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green cabbage) were obtained from local grocery store. For each head of ..... Acknowledgements. The authors thank Katherine Hopkins and Ernie Paroczay.
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Effect of curli expression and hydrophobicity of Escherichia coli O157:H7 on attachment to fresh produce surfaces J. Patel1, M. Sharma1 and S. Ravishakar2 1 USDA, Agricultural Research Service, Environmental and Microbial Food Safety Laboratory, Beltsville, MA, USA 2 Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ, USA

Keywords attachment, cabbage, curli, E. coli O157:H7, fresh produce, hydrophobicity, lettuce. Correspondence Jitendra Patel, USDA, Agricultural Research Service, Environmental and Microbial Food Safety Laboratory, 10300 Baltimore Avenue, BARC-East, Bldg. 201, Beltsville, MA 207052350, USA. E-mail: [email protected]

2010 ⁄ 1748: received 30 September 2010, revised 30 November 2010 and accepted 16 December 2010 doi:10.1111/j.1365-2672.2011.04933.x

Abstract Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation and attachment to cut and intact fresh produce surfaces. Methods and Results: Five Escherichia coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation and attachment to intact and cut fresh produce (cabbage, iceberg lettuce and Romaine lettuce) leaves. Biofilm formation was stronger when E. coli O157:H7 were grown in diluted tryptic soy broth (1 : 10). In general, strong curli-expressing E. coli O157:H7 strains 4406 and 4407 were more hydrophobic and attached to cabbage and iceberg lettuce surfaces at significantly higher numbers than other weak curliexpressing strains. Overall, E. coli O157:H7 populations attached to cabbage and lettuce (iceberg and Romaine) surfaces were similar (P > 0Æ05), indicating produce surfaces did not affect (P < 0Æ05) bacterial attachment. All E. coli O157:H7 strains attached rapidly on intact and cut produce surfaces. Escherichia coli O157:H7 attached preferentially to cut surfaces of all produce types; however, the difference between E. coli O157:H7 populations attached to intact and cut surfaces was not significant (P > 0Æ05) in most cases. Escherichia coli O157:H7 attachment and attachment strength (SR) to intact and cut produce surfaces increased with time. Conclusions: Curli-producing E. coli O157:H7 strains attach at higher numbers to produce surfaces. Increased attachment of E. coli O157:H7 on cut surfaces emphasizes the need for an effective produce wash to kill E. coli O157:H7 on produce. Significance and Impact of the Study: Understanding the attachment mechanisms of E. coli O157:H7 to produce surfaces will aid in developing new intervention strategies to prevent produce outbreaks.

Introduction Foodborne pathogens on fresh produce account for 9Æ5 million (12%) of the c. 76 million U.S. foodborne illnesses annually (CDC) at a cost c. $39 billion in medical and productivity losses (Schraff 2010). The number of foodborne illness outbreaks linked to fresh produce has increased during the past 15 years (Olsen et al. 2000). In addition to an increase in produce-associated outbreaks, number of illnesses per fresh produce outbreak (48) has Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

exceeded those of poultry (30)-, beef (27)- or seafoodassociated outbreaks (10) (DeWall 2007). Escherichia coli O157:H7 is frequently associated with outbreaks linked to fresh produce. In a 2006 spinach outbreak, E. coli O157:H7 contamination was linked to consumption of fresh, bagged product resulting in 205 cases of illness and 3 deaths (CDC 2006). Shredded lettuce contaminated with E. coli O157:H7 was also involved in an outbreak of infections in 2006, resulting in 71 cases, 53 hospitalizations and 8 cases of Haemolytic Uraemic Syndrome (HUS) (Team 1

E. coli attachment on fresh produce

2008). Lettuce contaminated with norovirus and enterotoxigenic E. coli was linked to 11 outbreaks in Denmark during January 2010, resulting in 260 illnesses (Ethelberg et al. 2010). Also in 2010, an outbreak linked to E. coli O145-contaminated Romaine lettuce resulted in more than 50 illnesses and 12 hospitalizations (CDC 2010). The attachment of bacterial cells to biotic or abiotic surface is the first step in contamination of food products. A better understanding of bacterial attachment mechanisms will be helpful in developing effective produce wash treatments for fresh produce. As fresh produce is consumed raw, effective produce wash is very critical to remove pathogens from fresh produce and to reduce subsequent illnesses associated with consumption of fresh produce. Attachment of bacteria to surfaces can be influenced by specific appendages (fimbriae, curli and other outer membrane proteins) and cell surface hydrophobicity (Goulter et al. 2009). Curli are involved in biofilm formation and bacterial auto-aggregation (Kim and Harrison 2009). Escherichia coli O157:H7 forms biofilms on food-processing surfaces as well as on produce surfaces like spinach, lettuce, cabbage and other fresh produce (Pawar et al. 2005). Previous studies reported a positive correlation between bacterial hydrophobicity and attachment to hydrophobic surfaces (Gallardo-Moreno et al. 2002a,b). Curli and cellulose production supported host colonization and biofilm formation of E. coli O157:H7 on abiotic surfaces (Saldan˜a et al. 2009). Takahashi et al. (2010) found significant correlation between hydrophobicity of Listeria monocytogenes and biofilm formation on polyvinyl chloride surface. Escherichia coli O157:H7 and L. monocytogenes attached preferentially to cut edges of Iceberg lettuce compared to intact tissues (Takeuchi et al. 2000). However, Salmonella Typhimurium did not attach preferentially to intact or cut lettuce surfaces. Listeria monocytogenes strains exhibited a preference to attach to cut cabbage tissues compared to intact tissues (Ells and Truelstrup Hansen 2006). In other studies, hydrophobicity in E. coli O157:H7 cells did not affect attachment to apple and lettuce surfaces (Boyer et al. 2007; Rivas et al. 2007). Salmonella spp. attachment to fresh produce varied among serovars as stronger attachment of Salmonella Senftenberg to basil leaves was observed compared to the attachment of Salm. Typhimurium (Berger et al. 2009). In our earlier study, Salmonella attachment varied with the type of produce and Salmonella enterica serovars (Patel and Sharma 2010). We evaluated the attachment of E. coli O157:H7 strains on intact and cut surfaces of cabbage, Iceberg and Romaine lettuce. The relationship between cell surface properties and E. coli O157:H7 attachment to fresh produce was investigated. 2

J. Patel et al.

Materials and methods Bacterial cultures and media Five E. coli O157:H7 isolates were used in the study: RM 4406, RM 4688, RM 1918 (clinical isolates from lettuce outbreaks), RM 4407 (clinical isolate from spinach outbreak) and RM 5279 (bagged vegetable isolate) were kindly provided by Robert Mandrel (U.S. Department of Agriculture, Agricultural Research Service, Albany, CA, USA). The strains were cultured from )80C stocks in tryptic soy broth (TSB) supplemented with 10% glycerol. Frozen cultures of each strain were partially thawed at room temperature (c. 22C) for 15 min, transferred into TSB (Acumedia, Lansing, MI, USA) and incubated at 37C for 24 h. Cells were centrifuged (7500 g, 10 min, 10C), and cell pellets were washed with phosphate-buffered saline (PBS, pH 7Æ2) twice. The cell pellets were resuspended in PBS to obtain an optical density at 600 nm (OD600) of 1Æ0 (Thermo Spectronic, Rochester, NY, USA). The cell density was adjusted to c. 6 log CFU ml)1 for attachment study on produce surfaces. The populations of individual strains were verified on tryptic soy agar by spot plate technique (Patel and Sharma 2010). Curli expression The Congo red binding assay was used to determine curli expression. Each strain grown in TSB at 20 and 37C was streaked onto Congo red indicator (CRI) agar, which contained 0Æ1% tryptone, 0Æ05% yeast extract, 1Æ5% agar (Becton Dickinson, Sparks, MD, USA), 0Æ004% Congo red and 0Æ002% Coomassie brilliant blue (Sigma Aldrich Co., St Louis, MO) (Kim and Harrison 2009), and incubated at 28C for 48 h. Curli-producing E. coli O157:H7 formed red colonies on CRI agar (from the binding of the dye with curli), while curli-negative E. coli O157:H7 formed colourless colonies on the medium. Hydrophobicity Hydrophobicity was determined using bacterial adherence to hydrocarbons (BATH) assay as described by Li and McLandsborough (1999). One millilitre of xylene was added to each of three tubes containing 4 ml of cells suspended in PBS. The tubes were vortexed for 2 min and incubated in a water bath at 37C for 30 min. The OD600 of aqueous layer was determined (Thermo Spectronic). Bacterial suspensions without xylene served as a control. The ratio of the absorbance of the bacterial assays to the control was used to calculate per cent hydrophobicity. Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

J. Patel et al.

Biofilm formation in 96-well polystyrene microtitre plate The cultures grown overnight at 37C and OD600 adjusted as described earlier were diluted 1 : 10 000 in growth media, transferred into sterile 96-well polystyrene microtitre plates (Fisher Scientific, Newark, DE, USA) at 200 ll per well and incubated under static conditions at 30C for 48 h. Growth media that were tested included LB (Luria–Bertani) broth, diluted LB (1 : 10) broth, TSB and diluted TSB (1 : 10). Eight wells were inoculated for each strain in each medium. Growth media devoid of bacterial inoculum served as a negative control. After 48-h incubation, microplate cultures were aspirated and washed five times with sterile distilled water in a microplate washer (BioTek Instruments, Winooski, VT, USA). The plates were air dried, and 200-ll crystal violet (0Æ41% w ⁄ v; Fisher Scientific) was added to each well and incubated at room temperature (22C) for 45 min. The wells were then aspirated and washed five times with sterile distilled water in microplate washer. After allowing the plates to air dry, 200 ll of 95% ethanol was added to each well. A multichannel pipette was used to mix the contents of the wells and to dissolve the crystal violet dye. A600 nm was then recorded for each well using a Microquant Microplate spectrophotometer (BioTek). Attachment and recovery of Escherichia coli O157:H7 from fresh produce Organic produce (Iceberg lettuce, Romaine lettuce and green cabbage) were obtained from local grocery store. For each head of produce, the two outermost leaf layers were removed aseptically and discarded. Intact (2-cm diameter, disc) and cut (2 cm · 0Æ5 cm, strip) surfaces (referred as coupons) for each produce commodity were obtained as described by Patel and Sharma (2010). An 11-ml aliquot of culture (6 log CFU ml)1) was transferred into each well of a sterile six-cell culture plate (Fisher Scientific) to determine bacterial attachment. The coupons were aseptically submerged individually into the well, and the culture plates were incubated at 10C for 24 h. At specific time intervals (0, 1, 4 and 24 h), coupons were removed from suspensions and dipped in a test tube containing 11-ml sterile PBS (pH 7Æ2) to remove residual cells carried over from the inoculum. Populations of loosely and strongly attached E. coli O157:H7 to produce surfaces were determined as described by Patel and Sharma (2010). Briefly, coupons were then transferred into a sterile 50-ml centrifuge tube (Fisher Scientific) containing 25-ml sterile PBS with 0Æ1% Tween 20 and vortexed for 20 s to remove loosely attached E. coli O157:H7 cells. In order to recover populations of strongly attached E. coli O157:H7, the vortexed coupons were Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

E. coli attachment on fresh produce

transferred into a 50-ml centrifuge tube containing 25-ml buffered peptone water (BPW; Becton Dickinson) and sonicated for 30 s using a PolyTron homogenizer (Kinematica, Lucerne, Switzerland). The PolyTron was sterilized in 70% ethanol between each coupon and rinsed twice in sterile distilled water to remove residual ethanol from homogenizer. Appropriately diluted homogenates (strongly attached bacteria) and wash solutions (PBS with 0Æ1% Tween 20, loosely attached bacteria) were then enumerated on sorbitol MacConkey agar (SMAC; Becton Dickinson). Typical E. coli O157:H7 sorbitol-negative colonies were counted after incubation of 24 h at 37C. Randomly selected colonies were confirmed by latex agglutination assay (Remel Inc., Lenexa, KS, USA). The attachment strengths (SR values) of E. coli O157:H7 were calculated as the percentage of the total population of bacteria associated with produce surface, which were strongly attached to produce surface (Ells and Truelstrup attached bacteHansen 2006). [SR = (strongly ria) ⁄ (strongly attached bacteria + loosely attached bacteria)]. Statistical analysis A randomized complete block design was used with three replicates per treatment. The total E. coli O157:H7 populations (loosely and strongly attached) calculated as log CFU cm)2 and the attachment strength values obtained at each sampling period were analysed by a two-way anova using ‘Proc Mixed’ (SAS 8.2, Cary, NC, USA) for interaction effects of the strain, produce and sampling period. Biofilm and hydrophobicity data were analysed for the effect of E. coli O157:H7 strain. In all cases, the level of statistical significance was P < 0Æ05. Results Curli expression and cell hydrophobicity of Escherichia coli O157:H7 Escherichia coli O157:H7 strains 4406 and 4407 previously grown at 20C showed strong curli expression (red colonies) on CRI agar, whereas these strains grown at 37C had a mixture results of curli-positive and curli-negative colonies (colourless). Escherichia coli O157:H7 strains 1918 and 5279 grown at 20 or 37C had mixed results, with more curli positive (red) in some replicates with fewer negative colonies (pink to colourless) on the agar. Curli expression was not observed with the strain 4688 grown at either temperature. The hydrophobicity of E. coli strains was affected by the individual strain and the growth phase (Table 1). Each strain in log phase (5 h, 37C) was significantly 3

E. coli attachment on fresh produce

J. Patel et al.

Table 1 Hydrophobicity of Escherichia coli O157:H7 strains grown at 37C and analysed after 4-h (log phase) and 18-h (stationary phase) incubation Per cent hydrophobicity* Growth phase

4406

4407

4688

1918

5279

Log Stationary

24Æ67 ± 8Æ23 bx 11Æ57 ± 2Æ39 by

31Æ87 ± 1Æ89 ax 16Æ38 ± 5Æ81 ay

29Æ66 ± 4Æ66 ax 9Æ35 ± 2Æ89 by

30Æ63 ± 3Æ23 ax 9Æ42 ± 2Æ69 by

19Æ26 ± 0Æ57 bx 7Æ91 ± 3Æ69 by

Results are mean values and standard deviation of three replicates. ab Means followed by different letters in a same row are significantly different (P < 0Æ05). xy Means followed by different letters in a same column are significantly different (P < 0Æ05).

(P < 0Æ05) more hydrophobic than the corresponding strain in stationary phase (18 h, 37C). The hydrophobicity of log-phase cultures of E. coli O157:H7 strains 4407, 4688 and 1918 (32, 30, and 31%, respectively) was significantly higher than the hydrophobicity of log-phase cultures of strain 4406 (25%) and strain 5279 (19%). In comparing the hydrophobicity of cultures in stationary phase, strain 4407 showed significantly greater hydrophobicity (16%) than four other E. coli O157:H7 strains (8–12%) examined.

biofilm (0Æ36 and 0Æ32, respectively) than strains 4406 (0Æ16) and 4407 (0Æ16) in diluted TSB (1 : 10). Similarly, strain 4688 produced significantly more biofilm (0Æ17) than strain 1918 (0Æ14) in TSB. All strains exhibited poor biofilm formation in LB growth media. Escherichia coli O157:H7 strains 5279 (0Æ12) and 4406 (0Æ11) formed significantly more biofilm compared to the biofilm formed by strains 1918 and 4688 (0Æ09) in LB. Biofilms formed by these strains in diluted LB media were not different (P > 0Æ05) from the control sample.

Biofilm formation on 96-well microtitre polystyrene plate

Escherichia coli O157:H7 attachment on produce surfaces

In general, biofilm formed by E. coli O157:H7 strains 4688 and 5279 (0Æ17) was similar to the biofilm formation by strain 1918 (0Æ15), 4406 and 4407 (0Æ13). However, biofilm formation on microtitre plates varied with strain in different growth media (Fig. 1). Biofilm formation by E. coli O157:H7 strains 4688, 1918 and 5279 was significantly higher in diluted TSB (1 : 10) than in TSB or LB. Biofilm formation in LB and diluted LB (1 : 10) was not different (P > 0Æ05) for all five E. coli O157:H7 strains examined. Escherichia coli O157:H7 strains 4688 and 5279 produced significantly more 0·50

OD600

0·40 0·30 0·20 0·10 0·00 4406

4407 4688 1918 E. coli O157:H7 strains

5279

Figure 1 Biofilm formation of Escherichia coli O157:H7 strains on the polystyrene surface. Biofilm formation was determined by crystal violet stain followed by optical density measurement at A600. ( ) TSB 1 : 10; ( ) TSB; ( ) LB (1 : 10) and ( ) LB.

4

Rapid attachment of all E. coli O157:H7 was observed on intact and cut produce surfaces. In general, differences in population of attached E. coli O157:H7 to Iceberg lettuce, Romaine lettuce and cabbage were not significant. Overall, attached populations of E. coli O157:H7 strains 4406 and 4407 were higher, but not significantly different from the attached populations of other E. coli O157:H7 strains on these commodities. Escherichia coli O157:H7 populations attached after 5 min (Time 0 h) to intact and cut surfaces were the following: 3Æ59 to 4Æ98 log CFU cm)2 for cabbage, 3Æ77 to 4Æ74 log CFU cm)2 for Iceberg lettuce and 3Æ80 to 4Æ86 log CFU cm)2 for Romaine lettuce (Table 2a–c). Initial attachment (0 h) of E. coli O157:H7 strain 4406 on intact (4Æ04 log CFU cm)2) and cut (4Æ98 log CFU cm)2) cabbage surface was higher (P > 0Æ05) than the initial attachment of other E. coli O157:H7 strains on corresponding intact or cut cabbage surface. Initial populations (0 h) of E. coli O157:H7 strains 1918 (4Æ40 log CFU cm)2), 4406 (4Æ98 log CFU cm)2), 4407 (4Æ63 log CFU cm)2) and 5279 (4Æ55 log CFU cm)2) attached to cut cabbage surfaces were significantly higher than the population of these strains (3Æ60, 4Æ04, 3Æ87, and 3Æ59 log CFU cm)2, respectively) attached to intact cabbage surface, respectively (Table 2a). The populations of E. coli O157:H7 attached to intact and cut cabbage increased with incubation time. Populations of all E. coli O157:H7 strains recovered at 24 h from intact or cut surfaces were significantly higher Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

J. Patel et al.

E. coli attachment on fresh produce

Table 2 Populations of Escherichia coli O157:H7 attached to intact and cut produce surfaces incubated at 10C to (a) cabbage (b) iceberg lettuce and (c) romaine lettuce over 24 h E. coli O157:H7 strain (a) Cabbage 1918 4406 4407 4688 5279 1918 4406 4407 4688 5279 (b) Iceberg lettuce 1918 4406 4407 4688 5279 1918 4406 4407 4688 5279 (c) Romaine lettuce 1918 4406 4407 4688 5279 1918 4406 4407 4688 5279

Produce surface

Intact

Cut

Intact

Cut

Intact

Cut

Log CFU cm)2 0h

1h

4h

24 h

3Æ60 4Æ04 3Æ87 3Æ83 3Æ59 4Æ40 4Æ98 4Æ63 4Æ49 4Æ55

± ± ± ± ± ± ± ± ± ±

0Æ67 0Æ47 0Æ52 0Æ37 0Æ58 0Æ50 0Æ72 0Æ69 0Æ28 0Æ48

bx bx bx bx bx cx bx cx bx cx

3Æ73 4Æ13 4Æ61 3Æ69 3Æ84 4Æ85 5Æ06 5Æ44 4Æ96 4Æ98

± ± ± ± ± ± ± ± ± ±

0Æ52 0Æ61 0Æ61 0Æ61 0Æ52 0Æ46 0Æ57 0Æ52 0Æ46 0Æ44

by bxy abx aby by bcx bx bcx bx bcx

4Æ18 4Æ86 4Æ60 4Æ09 4Æ17 5Æ53 6Æ37 5Æ90 5Æ35 5Æ90

± ± ± ± ± ± ± ± ± ±

0Æ28 aby 0Æ5 abx 0Æ55 abxy 0Æ41 aby 0Æ38 by 0Æ42 by 0Æ20 ax 0Æ45 bxy 0Æ5 by 0Æ42 abxy

5Æ19 5Æ72 5Æ65 5Æ15 5Æ44 6Æ93 7Æ10 7Æ24 7Æ09 6Æ71

± ± ± ± ± ± ± ± ± ±

0Æ09 0Æ33 0Æ33 0Æ24 0Æ41 0Æ55 0Æ49 0Æ18 0Æ37 0Æ63

ax ax ax ax ax ax ax ax ax ax

4Æ59 3Æ77 4Æ04 4Æ06 3Æ92 4Æ74 4Æ39 4Æ73 4Æ61 4Æ64

± ± ± ± ± ± ± ± ± ±

0Æ84 0Æ15 0Æ09 0Æ11 0Æ07 0Æ06 0Æ12 0Æ16 0Æ04 0Æ11

ax by axy axy ay bx bx bx bx bx

3Æ71 3Æ97 3Æ92 4Æ00 3Æ90 4Æ80 5Æ06 5Æ05 4Æ78 5Æ06

± ± ± ± ± ± ± ± ± ±

0Æ22 0Æ16 0Æ09 0Æ21 0Æ23 0Æ15 0Æ35 0Æ32 0Æ24 0Æ32

bx ax ax bx ax bx bx bx bx bx

4Æ44 4Æ23 4Æ09 4Æ54 4Æ24 5Æ18 4Æ92 4Æ96 4Æ89 5Æ11

± ± ± ± ± ± ± ± ± ±

0Æ14 0Æ05 0Æ25 0Æ56 0Æ28 0Æ06 0Æ13 0Æ27 0Æ06 0Æ08

ax ax ax ax ax bx bx bx bx bx

5Æ09 5Æ64 5Æ73 5Æ51 4Æ79 6Æ76 6Æ57 6Æ79 6Æ48 6Æ73

± ± ± ± ± ± ± ± ± ±

0Æ72 0Æ55 0Æ56 0Æ29 0Æ26 0Æ34 0Æ25 0Æ57 0Æ14 0Æ49

axy ax ax ax ay ax ax ax ax ax

5Æ04 4Æ19 3Æ80 3Æ93 4Æ03 4Æ59 4Æ86 4Æ72 4Æ67 4Æ68

± ± ± ± ± ± ± ± ± ±

1Æ37 0Æ13 0Æ08 0Æ21 0Æ17 0Æ04 0Æ16 0Æ22 0Æ23 0Æ12

ax ay by ay ay bx bx bx bx bx

3Æ83 3Æ79 4Æ02 3Æ55 3Æ69 4Æ80 4Æ97 4Æ92 4Æ77 4Æ78

± ± ± ± ± ± ± ± ± ±

0Æ16 0Æ14 0Æ18 0Æ11 0Æ15 0Æ17 0Æ12 0Æ09 0Æ12 0Æ12

ax bx ax bx bx bx ax bx bx bx

4Æ60 4Æ28 4Æ28 4Æ74 4Æ64 5Æ42 5Æ30 5Æ28 5Æ16 5Æ32

± ± ± ± ± ± ± ± ± ±

0Æ20 0Æ37 0Æ18 0Æ41 0Æ20 0Æ28 0Æ19 0Æ20 0Æ29 0Æ02

ax ax ax ax ax abx ax abx abx abx

4Æ91 5Æ64 5Æ38 5Æ61 5Æ11 6Æ26 6Æ25 6Æ51 6Æ31 6Æ54

± ± ± ± ± ± ± ± ± ±

0Æ64 0Æ85 0Æ70 0Æ77 0Æ50 0Æ41 0Æ81 0Æ33 0Æ72 0Æ39

ay ax axy ax axy ax ax ax ax ax

Results are expressed as mean log CFU cm)2 and standard deviation for three replicate experiments. Means followed by different letters within a row are significantly different (P < 0Æ05). xy Means followed by different letters in a column within a produce surface are significantly different (P < 0Æ05). abc

than the initial population (0 h) of specific E. coli O157:H7 strain on intact and cut cabbage. Populations of E. coli O157:H7 strains recovered from cut cabbage surfaces at 1, 4 and 24 h were significantly higher than the corresponding E. coli O157:H7 strain populations recovered from intact cabbage surfaces. Attachment of strain 4406 on intact (4Æ86 log CFU cm)2) and cut (6Æ37 log CFU cm)2) cabbage surfaces after 4 h was significantly higher than the attachment of strains 1918 and 4688 on intact and cut cabbage surfaces at 4 h. Initial attachment of E. coli O157:H7 varied from 3Æ77 to 4Æ59 log CFU cm)2 and 4Æ39 to 4Æ74 log CFU cm)2, on intact and cut Iceberg lettuce, respectively (Table 2b). Initial populations of strain 1918 (4Æ59 log CFU cm)2) Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

on intact Iceberg lettuce were significantly higher than the initial populations of strains 4406 and 5279 on intact Iceberg lettuce. There were no differences (P > 0Æ05) in attachment between strains on intact or cut surfaces at 1 and 4 h. All strains preferentially attached to cut Iceberg surfaces; however, the difference between initial attachment to intact and cut lettuce was similar (P > 0Æ05) with the exception of strains 4407 and 5279. The populations of all E. coli O157:H7 strains recovered at 0 and 24 h from cut Iceberg lettuce were significantly different. Numbers of attached E. coli O157:H7 also increased with time from 0 to 24 h on intact Iceberg lettuce, but the difference was not significant (P > 0Æ05). 5

E. coli attachment on fresh produce

J. Patel et al.

Table 3 Strength of bacterial attachment (SR value) of Escherichia coli O157:H7 strains to intact and cut fresh produce surfaces stored at 10C of (a) cabbage (b) iceberg lettuce and (c) Romaine lettuce E. coli O157:H7 strain (a) Cabbage 1918 4406 4407 4688 5279 1918 4406 4407 4688 5279 (b) Iceberg lettuce 1918 4406 4407 4688 5279 1918 4406 4407 4688 5279 (c) Romaine lettuce 1918 4406 4407 4688 5279 1918 4406 4407 4688 5279

Produce surface

Intact

Cut

Intact

Cut

Intact

Cut

SR values 0h

1h

4h

24 h

0Æ09 0Æ15 0Æ08 0Æ12 0Æ17 0Æ16 0Æ16 0Æ17 0Æ29 0Æ16

± ± ± ± ± ± ± ± ± ±

0Æ04 0Æ08 0Æ08 0Æ11 0Æ06 0Æ09 0Æ13 0Æ13 0Æ13 0Æ03

bx ax cx bx ax bx bx bx ax ax

0Æ21 0Æ12 0Æ19 0Æ17 0Æ12 0Æ16 0Æ29 0Æ21 0Æ13 0Æ13

± ± ± ± ± ± ± ± ± ±

0Æ07 0Æ08 0Æ17 0Æ07 0Æ08 0Æ03 0Æ09 0Æ03 0Æ03 0Æ06

abx ax bcx abx ax bx abx bx ax ax

0Æ43 0Æ18 0Æ42 0Æ27 0Æ21 0Æ16 0Æ46 0Æ43 0Æ25 0Æ33

± ± ± ± ± ± ± ± ± ±

0Æ23 ax 0Æ14 ax 0Æ32 abx 0Æ17 abx 0Æ16 ax 0Æ08 by 0Æ27 ax 0Æ3 abxy 0Æ06 axy 0Æ28 axy

0Æ45 0Æ34 0Æ58 0Æ41 0Æ12 0Æ50 0Æ30 0Æ49 0Æ38 0Æ35

± ± ± ± ± ± ± ± ± ±

0Æ07 0Æ18 0Æ26 0Æ15 0Æ10 0Æ15 0Æ16 0Æ28 0Æ23 0Æ30

ax axy ax ax ay a abx ax ax ax

0Æ59 0Æ36 0Æ35 0Æ34 0Æ36 0Æ29 0Æ22 0Æ25 0Æ13 0Æ20

± ± ± ± ± ± ± ± ± ±

0Æ35 ax 0Æ20 ax 0Æ08 ax 0Æ09 ax 0Æ09 ax 0Æ18 ax 0Æ14 ax 0Æ15 ax 0Æ1 ax 0Æ11 ax

0Æ27 0Æ22 0Æ25 0Æ29 0Æ17 0Æ31 0Æ27 0Æ39 0Æ24 0Æ28

± ± ± ± ± ± ± ± ± ±

0Æ16 0Æ05 0Æ10 0Æ07 0Æ11 0Æ17 0Æ14 0Æ18 0Æ16 0Æ22

ax ax ax ax bx ax ax ax ax ax

0Æ32 0Æ18 0Æ42 0Æ24 0Æ49 0Æ23 0Æ27 0Æ28 0Æ16 0Æ27

± ± ± ± ± ± ± ± ± ±

0Æ21 axy 0Æ1 ay 0Æ19 ax 0Æ12 axy 0Æ21 ax 0Æ06 ax 0Æ14 ax 0Æ12 ax 0Æ08 ax 0Æ12 ax

0Æ37 0Æ19 0Æ41 0Æ49 0Æ64 0Æ41 0Æ35 0Æ33 0Æ15 0Æ25

± ± ± ± ± ± ± ± ± ±

0Æ02 0Æ09 0Æ24 0Æ43 0Æ18 0Æ11 0Æ19 0Æ23 0Æ18 0Æ04

axy ay axy ax ax ax ax ax ax ax

0Æ71 0Æ40 0Æ60 0Æ43 0Æ49 0Æ37 0Æ32 0Æ20 0Æ19 0Æ32

± ± ± ± ± ± ± ± ± ±

0Æ25 0Æ11 0Æ09 0Æ25 0Æ15 0Æ15 0Æ13 0Æ04 0Æ15 0Æ16

0Æ24 0Æ22 0Æ25 0Æ19 0Æ25 0Æ27 0Æ30 0Æ26 0Æ26 0Æ19

± ± ± ± ± ± ± ± ± ±

0Æ21 0Æ13 0Æ18 0Æ07 0Æ18 0Æ13 0Æ08 0Æ11 0Æ13 0Æ04

bx ax ax ax ax ax ax ax abx ax

0Æ40 0Æ57 0Æ42 0Æ61 0Æ38 0Æ23 0Æ21 0Æ32 0Æ53 0Æ25

± ± ± ± ± ± ± ± ± ±

0Æ25 0Æ21 0Æ31 0Æ17 0Æ20 0Æ13 0Æ04 0Æ29 0Æ16 0Æ14

0Æ39 0Æ36 0Æ33 0Æ29 0Æ24 0Æ36 0Æ36 0Æ15 0Æ43 0Æ35

± ± ± ± ± ± ± ± ± ±

0Æ25 bx 0Æ20 ax 0Æ21 ax 0Æ41 ax 0Æ01 ax 0Æ32 ax 0Æ19 ax 0Æ11ax 0Æ10 abx 0Æ19 ax

abx ay axy ay axy ax ax ax bx ax

bx ax ax ax ax ay ay axy ax axy

Results are expressed as mean SR values and standard deviation. The SR values were calculated as: SR = (strongly attached bacteria) ⁄ (strongly attached bacteria + loosely attached bacteria). abc Means followed by different letters within a row are significantly different (P < 0Æ05). xy Means followed by different letters in a column within a produce surface are significantly different (P < 0Æ05).

Initial attachment of E. coli O157:H7 strain 1918 on intact Romaine lettuce (5Æ04 log CFU cm)2) was significantly higher than the attachment of other strains on intact Romaine lettuce (Table 2c). Similarly, initial populations of strain 4406 on cut Romaine lettuce (4Æ86 log CFU cm)2) were also higher; however, they were not significantly different from the populations of the other four strains. As with other produce, initial populations of strains 4406, 4407 and 4688 on cut Romaine lettuce were significantly higher than the populations of these strains on intact Romaine lettuce. Significantly higher populations of strains 4406 and 4407 were also reported on cut surfaces at 1, 4 and 24 h when compared 6

to intact surfaces. The populations of all E. coli O157:H7 strains recovered at 24 h were significantly greater than populations of corresponding strains recovered at 0 h on cut Romaine lettuce. Overall attachment strength (SR) of E. coli O157:H7 on cabbage surfaces (0Æ26) was significantly lower than its attachment strength on Romaine lettuce surfaces (0Æ34). Initial SR values for cabbage, Iceberg lettuce and Romaine lettuce ranged from 0Æ08–0Æ29, 0Æ13–0Æ59 and 0Æ20–0Æ71, respectively (Table 3a–c). The SR values of E. coli O157:H7 on cut cabbage surfaces were not significantly different from the SR values of these strains on intact cabbage surfaces at the corresponding sampling period Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

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(Table 3a). Initial SR values of E. coli O157:H7 strain 1918 on intact Romaine lettuce (0Æ71) were significantly higher than the initial SR values of strain 4688 (0Æ43) and 4406 (0Æ40) on intact Romaine lettuce (Table 3c). Similarly, the SR values of strains 4407 (0Æ42) and 5279 (0Æ49) obtained after 4-h incubation on intact Iceberg lettuce were significantly higher than the corresponding SR values of strain 4406 (0Æ18) (Table 3b). Occasional differences in SR values of these strains were also reported on cut Romaine lettuce surfaces. For example, the SR values of strain 4688 (0Æ53) obtained after 4-h incubation on cut Romaine lettuce were higher (P < 0Æ05) than the SR values of strains 1918 (0Æ23) and 4406 (0Æ21). The attachment strength increased with time on both intact and cut cabbage surfaces in most cases; however, the differences in SR values of these strains at 0 and 24 h were not significant. Likewise, the SR values of E. coli O157:H7 strains obtained after 24 h on Iceberg lettuce were higher (P > 0Æ05) than the initial SR values (0 h) except for strain 4406 on intact surface. No clear upward or downward trend in SR values for Romaine lettuce was observed with respect to incubation time. Discussion Curli are very thin, coiled, extracellular structures on cell surface of most E. coli, and their production can be influenced by low temperature, low osmolarity and growth phase (Boyer et al. 2007). Most pathogenic E. coli strains do not produce curli when grown at 37C (Olsen et al. 1993). However, Saldan˜a et al. (2009) reported curli expression of E. coli O157:H7 strains when grown at 37C in low-salt medium. Similarly, curli expression was also observed with number of E. coli O157 isolates grown at 37C (Goulter et al. 2010). Kim and Harrison (2009) observed consistent curli expression by E. coli O157:H7 strains grown at 22–26C, but weak expression when grown at 37C. In our study, E. coli O157:H7 strains 4406 and 4407 expressed strong curli production when grown at 20C and weak production when grown at 37C. Genetic differences among strains and growth conditions (media and temperature) may account for differences in curli expression observed in various studies (Uhlich et al. 2001). The relationship between curli expression and cell hydrophobicity has been variable. Prigent-Combaret et al. (2000) reported positive correlation between curliproducing E. coli and cell attachment to polystyrene surface, whereas Ryu et al. (2004) found that curli expression did not affect E. coli O157:H7 cell attachment to stainless steel surfaces. Poor correlation between curli and hydrophobicity of E. coli O157:H7 strains was also reported in other studies (Rivas et al. 2007; Kim and Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

E. coli attachment on fresh produce

Harrison 2009). Curli-producing E. coli O157:H7 strains were significantly more hydrophobic than the non-curli producing strains (Boyer et al. 2007). These authors reported a positive relationship between curli production and hydrophobicity, but this correlation did not affect attachment to lettuce. Escherichia coli O157:H7 cells grown in TSB were hydrophilic in nature and attached better to lettuce surfaces than those grown in nutrient broth (Hassan and Frank 2004). In our study, E. coli O157:H7 strains 4406 and 4407 expressed stronger curli production and were more hydrophobic in nature in comparison with the other strains. It is difficult to compare our results with other studies as cell hydrophobicity can be influenced by the growth condition (planktonic or sessile), bacterial strain variability and the hydrophobicity method used in the study. Further, the BATH hydrophobicity assay used in most studies only estimates bulk properties of numerous cells and interprets the hydrophobic interactions. A method such as atomic force microscopy to measure hydrophobic forces of single living bacterial cell may be helpful in determining the effect of cell hydrophobicity on attachment to biotic and abiotic surfaces. Enteric pathogens can persist for longer duration outside the host if they form biofilms or bacterial aggregates. Populations in biofilm are resistant to chemical sanitizers and other environmental stresses (temperature, ultraviolet light, desiccation) used in the food industry (Kroupitski et al. 2009). In our study, E. coli O157:H7 strains 4688, 1908 and 5279 formed stronger biofilm in diluted TSB (1 : 10) than in full-strength TSB and LB. Stronger biofilm in diluted TSB medium could be attributed to the induction of biofilm under starvation stress (Solomon et al. 2005). The LB medium contains twice the amount of salts (NaCl) to that present in TSB. Salt may interfere with multi-cellular behaviour of E. coli in expression of adhesive extracellular matrix components (Ro¨mling 2005), which may have resulted in weak biofilm formation of E. coli O157:H7 in LB medium as observed in our study. Kroupitski et al. (2009) reported maximum biofilm formation by Salmonella when grown in diluted TSB but not in diluted LB medium. The biofilm formation of L. monocytogenes varied with strain, incubation temperature and nutritional conditions and was weakly correlated with the cell hydrophobicity (Folsom et al. 2006). The relationship between curli production by E. coli O157 and attachment to hydrophobic or hydrophilic surfaces varied with the strain used (Goulter et al. 2010). In our study, the difference in E. coli O157:H7 biofilm formation when grown in TSB or LB may be attributed to the genetic background of strain, cell surface hydrophobicity or compositional difference between these media. In our study, we were unable to find specific trends between curli expression and biofilm formation on polystyrene plates. 7

E. coli attachment on fresh produce

We found rapid attachment of E. coli O157:H7 strains to intact and cut surfaces of fresh produce (cabbage, Iceberg lettuce and Romaine lettuce). Other studies have reported a rapid attachment (in 5 min) of L. monocytogenes (Ells and Truelstrup Hansen 2006) and Salmonella (Patel and Sharma 2010) to intact and cut produce surfaces. In our study, E. coli O157:H7 strains 4407 and 4406 that expressed curli and were more hydrophobic in nature attached at higher numbers to produce surfaces compared to weakly expressing or nonexpressing strains of E. coli O157:H7. Presence of hydrophobic waxy cuticle on intact produce surfaces would have facilitated more attachment of hydrophobic E. coli O157:H7 strains. In our study, overall populations of E. coli O157:H7 attached to cut surfaces were higher than the E. coli O157:H7 populations attached to intact surfaces. An increase in attachment to cut surface may be attributed to greater surface area and congregation of E. coli O157:H7 along the edges of cut surfaces (Ells and Truelstrup Hansen 2006). Increased E. coli O157:H7 attachment to cut produce surfaces is a challenge to produce industry as produce wash treatment will be less effective on fresh-cut produce. We observed a positive correlation between biofilm formation on polystyrene plates and attachment to produce for strain 5279 only. Rivas et al. (2007) suggested that biofilm formation by E. coli O157:H7 on polystyrene plates may not be appropriate to represent attachment to other surfaces. Our results are in contrast to Kroupitski et al. (2009) study that reported positive correlation between strong biofilm formation by Salmonella on polystyrene surfaces and better attachment to cut lettuce. Cell surface hydrophobicity was highly correlated with the attachment strength of Salmonella, E. coli and L. monocytogenes to the cantaloupe rind (Ukuku and Fett 2002). These authors found that rate of attachment decreased after initial attachment; however, the attachment strength increased with time. The initial attachment strength of Listeria spp. on cabbage surfaces was significantly lower when cells were grown at 37C compared to the attachment strength of cells grown at 22C (Ells and Truelstrup Hansen 2006). However, the attachment strength increased with time; the difference in attachment strength of Listeria spp. grown at 22 and 37C was not significant after 24 h. An increase in the attachment strength with time may be associated with the production of surface appendages during the incubation period and subsequent binding of E. coli O157:H7 with other E. coli O157:H7 already attached to produce surface. Overall attachment strength of E. coli O157:H7 was significantly higher on Romaine lettuce than E. coli O157:H7 attachment strength on cabbage surfaces. Similar results were reported in earlier studies with Salm. enterica serovars (Patel and Sharma 2010). 8

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In conclusion, bacterial attachment to produce surfaces is quite complex process and many attachment mechanisms may be involved. This study demonstrates relationship between curli expression, hydrophobicity and attachment of E. coli O157:H7 to intact and cut produce surfaces. Our results show differences in E. coli O157:H7 attachment to produce surfaces because of strain variation and type of surfaces. Stronger attachment of E. coli O157:H7 to cut produce surfaces over intact surfaces emphasizes the need for effective wash treatments to reduce potential contamination of pathogens. Escherichia coli O157:H7 attaches rapidly to produce surfaces, and attachment strength increases with time once attached; early intervention strategies are required to prevent crosscontamination and proliferation on these surfaces. The knowledge gained in this study will be helpful in developing novel intervention strategies for fresh produce wash treatments. Acknowledgements The authors thank Katherine Hopkins and Ernie Paroczay for technical assistance and Dr Bryan Vinyard for statistical analysis. References Berger, C.N., Shaw, R.K., Brown, D.J., Mather, H., Clare, S., Dougan, G., Pallen, M.J. and Frankel, G. (2009) Interaction of Salmonella enterica with basil and other salad leaves. ISME J 3, 261–265. Boyer, R.R., Sumner, S.S., Williams, R.C., Pierson, M.D., Popham, D.L. and Kniel, K.E. (2007) Influence of curli expression by Escherichia coli O157:H7 on the cell’s overall hydrophobicity, charge, and ability to attach to lettuce. J Food Prot 70, 1339–1345. CDC (2006) Ongoing multistate outbreak of Escherichia coli serotype O157:H7 infections associated with consumption of fresh spinach-United States. Morbid Mortal Wkly Rep 55, 1045–1046. CDC (2010) Investigation Update: multistate Outbreak of Human E. coli O145 Infections Linked to Shredded Romaine Lettuce from a Single Processing Facility. Available at: http://wwwcdcgov/ecoli/2010/ecoli_0145/ Accessed 5-12-2010. DeWall, C.S. (2007) Testifying before the House Committee on Energy and Commerce, Import Inspection Failures and what must be done. Presented at 110th Cong, 2nd session, July 17, 2007. Ells, T.C. and Truelstrup Hansen, L. (2006) Strain and growth temperature influene Listeria spp. attachment to intact and cut cabbage. Int J Food Microbiol 111, 34–42. Ethelberg, S., Lisby, M., Bottiger, B., Schultz, A.C., Villif, A., Jensen, T., Olsen, K.E., Scheutz, F. et al. (2010) Outbreaks Journal of Applied Microbiology ª 2011 The Society for Applied Microbiology No claim to US Government works

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