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1857 Journal of Food Protection, Vol. 70, No. 8, 2007, Pages 1857–1865 Copyright 䊚, International Association for Food Protection

Aerosol Studies with Listeria innocua and Listeria monocytogenes GUODONG ZHANG,1 LI MA,1 OMAR A. OYARZABAL,2 1Center

AND

MICHAEL P. DOYLE1*

for Food Safety, University of Georgia, Griffin, Georgia 30223; and 2Department of Poultry Science, Auburn University, Auburn, Alabama 36849, USA MS 06-556: Received 27 October 2006/Accepted 25 March 2007

ABSTRACT Aerosol studies of Listeria monocytogenes in food processing plants have been limited by lack of a suitable surrogate microorganism. The objective of this study was to investigate the potential of using green fluorescent protein–labeled strains of Listeria innocua as a surrogate for L. monocytogenes for aerosol studies. These studies were conducted in a laboratory bioaerosol chamber and a pilot food-processing facility. Four strains of L. innocua and five strains of L. monocytogenes were used. In the laboratory chamber study, Listeria cells were released into the environment at two different cell numbers and under two airflow conditions. Trypticase soy agar (TSA) plates and oven-roasted breasts of chicken and turkey were placed in the chamber to monitor Listeria cell numbers deposited from aerosols. A similar experimental design was used in the pilot plant study; however, only L. innocua was used. Results showed that L. monocytogenes and L. innocua survived equally well on chicken and turkey breast meats and TSA plates. No-fan and continuous fan applications, which affected airflow, had no significant effect on settling rates of aerosolized L. monocytogenes and L. innocua in the bioaerosol chamber or L. innocua in the pilot plant study. Listeriae cell numbers in the air decreased rapidly during the first 1.5 h following release, with few to no listeriae detected in the air at 3 h. Aerosol particles with diameters of 1 and 2 ␮m correlated directly with the number of Listeria cells in the aerosol but not with particles that were 0.3, 0.5, and 5 ␮m in diameter. Results indicate that L. innocua can be used as a surrogate for L. monocytogenes in an aerosol study.

Airborne microorganisms in food-processing facilities are extremely important because of the economic and health problems they may cause. Controlling microbial contamination in food processing plants can greatly reduce the chance of producing harmful food products (1, 2). Investigation of four poultry-slaughtering plants (two turkey and two duck) for airborne mesophilic and psychrotrophic bacteria, yeasts, and molds revealed that airborne microbial counts were highest in the shackling areas and decreased toward the packaging areas. Bacteria were the most common airborne microorganisms in these plants. Mesophilic bacteria counts ranged from 6 log CFU/m3 of air in the shackling area to 2.5 log CFU/m3 of air outside the plants. Yeast and mold counts were usually between 2.5 and 4.0 log CFU/m3 of air in the plants (16). Enterobacteriaceae and Escherichia coli were very common in defeathering areas of commercial poultry processing plants, and thermophilic campylobacters were most frequently isolated from air samples in the defeathering area, followed by the evisceration areas (26). Enterobacteriaceae, Micrococcaceae, streptococci, Flavobacterium, Alcaligenes, Acinetobacter, and Pseudomonadaceae were predominant in the air samples of the chilling facilities of the poultry processing plants (12). Studies of total aerobic bacteria, molds and yeasts, coliforms, and pseudomonads in the air in three shell egg processing operations (in-line, off-line, and mixed * Author for correspondence. Tel: 770-228-7284; Fax: 770-229-3216; E-mail: [email protected].

operations) (areas sampled were hen house, farm transition room, egg washers, egg dryer, packer heads, postprocessing cooler, nest-run cooler, loading dock, and dry storage) found that the highest counts for total aerobic bacteria (5.9 log CFU/ml of air), molds and yeasts (4.0 log CFU/ml of air), and coliforms (2.5 log CFU/ml of air) were in the hen house; the highest counts for pseudomonads were in the hen house (3.2 log CFU/ml of air) and behind the egg washer (3.5 log CFU/ml of air) (18). During the commercial production of Japanese quail, the highest counts for total aerobic bacteria (8.1 log CFU/ml of air), molds and yeasts (3.6 log CFU/ml of air), E. coli (1.9 log CFU/ml of air), and Enterobacteriaceae (2.3 log CFU/ml of air) occurred in the grow-out house; at the processing facility, the highest counts for total aerobic bacteria (6.8 log CFU/ml of air), E. coli (1.4 log CFU/ml of air), and Enterobacteriaceae (1.5 log CFU/ml of air) occurred in the areas where quail are hung or stunned and scalded or defeathered (19). In beef and pork processing establishments, high prevalence and populations of airborne microorganisms were reported, and associations between the microbial contamination of air and carcasses with the movements of workers were found (14, 15, 21, 23). It was reported that airborne microbial contamination in sausage processing plants could affect the shelf life of cooked ring sausages (17). Listeria monocytogenes is a widely distributed grampositive bacterium commonly present in the environment and less frequently in food. It has been isolated from soil, sewage, and vegetation (24). Listeriae survive well in cold

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and moist locations, including refrigeration and air handling systems, and thrive in a variety of foods, including soft cheeses, deli meats, and frankfurters (6, 7). The pathogen can be found on walls, floors, drains, ceilings, and equipment in food-processing facilities (3, 9, 22, 25). Listeriae can survive on nutritionally depleted dustlike particles for more than 151 days at 10⬚C and 88% relative humidity, as well as 82 days at 22⬚C and 0% relative humidity (8). L. monocytogenes cells on various surfaces and vents could easily get into the air in a food processing plant when air conditioners are on and fans are running all the time. Therefore, L. monocytogenes cells in the aerosol of food processing plants may be contributing to the contamination of foods. Because of its virulence, L. monocytogenes cannot be used in food processing plants to determine dissemination and contamination, and the lack of a suitable surrogate microorganism has limited the study of the transmission of listeriae in aerosols. The genus Listeria has six species, including L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi (4). L. innocua is very similar to L. monocytogenes in growth properties, biological characteristics, guanine-plus-cytosine DNA content, 16S rRNA sequence, electroporation transformation rate, and morphology (4, 20) but is not hemolytic and not pathogenic. L. innocua has been used as a surrogate in lieu of L. monocytogenes in several studies. Houtsma et al. (13) used L. innocua to predict L. monocytogenes growth rates in the presence of different sodium lactate and sodium chloride concentrations. Francis and O’Beirne (11) used L. innocua to study the effects of the indigenous microflora of minimally processed lettuce on the survival and growth of L. monocytogenes. Omary et al. (20) used L. innocua to study the growth of L. monocytogenes in shredded cabbage. However, L. monocytogenes was not included in these studies to validate the use of L. innocua as a surrogate of L. monocytogenes. Francis and O’Beirne (10), who compared the growth and survival of L. innocua and L. monocytogenes on minimally processed lettuce under different package atmospheres and storage temperatures, concluded that their behaviors were similar and used L. innocua for further studies on the effects of antimicrobial dip on the fate of L. monocytogenes on lettuce. Bourke and O’Beirne (5) compared the survival and growth of L. innocua with L. monocytogenes in packaged, shredded, dry coleslaw and determined that two strains of L. monocytogenes had survival characteristics comparable to L. innocua. However, they concluded that the use of L. innocua as a surrogate for L. monocytogenes required further investigation. The objective of this study was to determine the potential of using green fluorescent protein–labeled L. innocua strains as surrogates for L. monocytogenes for aerosol studies. MATERIALS AND METHODS Bacteria. Four strains of L. innocua (three strains from foodprocessing facility environments [E, 18, and Strep, provided by Silliker Laboratories, Chicago Heights, Ill., and ATCC 33090, isolated from the bovine brain]) and five strains of L. monocytogenes (J0161-1 [1/2a, human, Centers for Disease Control and Preven-

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tion, Atlanta, Ga.], H7550 [4b, human, Centers for Disease Control and Prevention], Scott A [4b, human, Centers for Disease Control and Prevention], 101M-1 [4b, beef and pork sausage, U.S. Department of Agriculture, Washington, D.C.], and F6854 [4b, frankfurter, U.S. Department of Agriculture]) labeled with jellyfish green fluorescent protein genes were used. Cultures were grown in Trypticase soy broth (TSB; Difco, Becton Dickson, Sparks, Md.) with 8 ␮g of erythromycin per ml at 35⬚C for 24 h and stored in TSB containing 30% glycerol at ⫺80⬚C. Erythromycin was added to maintain the plasmid with the green fluorescent protein gene. Broth cultures were prepared by subculturing from the frozen stock culture to TSB with 0.6% yeast extract and 8 ␮g of erythromycin per ml at 35⬚C for 24 h. Each culture was transferred three times before use. An equal volume of each bacterial strain was combined and used as a mixture of L. innocua or L. monocytogenes. Cell numbers were enumerated on Trypticase soy agar (TSA; Difco, Becton Dickinson) and incubated at 35⬚C for 24 h. Delicatessen meats. Oven-roasted breasts of chicken and turkey were used in both the bioaerosol chamber and food processing plant studies. These products were purchased from a local retail store, held at 4⬚C, and used within 1 week. The deli meats in unopened packages were sliced in the store. The meat slices were cut aseptically under a biological safety cabinet (class II, type A/B3, NuAire, Plymouth, Minn.) and placed into sterile plastic petri dishes (100 by 15 mm). Meats were tested for Listeria spp. before use according to the protocol described below. Ingredients on the labels of products included the following. (i) For ovenroasted breast of chicken, ingredients included chicken breast meat, water, salt, sugar, sodium phosphate, and extractives of spice and onion. (ii) For oven-roasted breast of turkey, ingredients included turkey breast, turkey broth, less than 2% salt, sugar, sodium phosphate, and flavoring. Experiments in a bioaerosol chamber. A Plexiglas bioaerosol chamber, 124 cm long by 51 cm wide by 51 cm high, was constructed for the laboratory study. Two gloves for manipulating plates and instruments were hermetically sealed to two ports. For additional safety, the bioaerosol chamber was placed inside a laminar flow hood throughout the study. All studies were conducted at 17.5⬚C with 75% relative humidity. Aerosolization of L. innocua or L. monocytogenes was created by a nebulizer (Mabis Mist Ultrasonic nebulizer, model 40-050-000, Mabis Healthcare, Inc., Lake Forest, Ill.) placed at the bottom center of the chamber. Particle size distribution in the aerosols was monitored every 30 min by an aerosol particulate monitor (model GT-321, Met One Instrument, Grants Pass, Oreg.). The monitor was placed at the bottom center of the chamber. Particle sizes of 0.3, 0.5, 1, 2, and 5 ␮m were monitored. Two minifans (air velocity at 9.7 m/min at the bottom center of the chamber) were attached to each end of the chamber, and one fan from each end was operated to create a more uniform aerosol. The second fan was a replacement if the first fan malfunctioned (Fig. 1). Listeriae culture (5 ml for each trial) was nebulized into the chamber for 10 min with fans operating before samples were exposed. Two Listeria cell numbers (105 and 103 CFU/liter of air) were used. Listeriae overnight culture diluents in 0.1% peptone water in concentrations of 105 (1.6 ⫻ 105 to 9.6 ⫻ 105) and 107 (1.2 ⫻ 107 to 8.1 ⫻ 107) CFU/ml were used to provide ca. 103 and 105 CFU/liter of air, respectively. The release rates were 2.4 ⫻ 105 (7.9 ⫻ 104 to 4.8 ⫻ 105) and 2.1 ⫻ 107 (6.1 ⫻ 106 to 4.0 ⫻ 107) CFU/min for 103 and 105 CFU/liter of air, respectively. Two fan conditions were compared: (i) no fan operating after samples were exposed and (ii) continuous fan blowing throughout the 3-h study. TSA plates, oven-roast-

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ed chicken breast, and oven-roasted turkey breast (in petri dish plates) were placed in the chamber, and the contents of the plates were exposed at different times to monitor the changes of Listeria cell number in the aerosol environment. All plates were placed in their original plastic covers before and after exposure to aerosol to prevent unanticipated exposure. Plates were placed in the chamber before aerosolization was initiated and removed from the chamber when the trial was completed. The chamber was not opened during the 3-h trial. For each trial, three TSA plates were unexposed and served as controls. Every 30 min, triplicate TSA plates and chicken and turkey meat samples were exposed, while previously exposed samples were placed in plastic covers. This was accomplished six times throughout each 3-h trial. Samples were identified as BC1 for the first 30 min of exposure time, BC2 for the second 30 min, through BC6 for the sixth 30 min. TSA plates were incubated at 35⬚C for 24 h and then enumerated for Listeria. Each turkey and chicken breast sample (ca. 20 g per slice) was macerated in a stomacher with 180 ml of University of Vermont broth (Difco, Becton Dickinson) and incubated at 30⬚C for 24 h. Enrichment cultures were streaked onto modified Oxford (Difco, Becton Dickinson) plates and incubated at 35⬚C for 24 h. Bacterial colonies were examined for fluorescence with a Leica X-Cite 120 Fluorescence Illumination System (Leica Microsystems, Inc., Bannockburn, Ill.). They were confirmed by the API Listeria kit (bioMe´rieux, Hazelwood, Mo.). All trials were repeated three times. Experiments in a small-scale food processing plant. Two separate trials were conducted in a pilot-scale food-processing facility (Department of Poultry Science, Auburn University, Auburn, Ala.), 6.7 m long by 6.7 m wide by 3 m high. A series of three sets of fans (five fans each set; model SA44-170, Witt Heat Transfer Products, Scottsboro, Ala.) with an airflow rate of 282

FIGURE 1. Layout of nebulizers, fans, and particle monitor in a bioaerosol chamber (A) and in a small-scale processing plant (B).

TABLE 1. Listeria monocytogenes– or Listeria innocua–positive plates following aerosolization in a bioaerosol chambera Fan was not operating when plates were exposed to aerosols (CFU/liter of air) 105 Sample group

TSAb

Turkeyc

Fan was continuously operating throughout the trial (CFU/liter of air)

103

105

103

Chickend

TSA

Turkey

Chicken

TSA

Turkey

Chicken

TSA

Turkey

Chicken

Listeria monocytogenes BC1e 9 BC2 9 BC3 9 BC4 9 BC5 9 BC6 8 Control 0

9 9 9 9 9 6 0

9 9 9 9 8 6 0

9 5 8 1 1 0 0

9 6 5 2 2 0 0

9 7 7 3 2 0 0

9 9 9 9 9 8 0

9 9 9 9 9 8 0

9 9 9 9 9 9 0

9 9 6 1 0 0 0

9 5 3 1 0 0 0

9 8 4 0 0 0 0

Listeria innocua BC1 9 BC2 9 BC3 9 BC4 9 BC5 9 BC6 9 Control 0

9 9 9 9 9 9 0

9 9 9 9 9 9 0

9 9 9 9 8 5 0

9 9 6 7 5 4 0

9 9 9 8 5 3 0

9 9 9 9 7 4 0

9 9 9 9 6 3 0

9 9 9 8 6 3 0

9 8 5 2 1 0 0

9 7 5 4 1 1 0

9 9 4 0 1 1 0

The total plates of sample medium for each sampling time ⫽ 9. Trypticase soy agar plates. c Oven-roasted turkey breast. d Oven-roasted chicken breast. e BC1, first 30 min of exposure; BC2, second 30 min; through BC6, sixth 30 min. a b

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TABLE 2. Listeria innocua–positive plates in a food-processing facility with fans continuously operating 0.5b Sample groupa

Tur- ChickTSAc keyd ene TSA

1.5

2.5

Tur- Chickkey en TSA

Tur- Chickkey en

Trial 1 FP1f FP2 FP3 FP4 FP5 Control

9 9 5 3 2 0

9 9 5 1 0 0

9 9 8 3 0 0

9 9 5 5 1 0

9 9 5 2 0 0

9 9 5 3 2 0

9 9 6 4 2 0

9 9 4 1 1 0

9 9 5 2 0 0

Trial 2 FP1 FP2 FP3 FP4 FP5 Control

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

3 3 3 3 3 0

a

In trial 1, L. innocua (106 CFU/ml) was aerosolized for 40 min into the pilot plant by a nebulizer, which was discontinued immediately before exposing samples to the aerosol. The total plates of sample medium per sampling time ⫽ 9. In trial 2, L. innocua (104 CFU/ml) was aerosolized into the plant by a nebulizer throughout the trial. The total plates of sample medium per sampling time ⫽ 3. b Distance (expressed in meters) of TSA or meat plates from the wall with fans. c Trypticase soy agar plates. d Oven-roasted breast of turkey. e Oven-roasted breast of chicken. f FP1, first 30 min of exposure; FP2, second 30 min; though FP5, fifth 30 min. m3 per min were located 2 m above the floor on three sides of the walls of the processing facility. The nebulizers (Medline, Sport Mist II, model HCS30004, Medline Industries, Inc., Mundelein, Ill.) for aerosolization were hung immediately in front of and below the middle fan (Fig. 1). The temperature in the pilot plant was 13⬚C with 37% relative humidity. Persons entering the pilot plant while the study was in progress wore NexGen coveralls (DuPont Personal Protection, DuPont, Wilmington, Del.) and respirators (2M 6100/07024, 3M Occupational Health and Environmental Safety Division, St. Paul, Minn.). For trial 1, L. innocua in 0.1% peptone water (106 CFU/ml distributed at a rate of 1.43 ml/10 min) was aerosolized for 40 min into the pilot plant by a nebulizer with all three sets of fans operating. The nebulizer was discontinued after 40 min, but the fans continued to operate during the entire trial. Plates with TSA, oven-roasted breast of chicken, and oven-roasted breast of turkey were placed 0.5, 1.5, and 2.5 m in front of the wall with fans. Every 30 min, three plates each of previously unexposed TSA and chicken and turkey meat speci-

mens were exposed, and previously exposed samples were removed. This was done five times throughout each 2.5-h trial. Samples were identified as FP1 for the first 30 min of exposure, FP2 for the second 30 min, through FP5 for the fifth 30 min. TSA plates and meat specimens were prepared and incubated for the detection of L. innocua according to the protocol described above. In trial 2, L. innocua in 0.1% peptone water (104 CFU/ml distributed at a rate of 1.43 ml/10 min) was aerosolized into the plant by a nebulizer with all fans operating throughout the trial. After 30 min of aerosolization, TSA plates and meat specimens were exposed. Listeria testing was conducted according to the protocol described above. Statistical analysis. PROC CORR was used for analysis of Pearson correlation coefficients (SAS 9.1.3; SAS Institute Inc., Cary, N.C.).

RESULTS Contamination of TSA and chicken and turkey breast meat plates by aerosolized L. monocytogenes and L. innocua. Contamination of TSA and chicken and turkey breast meat plates through aerosolized L. monocytogenes and L. innocua during the time of exposure is illustrated in Tables 1 and 2. All control plates in plastic covers, which were placed in the chamber and never exposed to the aerosol, were negative for green fluorescent protein–labeled Listeria. For L. monocytogenes studies, in which the fan was not operating when the plates were exposed to aerosol with 105 CFU/liter of air, most samples were L. monocytogenes positive throughout the 3-h exposure time. For aerosols with 103 CFU/liter of air, all plates were positive at BC1, and the number of Listeria-positive plates decreased gradually to the nondetectable level at the 3-h sampling time. With continuous fan operations throughout the trial, all 162 TSA and chicken and turkey meat plates, except for 2, were L. monocytogenes positive when exposed to aerosol with 105 CFU of L. monocytogenes per liter of air. For aerosol with 103 CFU/liter of air, all plates exposed for the first 30 min (BC1) were L. monocytogenes positive, whereas no L. monocytogenes was detected at the 2.5-h sampling time (BC5) (Table 1). With no fan operating when the plates were exposed to aerosol at 105 CFU of L. innocua per liter of air, all samples were positive throughout the 3-h study. However, with 103 CFU of L. innocua per liter of air, most plates were Listeria positive for up to 2 h of sampling, whereas less than half of the plates were Listeria positive at 3 h of sampling. With fans operating continuously throughout the 3-h trial, the number of L. innocua–positive plates decreased gradually from the 30-min to 3-h samplings for trials with either 105 or 103 CFU of L. innocua per liter of air. However, L. innocua–positive plates decreased consis→

FIGURE 2. Settling rates of L. monocytogenes and L. innocua with different initial cell numbers in a bioaerosol chamber. (A) 103 CFU of Listeria per liter of air, no forced airflow (fans not operating). (B) 105 CFU of Listeria per liter of air in the chamber, no forced airflow. (C) 103 CFU of Listeria per liter of air, continuous airflow with fans. (D) 105 CFU of Listeria per liter of air, continuous airflow with fans. 䡵, L. monocytogenes; ⽧, L. innocua; BC1, first 30 min of exposure; BC2, second 30 min; through BC6, sixth 30 min.

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FIGURE 3. Settling rates of L. innocua in a small-scale processing plant with continuous airflow with fans operating. (A) 106 CFU of L. innocua per ml released into the air prior to exposure for 40 min (trial 1). (B) 104 CFU of L. innocua per ml being continuously released into the air throughout the trial (trial 2). Distance from the wall with fans was as follows: ⽧, 0.5 m; 䡵, 1.5 m; 䉱, 2.5 m. FP1, first 30 min of exposure of TSA and turkey and chicken meat plates; FP2, second 30 min; through FP5, fifth 30 min.

tently to a low level of contamination (0 of 9 or 1 of 9 L. innocua positive) at the 2.5- and 3.0-h sampling times (Table 1). Results from trial 1 in the food processing plant studies showed that all plates at all distances were L. innocua positive at the 30-min and 1-h sampling times (FP1 and FP2) and consistently decreased to a low incidence of contamination (0 of 9 or 2 of 9 L. innocua positive) at the 2.5-h sampling time (Table 2). All plates at all distances during the 2.5-h trial were L. innocua positive for trial 2 (Table 2). Settling rates of aerosolized L. monocytogenes and L. innocua. Listeria cell numbers in the aerosol were determined by colony counts on TSA plates. Under all con-

ditions in the bioaerosol chamber study, i.e., no or continuous operating fans and either 105 or 103 CFU of Listeria per liter of air, Listeria cell numbers in the aerosol decreased dramatically during the first hour after the aerosol was released (BC1 and BC2) but declined slowly during the following 2 h (BC3 through BC6). By BC6 (3 h after aerosolization was stopped), there were still low numbers of Listeria cells in the air (Fig. 2). Listeria cell numbers were too high to count during the first 30 min following the release of the aerosol when 105 CFU/liter of air was used; hence, no data were collected. Graphs of settling rates of L. monocytogenes and L. innocua for both fan conditions and cell numbers either paralleled or overlapped each other (Fig. 2). →

FIGURE 4. Correlation of settling rates at 17.5⬚C with 75% relative humidity between L. monocytogenes and L. innocua at different initial Listeria cell numbers aerosolized into a bioaerosol chamber. (A) 105 CFU of Listeria per liter of air in the chamber, continuous airflow with fans operating. (B) 105 CFU of Listeria per liter of air in the chamber, no forced airflow (fans not operating). (C) 103 CFU of Listeria per liter of air in the chamber, continuous airflow with fans operating. (D) 103 CFU of Listeria per liter of air in the chamber, no forced airflow (fans not operating).

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TABLE 3. Relationship (Pearson correlation coefficients) between different particle sizes and Listeria cell numbers in the aerosol in a bioaerosol chamber a Particle size (␮m): Trialb

0.3

0.5

1

2

teria cell numbers in the aerosol (P ⱕ 0.01). In all eight trials, highly significant positive correlations (Pearson correlation coefficients, ⬎0.6900, P ⱕ 0.01) between numbers of 1- and 2-␮m particles and Listeria cell numbers in the aerosol were observed (Table 3).

5

DISCUSSION

L. monocytogenes 5 C fan 0.2527 5 No-fan 0.3031 3 C fan 0.3368 3 No-fan ⫺0.0814

0.5721*c 0.6124* 0.6791** 0.4958*

0.7499** 0.8504** 0.8766** 0.8828**

0.7505** 0.2270 0.8659** ⫺0.0366 0.6924** 0.6900* 0.8263** 0.6820*

L. innocua 5 C fan 0.4462 5 No-fan ⫺0.7044* 3 C fan ⫺0.2355 3 No-fan ⫺0.5431*

0.7720** 0.3396 0.3916 0.2121

0.8311** 0.8527** 0.9334** 0.8415**

0.6950** 0.8189** 0.9267** 0.8418**

0.4907 0.4485 0.7855** 0.4652

a

Values are expressed as CFU per plate. L. monocytogenes or L. innocua at cell numbers of 105 CFU/ liter of air (5) or 103 CFU/liter of air (3) with fans continuously operating (C fan) or no fan operating (No-fan). c * Significant at P ⱕ 0.05; ** significant at P ⱕ 0.01. b

Similar trends were observed in the small-scale food processing plant studies (Fig. 3). In trial 1, in which the release of L. innocua into the plant was stopped immediately before exposure of samples to the aerosol, L. innocua cell numbers in the air decreased substantially during the first hour (FP1 and FP2) and gradually thereafter with time, similar to the results from the bioaerosol chamber. There was no apparent difference in L. innocua cell numbers in the aerosol at a distance ranging from 0.5 to 2.5 m from the wall with fans (Fig. 3). In the second small-scale processing plant trial, in which L. innocua was continuously released into the plant during the entire trial, L. innocua cell numbers in the air decreased at a much slower rate than during the first trial (Fig. 3). Although there were slightly higher cell numbers at a distance of 2.5 m, there was no significant difference in cell numbers at distances of 0.5 to 2.5 m. Correlation of settling rates between L. monocytogenes and L. innocua in a bioaerosol chamber. Results of regression and correlation analyses are shown in Figure 4. Correlation coefficients of cell numbers (per plate) of L. monocytogenes and L. innocua at different time intervals (BC1 through BC6) were all above 0.99 for both fan conditions and different cell numbers in the aerosol in the bioaerosol chamber. Relationship between numbers of different sizes of particles and Listeria cell numbers in the aerosol in the bioaerosol chamber. In eight trials with different airflow conditions, different Listeria cell numbers, and different bacteria, two trials had negative correlations between numbers of 0.3-␮m particles and Listeria cell numbers in the aerosol (P ⱕ 0.05); five trials had positive correlations between numbers of 0.5-␮m particles and Listeria cell numbers in the aerosol (P ⱕ 0.01), and three trials had positive correlations between numbers of 5-␮m particles and Lis-

When Listeria colonies were on TSA plates, Listeria usually was also present on turkey and chicken breast meat specimens. When no Listeria colonies were on TSA plates, Listeria usually was not present on turkey and chicken breast meat specimens. These results showed that L. monocytogenes and L. innocua survived as well on chicken and turkey breast meats as on TSA plates (Tables 1 and 2). Chicken and turkey breast meats are more expensive and more difficult to handle for scientific study in comparison with TSA plates. Also, very few background microflora contaminations were observed on TSA plates exposed to aerosol in the chamber. Therefore, TSA plates could be used in lieu of turkey and chicken breast meat for studying aerosol contamination by L. innocua or L. monocytogenes. With the 3-h study in the bioaerosol chamber, there was no significant difference between having a continuous fan or no fan operating on the settling rates of aerosolized L. monocytogenes and L. innocua. Most of the Listeria cells settled down during the first hour of release into the air, regardless of the number of cells released into the air, although slightly more Listeria cells remained in the air for higher concentration treatments (105 CFU/liter of air) in the following hours (Fig. 2). The settling rates of aerosolized L. monocytogenes and L. innocua were highly correlated (Fig. 4) and were very similar. These results indicate that L. monocytogenes and L. innocua cells respond similarly in aerosols in their settling characteristics. Aerosol particles with diameters of 1 and 2 ␮m exhibited a significantly positive correlation with the number of Listeria cells in the aerosol. The lower the numbers of particles with diameters of 1 and 2 ␮m, the lower the Listeria cell numbers in the aerosol. However, particles 0.3, 0.5, and 5 ␮m in diameter did not consistently correlate with Listeria cell numbers in the aerosol (Table 3). Bacteria of the genus Listeria are nonbranching, regular, short (0.5 to 2 by 0.4 to 0.5 ␮m) gram-positive rods that occur singly or in short chains (4). It is very unlikely to have Listeria cells in particles equal to or less than 0.5 ␮m. Conversely, larger particles (5 ␮m) may contain one or multiple Listeria cells in one particle, and their numbers are not proportional to the number of Listeria cells in the aerosol. This may explain why 0.3-, 0.5-, and 5-␮m particles did not correlate well with Listeria cell numbers in the aerosol. The data showed that 1- and 2-␮m particles could be used to predict Listeria cell numbers in the aerosol. In the pilot plant trial, L. innocua cells survived for at least 2.5 h, similar to what occurred in the bioaerosol chamber. Cells were relatively evenly distributed in the aerosol within 2.5 m from the wall with or without fans operating when TSA and meat specimens were exposed to the aerosol. This suggests that if there is Listeria aerosol contamination in a food-processing facility, a similar likelihood of

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contamination will occur in foods that are 1 to 2.5 m from the contamination source, whether fans are on or off. On the basis of the similarities of L. innocua and L. monocytogenes in their survival and settling rates in the aerosol, we concluded that L. innocua could be used as a surrogate for L. monocytogenes in aerosol studies.

L. INNOCUA IN AEROSOL STUDIES

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ACKNOWLEDGMENTS

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This study was partially funded by a grant from the American Meat Institute Foundation. We thank Vijayalakshmi Mantripragada, Brent Harrison, Tiffany Green, and Kenneth Floyd for technical assistance.

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