweden

Received 26 August 2005; received in revised form 3 November 2005; accepted 5 January 2006

Abstract Although several studies provide evidence that the formation of biofilms by human pathogens on plant tissue is possible, to date there is no direct evidence that biofilms enhance the resistance of plant-associated pathogens to disinfectants or biocides. We hypothesized that biofilm formation would enhance the adhesion and survival of Salmonella on leafy vegetables. To test our hypothesis, we compared the adhesion and persistence of Salmonella Typhimurium and its biofilm-deficient isogenic mutant. Following inoculation of parsley and rinsing with water or chlorine solution, both strains had similar survival properties, and up to 3-log reduction were observed, depending on chlorine concentration. This indicates that the biofilm matrix of Salmonella likely does not play a significant role in initial adhesion and survival after disinfection. After a week of storage the biofilm producing strain survived chlorination significantly better than the biofilm-deficient mutant. However, the recovery of the mutant was still elevated, indicating that although the biofilm matrix has a role in persistence of Salmonella after chlorination treatment of parsley, this is not the most important mechanism, and other mechanisms, probably the ability to penetrate the plant tissue or the pre-existing biofilms, or production of different polysaccharides other than cellulose, provide the protection. © 2006 Elsevier B.V. All rights reserved. Keywords: Produce; Disinfection; Food-borne pathogen; Food safety; Resistance

1. Introduction The incidence of human pathogens on fresh produce is a serious concern in industrialized countries. Salmonella is among the most commonly isolated pathogens associated with fresh fruits and vegetables. Outbreaks of salmonellosis have been linked to a wide variety of fresh produce including alfalfa sprouts (Mahon et al., 1997), lettuce, fennel (Ercolani, 1976), cilantro (Campbell et al., 2001), cantaloupes (Mohle-Boetani et al., 1999), unpasteurized orange juice (Cook et al., 1998), tomatoes (Hedberg et al., 1999), melons, mango, celery (Burnett and Beuchat, 2000) and parsley (CDC, 2000). Contaminated fresh parsley has also been linked to outbreaks of Shigella ⁎ Corresponding author. Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel. Tel.: +972 4 8292940; fax: +972 4 8293399. E-mail address: [email protected] (S. Yaron). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.01.012

sonnei, enterotoxigenic Escherichia coli (Naimi et al., 2003) and verotoxinogenic Citrobacter freundii (Tschape et al., 1995). Contamination of fruits and vegetables may occur at various stages during production, harvest, processing, and transport. Attached microorganisms (pathogens and spoilage bacteria) are not easily removed by washing with water or antibacterial agents. Chemical treatments, such as calcium or sodium hypochlorite, hydrogen peroxide, ethanol, and a variety of detergents partially reduced (if any) the populations of the pathogens (Beuchat, 1997; Gandhi et al., 2001). At present, chlorine at a concentration of 50–200 mg/L is the primary postharvest sanitizing agent in routine use in the fresh produce industry (Beuchat et al., 1998). This concentration of chlorine is usually ineffective at eliminating pathogens from leafy vegetables. Chlorine treatment at a concentration of 200 mg/L of inoculated lettuce, for instance, reduced less than 2 log of either Listeria monocytogenes, E. coli O157:H7 or Salmonella population (Lang et al., 2004). The reason for the lack of

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sanitizer effectiveness is still unknown, but was thought to be due to reduction of the oxidizing power of the chlorine by the high organic load of the plants (Burnett and Beuchat, 2000), or lower accessibility of the target pathogen, that could be achieved by either internalization of the organisms into the plant tissue, or aggregation and biofilm production on the plants. Biofilms are assemblages of microorganisms adherent to each other and/or to a surface and embedded in a matrix of exopolymers (Costerton et al., 1999). Due to the protection afforded to cells enclosed within this matrix, chemical sanitizers are generally unable to eliminate most biofilm-associated bacteria. Our previous research revealed that Salmonella Typhimurium embedded in the biofilm matrix resisted sodium hypochlorite at concentrations above 500 mg/L, while planktonic cells were sensitive to less than 50 mg/L (Scher et al., 2005). Those results, coupled with the recent observation that most isolates of Salmonella spp. originating from produce are able to synthesize the main components of the biofilm matrix (curli and cellulose) (Solano et al., 2002; Romling et al., 2003; Zogaj et al., 2003; Solomon et al., 2005), led us to hypothesize that Salmonella cells in the form of biofilms survive and resist disinfection treatments on leafy vegetables. The goal of our current research was to investigate the survival of Salmonella Typhimurium on parsley after disinfection, and compare it to the survival of a mutant that is deficient in biofilm formation. 2. Materials and methods 2.1. Strains and plasmids Salmonella enterica serovar Typhimurium ATCC14028 and its mutants MAE52 and MAE190 have been described previously (Zogaj et al., 2001). MAE52 forms biofilms at both 37 and 28 °C, while the WT strain produces a biofilm only at 28 °C. MAE190 is a mutant of MAE52 that does not express the gene products of bcsA (bacterial cellulose synthesis A) and agfBA (also called csgBA for curli subunit genes A and B). Thus it does not produce the two main extracellular matrix compounds, cellulose and curli, and a biofilm is not formed (Zogaj et al., 2001). Bacteria were transformed by electroporation using the pGFP plasmid (Clontech, Palo Alto, Calif.) to obtain stable green fluorescent protein (GFP)-labeled cells as previously described (Scher et al., 2005). Transformed colonies were stored at − 80 °C in Luria–Bertani (LB) supplemented with 20% glycerol. 2.2. Contamination of parsley During harvest, parsley and other leafy greens, fruits and vegetables are commonly submerged in a rinse tank to remove insects, pesticide residues and visible soil contamination (Sivapalasingam et al., 2003; Duffy et al., 2005). Pathogens in the rinse water would have ample opportunity to adhere to the plant tissue, thus we decided to study the adhesion and survival properties of Salmonella inoculated onto parsley during rinsing.

Parsley (flat Petroselinum crispum) plants were grown in a greenhouse at the ecological garden at the Technion. Plants were harvested when they reached a height of approximately 20–30 cm. Bunches were rinsed with water to remove soil and dust, air-dried and stored in 400-ml sterile stomacher bags at 4 °C for up to 24 h. Bacterial cultures were prepared by inoculation of a colony into LB broth supplemented with ampicillin (100 mg/L) and incubation overnight at 37 °C with aeration. Overnight cultures were diluted (1 : 100) in 50 ml fresh LB broth and were incubated for 2.5 h at 37 °C. Cells were harvested by centrifugation (4000 g for 20 min at 4 °C) and resuspended in 250 ml sterile saline solution (0.85% NaCl) to yield a final concentration of ca. 7 log CFU/ml. The pre-washed parsley samples (25 g each) were inoculated by immersion in the bacterial suspension (50 ml) for 60 min at room temperature. Plants were then removed from the inoculum and air-dried in a laminar flow biosafety cabinet for 1 h. 2.3. Disinfection of contaminated parsley Immediately after contamination (i.e. about 1 h after removal from the bacterial suspension) parsley samples were immersed in 100-ml deionized sterile water (control) or chlorinated water containing sodium hypochlorite at concentrations of 100, 200, 800 and 1600 mg/L in stomacher bags. Bags were positioned such that entire bunches were submerged in the water or chlorine solution. After treating for 5 min, water or chlorine solution was decanted, and parsley was washed twice with deionized sterile water. Fresh sterile saline (100 ml) was added and bags were pummeled for 3 min in a stomacher at normal speed. Cells were serially diluted (1 : 10) and plated on LB agar plates (for total viable cell counts) or LB supplemented with ampicillin (for enumeration of GFP-fluorescent cells). Fluorescent colonies were illuminated with ultraviolet light, and colonies exhibiting bright green fluorescence were counted. Control samples were uncontaminated parsley and contaminated parsley that was not immersed with either sterile water or chlorine solution. For stored samples, parsley was contaminated as described above, and dried in a laminar flow biosafety cabinet for 5 h for prevention of any moistness that might facilitate spoilage during storage. The bags were sealed and stored in the refrigerator (4 °C) or on the benchtop (at ambient temperature, ∼25 °C) for 7 days. No microbial spoilage was observed after 7 days of storage when fresh brunches from the greenhouse were used. After storage, the parsley samples were immersed in 100 ml water or 100 mg/L chlorine solution for 5 min, washed twice with sterile water, resuspended in saline and stomached for 3 min. Suspension was serially diluted and plate counted as described. 2.4. Statistic analysis All experiments were conducted at least 4 independent times in duplicates. Statistical significance was determined by Tukey–Kramer test using One Way Analysis of Variance

A. Lapidot et al. / International Journal of Food Microbiology 109 (2006) 229–233 Table 1 Reduction of Salmonella from parsley after washing with chlorine (0–1600 ppm) immediately following contamination Strain/ chlorine (mg/L) WT MAE52 MAE190

Table 2 Survival of MAE52 and MAE190 on parsley after incubation at 4 and 25 °C for 7 days

Log reduction (log CFU/g)1 0 1.2a 0.9a 1.0a

100 1.7b 2.0b 1.9b

200 2.0b 1.9b 1.7b

231

Population (log CFU/g parsley)1 800 2.5c 2.2c 2.6c

1600 3.0c 2.9c 2.6c

Strain/conditions MAE52 MAE190

Time 0

7 days after incubation at

7.4 7.2

4 °C 7.0 6.9

25 °C 7.3 7.0

1 1

Numbers represent the average reduction (log CFU per gram parsley) after treatment with water (control) or chlorine (100–1600 mg/L) compared with the population recovered from non-treated contaminated parsley of 4 independent experiments. Values followed by the same letter are not significantly different (P b 0.05).

(ANOVA). A P value b 0.05 was accepted as indicating significance. 3. Results and discussion Although several studies provide evidence that the formation of biofilms by human pathogens on plant tissue is possible (Takeuchi et al., 2000; Wade et al., 2003; Rayner et al., 2004), to date there is no direct evidence that biofilms enhance the resistance of plant-associated bacteria to disinfectants or biocides (Morris and Monier, 2003). To prove or disprove the hypothesis that biofilm formation enhances the adhesion of Salmonella to parsley and increases its survival following disinfection with chlorine, we compared the adhesion and persistence of Salmonella Typhimurium and a biofilm-deficient mutant. Initially, we determined the role of cellulose and curli in a loose and strong adhesion of the cells to parsley. Parsley samples were dipped in water containing each of the Salmonella strains, and viable cells that remained on the plant following a mild washing step with water were plate-counted. The WT and the mutants (that either constitutively produce the biofilm matrix components, or absent the ability to form biofilm) bound the parsley in an average of ca. 107 CFU/g parsley, and rinsing with water removed about 1 log of the attached cells (Table 1). These numbers are similar to previously reported levels of Salmonella on inoculated parsley. Duffy et al. reported that about 0.8 log (of 105 CFU/g) were removed by a simple washing (Duffy et al., 2005). In another experiment, Lang et al. showed a reduction of 0.5 log CFU of a population of a mixture of Salmonella strains recovered from dip-inoculated parsley after rinsing with water (Lang et al., 2004). Our results indicate that the ability to produce cellulose or curli does not provide any advantage during the initial step of adhesion, and that the WT as well as a biofilm-deficient mutant were strongly attached to parsley. The use of chlorinated water for the washing of whole or cut produce has a mild sanitizing effect and reductions in microbial levels were generally less than 103 CFU following treatment with chlorine at up to 2000 mg/L (Beuchat et al., 1998). Indeed, when we washed contaminated parsley with increasing concentrations (100–1600 mg/L) of chlorine, population reduction was very

Numbers are an average of at least 4 independent experiments.

low. As can be seen in Table 1, an additional 1-log reduction (comparing to rinsing with water) was observed after treatment with 100–200 mg/L chlorine, and additional 2 logs were reduced with the higher concentrations (800–1600 mg/L). The highest chlorine concentration reduced Salmonella levels (including tightly and loosely bound cells) by 3 logs. We did not observe any significant difference (P b 0.05) between the WT and the mutants, indicating that a short time after contamination (about 1 h), the ability to form exopolymers-matrix does not have a significant advantage, and other mechanisms such as aggregation, internalization into the plant tissue, or integration into existing biofilms protect the cells. The practical importance of these results is the realization that screening for the presence or expression of the biofilm-encoding genes will not predict the survivability of foodborne pathogens a short time after contamination of leafy produce such as parsley, and that efforts should target the adhesion and internalization mechanisms of the bacteria. Parsley is usually not consumed at the moment of picking from the field, or short time after exposure to the pathogen during processing. It is possible that biofilm matrix is not produced immediately after the exposure, but during processing or storage. Thus, we stored the contaminated parsley either in the refrigerator (4 °C) or at room temperature (∼25 °C) for 7 days. As can be seen in Table 2 number of Salmonella cells remained more or less constant during the storage at either 4 or 25 °C, without any significant differences between the mutants. After a week, attached cells that were able to produce the biofilm matrix (MAE52) were significantly (P b 0.05) more resistant to the disinfection treatments (Table 3). In the case of the biofilm-deficient mutant (MAE190) that was stored at 4 °C, numbers of recovered cells after rising with water or chlorine solution (Table 3) were similar to those recovered 1 h after Table 3 Recovery of MAE52 and MAE190 from stored parsley after rinsing with chlorine and water Storage temp (7 days) Treatment MAE52 MAE190 1

Log reduction (log CFU/g)1 4 °C Water 0.6a 1.0b

25 °C 100 mg/L chlorine 1.1b 1.7c

Water b0.1d 0.3a

100 mg/L chlorine 0.6a 1.4c

Numbers represent the average reduction (log CFU per gram parsley) after treatment with water (control) or chlorine (100 mg/L) compared with the population recovered from non-treated contaminated parsley of 4 independent experiments. Values followed by the same letter are not significantly different (P b 0.05).

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contamination (Table 1). On the other hand, storage of this mutant (MAE190) at 25 °C improved the adhesion of the cells to the plant (higher recovery after rising with water), but did not afford significant protection to chlorination. MAE52, which is potentially able to form biofilm, strongly attached the plant, and had significantly improved recovery after chlorination, independent of the storage temperature. These results indicate that production of curli and cellulose has a long-term role in the adhesion of cells to the parsley. Moreover, the ability to form the two main components of the biofilm matrix has an important role in persistence on the plant after chlorination, if treatment is conducted in sufficient time after exposure to the pathogen. Results also indicate that although curli and cellulose have a role in persistence of the cells after chlorination treatment, this is not the most important mechanism, since less than 2-log reduction was observed after chlorination of stored parsley contaminated with the MAE190 mutant. It seems that other mechanisms such as penetration of the waxy cuticle and the internal leaf tissue, integration into the pre-existing biofilms produced by the natural nonpathogenic microorganisms, or production of other polysaccharides (Stevenson et al., 2000; White et al., 2003), have much more importance in survival on the plants. Microscopic observations revealed Salmonella cells near the stomata and within cracks in the cuticle of the parsley leaves (Duffy et al., 2005). Moreover, Gandhi et al. demonstrated bacteria presented at a depth of 12 μm within intact alfalfa sprout tissue, indicating that bacteria are able to penetrate and survive in the plant tissue (Gandhi et al., 2001). Brandl and Mandrell investigated the adhesion of Salmonella Thompson on cilantro plants that developed lesions, and reported that Salmonella Thompson was present at extremely high densities in some lesions, and that whereas cells were present on the surface of the cuticle of healthy leaf tissue, they internalized the plant tissue after gaining access through the disrupted cuticle of the damaged regions (Brandl and Mandrell, 2002). Fett (Fett, 2000) examined and observed biofilms on different parts of alfalfa, broccoli, cloves and sunflower sprouts. He concluded that naturally occurring biofilms may afford protected colonization sites for human pathogens. Estimates of biofilm abundance showed that bacteria in biofilms constitute 10– 40% of the bacterial population on broad-leaf endive and parsley (Morris et al., 1998). However no information is available on the behavior of Salmonella or other pathogenic bacteria in biofilms formed by microflora associated with raw produce. The significance of those mechanisms was higher after storage at 25 °C, since rising with water at those conditions had very little effect (reduction of 0.1–0.3 log CFU of the attached mutant cells). It is not clear at this stage, however, whether the temperature effects are dependant on the bacteria (better mobility, expression of enzymes or adhesion factor), the physiological state of the plants, or other elements that are dependant on the existing microflora. To summarize this study, we found that biofilms are likely to influence the effectiveness of strategies to control food-borne pathogens on parsley. However, other protective mechanisms afford more significant protection effect. We also suggest that biofilm formation strengthens the adhesion and provides

protection against disinfection after storage of the contaminated produce, and not immediately after contamination. Consequently, it is important to investigate the other mechanisms that allow the bacterial protection under stress conditions on the plants, since our knowledge of the fitness of these enteric pathogens on plant surfaces is greatly lacking. Acknowledgment This research was supported by Henri Gutwirth Fund. We thank the Technion ecological garden team and Itamar Zelas for their technical assistance and Dr. Ethan Solomon for valuable comments on the manuscript.

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