William F. Fett

Behavior of Listeria monocytogenes inoculated on cantaloupe surfaces and efficacy of washing treatments to reduce transfer from rind to fresh-cut pieces.

Attachment and survival of Listeria monocytogenes on external surfaces (rind) of inoculated cantaloupe, resistance of the surviving bacteria to chlorine or hydrogen peroxide treatments, transfer of the pathogen from unsanitized and sanitized rinds to fresh-cut tissues during cutting and growth, and survival of L. monocytogenes on fresh-cut pieces of cantaloupe were investigated. Surface treatment with 70% ethanol to reduce the native microflora on treated melon, followed by immersion in a four-strain cocktail of L monocytogenes (10(8) CFU/ml) for 10 min, deposited 4.2 log10 CFU/cm2 and 3.5 log10 CFU/cm2 of L monocytogenes on treated and untreated cantaloupe rinds, respectively. L. monocytogenes survived on the treated or untreated cantaloupe rinds for up to 15 days during storage at 4 and 20 degrees C, but populations declined by approximately 1 to 2 log10 CFU/cm2. Fresh-cut pieces prepared from inoculated whole cantaloupes stored at 4 degrees C for 24 h after inoculation were positive for L. monocytogenes. Washing inoculated whole cantaloupes in solutions containing 1,000 ppm of chlorine or 5% hydrogen peroxide for 2 min at 1 to 15 days of storage at 4 degrees C after inoculation resulted in a 2.0- to 3.5-log reduction in L. monocytogenes on the melon surface. Fresh-cut pieces prepared from the sanitized melons were negative for L. monocytogenes. After direct inoculation onto fresh-cut pieces, L. monocytogenes survived, but did not grow, during 15 days of storage at 4 degrees C. Growth was evident by 4 h of storage at 8 and 20 degrees C. It is concluded that sanitizing with chlorine or hydrogen peroxide has the potential to reduce or eliminate the transfer of L. monocytogenes on melon surfaces to fresh-cut pieces during cutting.

Naturally Occurring Biofilms on Alfalfa and Other Types of Sprouts

Scanning electron microscopy was used to examine the cotyledons, hypocotyls, and roots of alfalfa, broccoli, clover, and sunflower sprouts purchased from retail outlets as well as alfalfa sprouts grown in the laboratory using a tray system equipped with automatic irrigation. Biofilms were observed on all plant parts of the four types of commercially grown sprouts. Biofilms were also commonly observed on alfalfa sprouts grown in the laboratory by 2 days of growth. Rod-shaped bacteria of various sizes were predominant on all sprouts examined both as free-living cells and as components of biofilms. Occasionally, cocci-shaped bacteria as well as yeast cells were also present in biofilms. The microbes contained in the biofilms appeared to be attached to each other and to the plant surface by a matrix, most likely composed of bacterial exopolysaccharides. Biofilms were most abundant and of the largest dimensions on cotyledons, sometimes covering close to the entire cotyledon surface (approximately 2 mm in length). Naturally occurring biofilms on sprouts may afford protected colonization sites for human pathogens such as Salmonella and Escherichia coli O157:H7, making their eradication with antimicrobial compounds difficult.

Relationship of cell surface charge and hydrophobicity to strength of attachment of bacteria to cantaloupe rind.

The cantaloupe melon has been associated with outbreaks of Salmonella infections. It is suspected that bacterial surface charge and hydrophobicity may affect bacterial attachment and complicate bacterial detachment from cantaloupe surfaces. The surface charge and hydrophobicity of strains of Salmonella, Escherichia coli (O157:H7 and non-O157:H7), and Listeria monocytogenes were determined by electrostatic and hydrophobic interaction chromatography, respectively. Initial bacterial attachment to cantaloupe surfaces and the ability of bacteria to resist removal by washing with water were compared with surface charge and hydrophobicity. Whole cantaloupes were submerged in inocula containing individual strains or in cocktails containing Salmonella, E. coli, and L. monocytogenes, either as a mixture of strains containing all three genera or as a mixture of strains belonging to a single genus, for 10 min. Inoculated cantaloupes were dried for 1 h in a biosafety cabinet and then stored for up to 7 days at 4 degrees C. Inoculated melons were washed with water, and bacteria still attached to the melon surface, as well as those in the wash water, were enumerated. Initial bacterial attachment was highest for individual strains of E. coli and lowest for L. monocytogenes, but Salmonella exhibited the strongest attachment on days 0, 3, and 7. When mixed-genus cocktails were used, the relative degrees of attachment of the three genera ware altered. The attachment of Salmonella strains was the strongest. but the attachment of E. coli was more extensive than that of L. monocytogenes on days 0, 3, and 7. There was a linear correlation between bacterial cell surface hydrophobicity (r2 = 0.767), negative charge (r2 = 0.738), and positive charge (r2 = 0.724) and the strength of bacterial attachment to cantaloupe surfaces.

Analysis of native microflora and selection of strains antagonistic to human pathogens on fresh produce.

The native microflora of three types of produce (green bell peppers, Romaine lettuce, and prepeeled baby carrots) and two types of sprouting seeds (alfalfa and clover) were investigated. Aerobic plate count (APC) for each produce or seed type as determined on Pseudomonas agar F (PAF) with incubation at 28 degrees C was in the range of 4 to 7 log CFU per g of tissue or seed. There was no significant difference (P > or = 0.05) in APC when the determinations were made with three agar media including PAF, brain heart infusion agar, and plate count agar. However, the APC as determined from plates that were incubated at 28 degrees C was significantly (P < or = 0.05) higher than with incubation at 37 degrees C. Fluorescent pseudomonads accounted for 23 to 73% of APC and 6 to 18% of APC recovered from carrots, pepper, and lettuce were pectolytic. Forty-eight strains of pectolytic bacteria were randomly isolated and identified, respectively, as members of the genera of Pseudomonas, Erwinia, Bacillus, Xanthomonas, or Flavobacterium. Lactic acid bacteria and/or yeast were consistently isolated from baby carrots, lettuce, and sprouting seeds (alfalfa or clover) but not from green bell peppers. Approximately 120 strains of indigenous microflora were tested for their ability to inhibit the growth of Salmonella Chester, Listeria monocytogenes, Escherichia coli, or Erwinia carotovora subsp. carotovora on PAF. Six isolates capable of inhibiting the growth of at least one pathogen were isolated and identified, respectively, as Bacillus spp. (three strains), Pseudomonas aeruginosa (one strain), Pseudomonas fluorescens (strain A3), and yeast (strain D1). When green pepper disks were inoculated with strains A3 and D1, the growth of Salmonella Chester and L. monocytogenes on the disks was reduced by 1 and 2 logs, respectively, over a period of 3 days. Application of strains A3 and D1 as potential biopreservatives for enhancing the quality and safety of fresh produce is discussed.

Inactivation of Escherichia coli O157:H7 on inoculated alfalfa seeds with ozonated water and heat treatment

Alfalfa seeds inoculated with a five-strain mixture of Escherichia coli O157:H7 were immersed in water containing 4, 8, 10, and 21 ppm of ozone for 2, 4, 8, 16, 32, and 64 min at 4 degrees C. Direct ozone sparging of seeds in water was used as an alternative mode of ozone treatment. Ozone-sparged seeds were also subsequently exposed to heat treatment at 40, 50, and 60 degrees C for 3 h. Populations of E. coli O157:H7 on untreated and treated seeds were determined by spread plating diluted samples on tryptic soy agar supplemented with 50 microg/ml of nalidixic acid. Since E. coli O157:H7 was released from inoculated seeds during treatment with ozone, the rate of release of cells from inoculated seeds soaked in 0.1% peptone water for up to 64 min was also determined. The overall reduction of E. coli O157:H7 on seeds treated with ozonated water without continuous sparging ranged from 0.40 to 1.75 log10 CFU/g (59.6 to 98.2%), whereas reductions for control seeds were 0.32 to 1.03 log10 CFU/g (51.7 to 90.5%). Treatment with higher ozone concentrations enhanced inactivation, but contact time longer than 8 min did not result in significantly higher reductions (P > 0.05). For seeds treated by ozone sparging, a 1.12-log10 CFU/g (92.1%) reduction was achieved using a 2-min contact time, and a 2.21-log10 CFU/g (99.4%) reduction was achieved with a 64-min contact time. The corresponding reductions for control seeds were 0.71 log10 CFU/g (79.5%) and 2.21 log10 CFU/g (99.4%), respectively. Treatment of ozone-sparged seeds at 60 degrees C for 3 h reduced the population to an undetectable level by direct plating (4 to 4.8 log10 CFU/g), although survivors were detected by enrichment. Ozone did not have a detrimental effect on seed germination percentage.

Effect of Nisin in Combination with EDTA, Sodium Lactate, and Potassium Sorbate for Reducing Salmonella on Whole and Fresh-Cut Cantaloupe

Nisin (50 microg/ml), EDTA (0.02 M, disodium salt), sodium lactate (NaL, 2%), and potassium sorbate (KS, 0.02%) were tested individually and in various combinations as sanitizer treatments for reducing Salmonella on whole and fresh-cut cantaloupe. Whole cantaloupe and fresh-cut pieces were inoculated with a five-strain cocktail of Salmonella to give 4.76 +/- 0.23 log CFU/cm2 and 3.42 +/- 0.13 log CFU/g, respectively. Inoculated whole melons and fresh-cut pieces were stored at 5 degrees C for 7 days. Washing treatments were applied to inoculated whole melons at days 0, 3, and 7 of storage, and surviving bacterial populations were determined. The effect of the washing treatments on transfer of Salmonella to fresh-cut pieces prepared immediately after treatment was also determined. Directly inoculated fresh-cut pieces were treated at day 0, and surviving bacteria were enumerated at days 0, 3, and 7 of storage. The combination treatments of nisin-EDTA, nisin-NaL, nisin-KS, NaL-KS, and nisin-NaL-KS all resulted in reductions of approximately 3 log CFU/cm2 at day 0 for whole melons. When tested alone, all compounds, along with water washes, were ineffective. After 3 and 7 days of storage, the five combination washing treatments were less effective, resulting in reductions of approximately 2 log CFU/cm2. None of the combination treatments completely eliminated transfer of pathogen survivors to fresh-cut pieces. The combination treatments nisin-NaL, nisin-KS, NaL-KS, and nisin-NaL-KS, but not nisin-EDTA, gave significant (P < 0.05) reductions of Salmonella directly inoculated onto fresh-cut pieces. Washing with nisin-NaL-KS was significantly (P < 0.05) more effective than the other three combination treatments, resulting in a reduction of 1.4 CFU/g. Inhibition by the four effective treatments carried over from day 0 through day 7 of storage, with no increase in the population of Salmonella on the stored fresh-cut pieces. Sensory evaluations indicated that treatment of fresh-cut pieces with nisin-NaL and NaL-KS, but not nisin-KS or nisin-NaL-KS, were acceptable in terms of appearance, odor, and overall acceptability. After the required regulatory approval, treatment of whole cantaloupe with nisin in combination with EDTA, NaL, KS, or NaL and KS and of fresh-cut pieces with nisin-NaL or NaL-KS could help ensure the microbiological safety of fresh-cut cantaloupe.

Cutinase production byStreptomyces spp.

Forty-fiveStreptomyces strains, including representatives of the plant pathogensS. acidiscabies, S. scabies, andS. ipomoea, were screened for ability to produce enzymes (cutinases) capable of hydrolyzing the insoluble plant biopolyester cutin. Initially, all strains were tested for production of extracellular esterase in liquid shake (250 rpm) cultures at room temperature in defined (glycerol-asparagine) or complex (tryptone-yeast extract with or without addition of mannitol) broth media supplemented with either tomato or apple cutin. Esterase activity was determined by a spectrophotometric assay utilizing the model substratep-nitrophenyl butyrate. Of the five strains exhibiting highest esterase activity, four (S. acidiscabies ATCC 49003,S. “scabies” ATCC 15485 and IMRU 3018, andS. badius ATCC 19888) were confirmed to produce enzymes with cutin-degrading activity (cutinases). Confirmation of extracellular cutinase production was accomplished by use of a new high-performance liquid chromatography method for separation and quantification of released cutin monomers. Monomer identification was confirmed by GC/MS analyses. Cutinase production was induced 2- to 17-fold by inclusion of cutin in the media. To our knowledge this constitutes the first report of cutinase production byStreptomyces spp. other thanS. scabies.

Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts.

A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were also determined. The treatment (10 or 20 min) of seeds in water (4 degrees C) containing an initial concentration of 21.8 +/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05) reduction in populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for 20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/- 0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05) reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water. Treatments (2 min) with up to 23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and natural microbiota compared to soaking in water (control) but did not enhance the lethality compared to the sprouts not treated with continuous sparging. The treatment of sprouts with ozonated water (20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the sensory quality during subsequent storage at 4 degrees C for 7 to 11 days. Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth, and biofilm formation that provide evidence of factors contributing to the difficulty of killing microorganisms by treatment with ozone and other sanitizers.

Reduction of the native microflora on alfalfa sprouts during propagation by addition of antimicrobial compounds to the irrigation water

Alfalfa and other types of sprouts are known to harbor large populations of native microorganisms. As some of these microbes may be causes of reduced shelf life of the product (plant pathogens and other spoilage organisms) and sprouts may, on occasion, harbor bacteria pathogenic towards humans, the addition of antimicrobial compounds to the irrigation water may be warranted. In this study, we tested the efficacy of several antimicrobial compounds for reducing the native microbial populations on alfalfa sprouts during propagation. These compounds included H2O2, peroxyacetic acid+hydrogen peroxide (Tsunami 100), acidified NaClO2, NaClO2 (Aquatize), EDTA, Na3PO4 and NaOCl. When added to the irrigation water at vanous concentrations, none of the antimicrobial compounds reduced the levels of any class of native microflora by more than 1 log10 without evidence of phytotoxicity.

Effects of cell surface charge and hydrophobicity on attachment of 16 Salmonella serovars to cantaloupe rind and decontamination with sanitizers.

Adherence of bacteria to cantaloupe rind is favored by surface irregularities such as roughness, crevices, and pits, thus reducing the ability of washing or sanitizer treatments to remove or inactivate attached cells. In this study, we compared the surface charge and hydrophobicity of two cantaloupe-related outbreak strains of Salmonella Poona (RM2350 and G-91-1595) to those of 14 additional Salmonella strains using electrostatic and hydrophobic interaction chromatography. The relative abilities of the 16 strains to attach to cantaloupe surfaces and resist removal by washing with water, chlorine (200 ppm), or hydrogen peroxide (2.5%) for 5 min after a storage period of up to 7 days at 5 to 20 degrees C also were determined. Whole cantaloupes were inoculated with each pathogen at 8.36 log CFU/ml, dried for 1 h inside a biosafety cabinet, stored, and then subjected to the washing treatments. Only the positive surface charge of the two cantaloupe-related strains of Salmonella Poona was significantly higher (P < 0.05) than that of the other strains. Initial bacterial attachment to cantaloupe surfaces ranged from 3.68 to 4.56 log CFU/cm2 (highest values for Salmonella Michigan, Newport, Oranienburg, and Mbandaka). The average percentage of the total bacterial population strongly attached to the cantaloupe surface for the Salmonella serovars studied ranged from 0.893 to 0.946 at 5 degrees C and from 0.987 to 0.999 at 25 degrees C. Washing inoculated melons with water did not produce a significant reduction in the concentration of the pathogens (P > 0.05). Chlorine and hydrogen peroxide treatments caused an average 3-log reduction when applied 20 to 40 min postinoculation. However, sanitizer treatments applied 60 min or more postinoculation were less effective (approximately 2.5-log reduction). No significant differences were noted in sanitizer efficacy against the individual strains (P > 0.05). The two cantaloupe-related outbreak Salmonella Poona strains did not significantly differ from the other Salmonella strains tested in negative cell surface charge or hydrophobicity, were not more effective in attaching to whole melon surfaces, and were not more resistant to the various washing treatments when present on rinds.