Fate of Listeria monocytogenes in Bovine Manure Amended Soil

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1 1676 Journal of Food Protection, Vol. 67, No. 8, 2004, Pages Copyright, International Association for Food Protection Fate of Listeria monocytogenes in Bovine Manure Amended Soil XIUPING JIANG, MAHBUB ISLAM, JENNIE MORGAN, A MICHAEL P. DOYLE* Center for Food Safety, University of Georgia, 1109 Experiment Street, Griffin, Georgia , USA MS : Received 22 October 2003/Accepted 6 March 2004 ABSTRACT The survival and growth of Listeria monocytogenes in soil amended with bovine manure was studied under different environmental conditions of temperature, nutrients, and soil microflora. Autoclaved soil was compared with unautoclaved soil for assessing the influence of competitive soil microflora on the survival of L. monocytogenes. Initial L. monocytogenes cell numbers of 5 to 6 log CFU/g survived for up to 43, 43, and 14 days in manure-amended autoclaved soil at 5,, and C, respectively. In manure-amended unautoclaved soil, the pathogen was detectable for up to 43,, and days at 5,, and C, respectively. L. monocytogenes was inactivated more rapidly in autoclaved soil amended with manure at a manure/soil ratio of than in the more dilute (0) manure in soil samples at both and C. However, in manure-amended unautoclaved soil, L. monocytogenes survived longer in samples with ratios of than in the more dilute (0) manureamended soil. The persistence of L. monocytogenes for several weeks in manure-amended soil suggests listeriae could be transmitted through soil to fresh produce or to shoes, clothing, and hands of field workers, especially during the cold months. Listeria monocytogenes is widespread in the environment and frequently contaminates soil, silage, water, and pastures. Animals can carry the pathogen without appearing ill and thereby contaminate foods of animal origin such as meats and dairy products. Vegetables can become contaminated from the soil or from manure used as fertilizer (3, 17). An outbreak in 1981 in Nova Scotia resulted in 41 cases of listeriosis, including 18 deaths; 83% of the cases were perinatal (). The outbreak was traced to L. monocytogenes on coleslaw that had been made from cabbage grown in a field fertilized with manure from Listeria-infected sheep. L. monocytogenes can grow on a variety of vegetables even at refrigeration temperatures (3). The prevalence of L. monocytogenes in feces of dairy cattle ranges from a few percentage points up to 52% (9, 16). Husu (9) reported that the highest seasonal prevalence of L. monocytogenes in feces of dairy cattle was in December (16.1%) and the lowest was in September (0.9%). On average, 34.5% of the animals in 249 herds shed L. monocytogenes in feces. Generally, when shed in cattle feces, L. monocytogenes is present in low cell numbers. Fenlon et al. (8) reported that cattle fed silage shed L. monocytogenes in feces at 3 to 26 CFU/g. Few researchers have investigated the fate of listeriae in animal manure when applied to the agricultural land. Van Renterghem et al. (18) studied the fate of L. monocytogenes in soil and manure at C and determined that the maximum length of survival of L. monocytogenes in pig manure, soil, and cattle manure was 3 to 4, 6 to 8, and 6 to 8 weeks, respectively. The potential for transmission of L. monocytogenes from contaminated manure and soil to produce may be * Author for correspondence. Tel: ; Fax: ; mdoyle@uga.edu. Present address: Department of Food Science and Human Nutrition, Clemson University, Clemson, SC 29634, USA. great for vegetables grown in soils amended with raw or inadequately treated manure that contains large populations of Listeria. However, research is needed on Listeria survival in manure applied to soil, treatments to reduce Listeria levels in manure, and assessment of the risk of crosscontamination of food crops from Listeria-contaminated manure used as a soil amendment. The objective of this study was to determine the effect of environmental factors on the fate of L. monocytogenes in manure-amended soil. Such information may be useful for establishing conditions to assure the safety of manure application. MATERIALS A METHODS Preparation of L. monocytogenes cultures for inoculation into manure. For inoculation of manure, a five-strain mixture of L. monocytogenes was used: strain Scott A (human isolate, serovar 4b), strain LCDC (cabbage isolate, serovar 4b), strain H7550 (hot dog isolate), strain 201, and strain DA3. To facilitate enumeration of these isolates, all five strains were labeled with jellyfish green fluorescent protein (GFP). The competent bacterial cells were electroporated in a Gene Pulser II (BioRad, Hercules, Calif.) with plasmid vector pnf8 (kindly provided by Dr. Nicolas Fortineau, Laboratoire de Microbiologie, Institut National de la Sante et de la Recherché Medicale U 411, Paris, France), and an electrical pulse ( 4 ms) was applied at 2.5 kv and 25 F with the pulse controller adjusted to 200 ohms. Transformants were selected from isolated colonies grown on Luria-Bertani plates containing 8 g/ml erythromycin. The resulting erythromycin-resistant transformants emitted green fluorescence under a handheld UV light. Each GFP-labeled L. monocytogenes strain was inoculated into 10 ml of Trypticase soy broth (TSB; Difco, Becton Dickinson, Sparks, Md.) containing 8 g/ml erythromycin and incubated at 37 C for 16 to 18 h with agitation (0 rpm). A 0.5-ml suspension of each isolate was transferred to 100 ml of TSB containing erythromycin (8 g/ml) and incubated for 16 to 18 h with agitation (0 rpm). The bacteria were harvested by centrifugation (4,000 g for 20 min) and then washed three times in 0.1%

2 J. Food Prot., Vol. 67, No. 8 LISTERIA MONOCYTOGENES IN MANURE 1677 peptone water. Each strain was adjusted with 0.1% peptone water to an optical density at 630 nm of 0.7 (ca CFU/ml) and combined in equal volume to obtain a five-strain mixture prior to inoculation of the manure. L. monocytogenes counts in the fivestrain mixture were determined by plating on Trypticase soy agar (TSA; Difco, Becton Dickinson) plates containing 8 g/ml erythromycin (TSA-E) or modified Oxford agar with supplements (MOX; Difco, Becton Dickinson). Fate of L. monocytogenes in soil amended with bovine manure. Sandy loam soil samples were collected from a field in Tifton, Ga., that was used to grow vegetables. The soil samples had a ph of 7.0 and a 7% moisture content (MC) and contained P, K, Ca, and Mg at 140, 123, 724, and 85 lb/acre (7, 138, 811, and 95 kg/ha), respectively. Fresh cow manure was collected from Ruck s dairy farm near Griffin, Ga. Clean plastic containers and utensils used for collecting the manure were sanitized with a solution of 600 ppm sodium hypochlorite before use. Manure from a group of healthy dairy cattle was pooled and frozen for at least 24 h to kill insects. The five-strain mixture of GFP-labeled L. monocytogenes was inoculated into the bovine manure at ca CFU/g. The culture mixture (10 ml) was added into 1 kg of manure in a mixer (KitchenAid, St. Joseph, Mich.) and then mixed for 5 min at medium speed. The inoculated manure was held at 23 C in sterile polypropylene trays as described previously (10). After 20 to 24 h of incubation, the inoculated manure was mixed with unautoclaved or autoclaved (1 C for 20 min on three successive days) soil at a ratio of 1 part of manure to 10 or 100 parts of soil (wt/wt), i.e., or 0. The manure-amended soil was then held inside a polyethylene plastic box with a lid (59 by 42 by 30 cm) at,, and 5 C. Enumeration and detection of L. monocytogenes in manure-amended soil. Duplicate soil samples (ca. 40 g each) were obtained from each treatment tray at each sampling interval and assayed for L. monocytogenes counts, ph, and MC. Each sample (5 g) was added to 45 ml of 0.1% peptone water in a sterile Whirl- Pak bag and pummeled in a Stomacher (Seward, London, UK) for 1 min at medium speed. Dilutions were prepared using 0.1% peptone water, and 0.1-ml aliquots of each dilution were spread in duplicate onto TSA, TSA-E, and MOX agar supplemented with Listeria supplements. Plates were incubated at 37 C for 48 h for determination of L. monocytogenes counts (MOX and TSA-E) and aerobic bacterial counts (TSA). The GFP-labeled L. monocytogenes colonies were counted under a UV light at 365 nm. When L. monocytogenes could not be detected by the direct plating method (L. monocytogenes detection limit was 50 CFU/ g), a selective enrichment culture method was used. A manureamended soil sample (1 g) was added to 99 ml of selective enrichment broth (TSB containing 8 g/ml erythromycin) and incubated at 37 C for 24 h with agitation (0 rpm). Dilutions of cultures were surface plated on both TSA-E and MOX agar plates. Other analyses. For ph determination, manure-amended soil (5 g) was added to 250 ml of distilled water in an Erlenmeyer flask. The suspension was stirred for 5 min and then allowed to settle for 5 min. The ph of the liquid was determined with an Accumet Basic ph meter (Fisher Scientific, Pittsburgh, Pa.). For MC analysis, manure-amended soil (5 g) was weighed and dried at 105 C for 24 h in a conventional drying oven (Precision Scientific, Winchester, Va.) and then weighed. Statistical analysis. Microbiological data were converted to log CFU per gram for analysis. An analysis of variance (ANOVA) for a completely randomized design with repeated measures across dates was conducted to determine whether general differences existed among the four treatment means. Specific comparisons between any pair of treatment means at any date were accomplished with Fisher s least significant difference (LSD) test. All calculations were performed using the GLM procedure of the Statistical Analysis System (SAS 2001, Cary, N.C.). The analysis was repeated for each of the six temperature and soil conditions. RESULTS L. monocytogenes populations in manure-amended autoclaved soil formulated at 0 (manure/soil) increased by ca. 1.0 log CFU/g at 5 C for 2 weeks compared with no growth in the formulation (Fig. 1A). During the following 2 weeks, L. monocytogenes populations in all samples of manure-amended autoclaved soil decreased very slowly at approximately the same rate. The pathogen survived up to 30 and 43 days in autoclaved 0 and samples, respectively. In contrast, there was no growth of L. monocytogenes in the 0 unautoclaved soil held at 5 C (Fig. 1B). L. monocytogenes was inactivated slowly up to 30 days and was nonculturable by 36 and 50 days in the 0 and samples, respectively. The efficacy of the two plating media, MOX and TSA-E, for enumerating L. monocytogenes cells from manure-amended autoclaved soil at 5 C was compared using an ANOVA with T grouping (Table 1). Significantly higher L. monocytogenes counts (P 0.05) were obtained on MOX than on TSA-E. L. monocytogenes populations increased within 3 days at C by ca. 0.1 and 1 log CFU/g in autoclaved soil formulated at and 0, respectively (Fig. 1C). Thereafter, L. monocytogenes died off rapidly in autoclaved soil samples and was not detectable by 14 days. However, the pathogen survived in 0 autoclaved soil samples for 43 days. L. monocytogenes did not grow in manure-amended unautoclaved soil held at C (Fig. 1D). L. monocytogenes populations decreased by 5 log CFU/g within 20 days in both samples and were no longer detectable thereafter, even by enrichment culture. At C, L. monocytogenes populations increased by ca. 1 log CFU/g within 3 days in the 0 autoclaved soil, whereas there was a slight increase (ca. 0.1 log CFU/g) in the formulation (Fig. 1E). However, L. monocytogenes survived for only 14 days in all manure-amended autoclaved soil samples. Similar survival patterns for L. monocytogenes were observed for the and 0 unautoclaved soil samples held at C compared with those held at C (Fig. 1F). Similar to results from studies at C, there was no growth of L. monocytogenes in manureamended unautoclaved soil samples. The pathogen died off rapidly, with similar rates in both and 0 samples at C (Fig. 1F) and was no longer detectable by direct plating in 14-day samples for both formulations. However, enrichment culture was positive for L. monocytogenes in the 0 sample only at day. The L. monocytogenes survival data in Figure 1 were analyzed statistically across all dates to determine whether general differences existed among the two treatment means. The LSD values (P 0.05) between and 0 treatment means at any date were 1.17, 0.24, 0.19, 0.43, 0.60, and 0. log CFU/g for Figure 1A, 1B, 1C, 1D, 1E, and

3 1678 JIANG ET AL. J. Food Prot., Vol. 67, No. 8 FIGURE 1. Survival of L. monocytogenes at 5 C in manure-amended autoclaved (A) and unautoclaved (B) soil, at C in manureamended autoclaved (C) and unautoclaved (D) soil, and at C in manure-amended autoclaved (E) and unautoclaved (F) soil. L. monocytogenes was enumerated on modified Oxford agar from manure/soil formulations of (#) and 0 ( ). A right arrow indicates below the detection limit; a left arrow indicates detection by enrichment culture only. Each point represents the average of duplicate trials.

4 J. Food Prot., Vol. 67, No. 8 LISTERIA MONOCYTOGENES IN MANURE 1679 TABLE 1. L. monocytogenes counts in cow manure amended autoclaved soil held at 5 C a Counts on day: Dilution Enumeration b medium B B A B A B A B A A A B A B TSA-E MOX B B A B A B A B A A A B 0 TSA-E MOX a Mean standard deviation (log CFU/g). Means within a column with different letters for the same treatment are significantly different (P 0.05). b Manure/soil ratio. 1F, respectively. L. monocytogenes died off significantly more rapidly (P 0.05) in 0 than in unautoclaved soil samples at all three temperatures. In contrast, L. monocytogenes multiplied and survived significantly better (P 0.05) in 0 than in autoclaved soil samples at and C. At each sampling time, the MC and ph values of the manure-amended soil were determined. After adding manure to both autoclaved and unautoclaved soil, the samples had higher MC (ca. 17%) and ph (ca. 7.4) than did 0 samples (MC 11% and ph 6.8) (Tables 2 and 3). The MC of samples held at 5 C decreased more slowly, from ca. 17 to %, during 50 days of storage than did that of samples held at or C. There was a rapid loss of moisture from all samples held at or C, to as low as ca. 2.7% at 43 days in one 0 sample held at C. During the first 1 to 2 weeks, the ph values of all manure-amended soil samples increased about 0.1 to 0.8 ph unit but thereafter decreased with increased storage time. The autoclaved soil samples maintained a slightly higher ph than did the 0 autoclaved soil samples. Overall, the ph values of the soil samples were in the range of 6.1 to 8.1 throughout the experiment. DISCUSSION GFP is a useful marker for studying the fate of bacteria in complex environments such as soil, water, manure, and food products (4, 5, 10). In this study, the GFP-labeled L. monocytogenes colonies weakly emitted green fluorescence under handheld UV light (probably because of the low expression level of the gfp gene in manure-amended soil). Because L. monocytogenes counts on TSA-E were lower than those on MOX, all the data shown in Figure 1 were based on counts on MOX. L. monocytogenes has been isolated from soil, mud, silage, water, animal feces, and fresh vegetables (3). Previous studies indicated that soil is a favorable medium for the growth and survival of L. monocytogenes (14). The pathogen survived for up to 400 days at 20 C in autoclaved dry chalky soil and decreased by only ca. 1 log CFU/g at 4 C by 1,500 days. The investigators in that study concluded that both a slightly acidic soil and low exterior temperature (4 C) increases the survival of L. monocytogenes, whereas a low-acid nutrient-rich soil decreased survival of the pathogen to within 160 days. In a few studies, the fate of L. monocytogenes in manure or manure-amended soil has been examined. Al-Ghazali et al. (1) isolated L. monocytogenes from a sewage treatment plant at all stages of treatment, and the pathogen population in final discharge ranged from 3 to 39 CFU/ ml. Watkins and Sleath (19) determined that L. monocytogenes populations were maintained at their initial levels for at least 8 weeks in sewage sludge applied to agricultural soils. Dowe et al. (6) evaluated the effect of soil type, inoculum level, microbial competition, manure, and nutrients on the survival of L. monocytogenes. Soil amended with chicken manure promoted better growth of L. monocytogenes than did liquid hog manure or inorganic fertilizer only when the competitive bacterial flora was reduced by

5 1680 JIANG ET AL. J. Food Prot., Vol. 67, No. 8 TABLE 2. Moisture content and ph of manure-amended autoclaved soil during storage Dilution a Values on day: Moisture content (%) b ph a Manure/soil ratio. b, not determined. autoclaving. Competition from indigenous soil microorganisms inhibited L. monocytogenes populations in manureamended soil. Van Renterghem et al. (18) determined that the maximum survival of L. monocytogenes in pig and cattle manure was 3 to 4 weeks and 6 to 8 weeks, respectively, at C. However, the pathogen was detected 3 months later in three of six radish samples grown in soil inoculated with the pathogen in pig manure. They suggested that the manure-amended soil lacked saprophytic conditions (containing dead or decaying vegetation), which are considered conducive to the survival and growth of L. monocytogenes. In our study, L. monocytogenes survived for about 2 to 3 weeks in both autoclaved and unautoclaved manure-amended soil (without plant root materials) at or C and for 6 to 7 weeks at 5 C. The relative rapid decline of L. monocytogenes populations in manure-amended soil at or C may be due to the rapid growth of competitive manureborne or soilborne bacteria at warmer temperatures. In our study, autoclaved soil was used as a control to minimize the influence of indigenous soil microorganisms on the survival and growth of L. monocytogenes. The 1: 100 autoclaved soil samples should have contained ca. 10 times less nutrients and microorganisms, including L. monocytogenes from manure, than the autoclaved soil samples. The rapid die-off of L. monocytogenes at and C in the autoclaved soil samples is likely a result of the antagonistic effect of microorganisms in the manure. Unlike the fate of L. monocytogenes in the manure-amended autoclaved soil, listeriae in both the and 0 unautoclaved soil samples did not multiply, and their populations declined at approximately the same rate at all three temperatures. There was significant initial growth of L. monocytogenes in the 0 autoclaved soil samples. The autoclaved soil may have had fewer competitive microorganisms to impede the growth of L. monocytogenes and more nutrients that were readily available for the growth of L. monocytogenes. Dowe et al. (6) reported that L. monocytogenes, TABLE 3. Moisture content and ph of manure-amended unautoclaved soil during storage Temperature Temperature Dilution a Values on day: Moisture content (%) b ph a Manure/soil ratio. b, not determined.

6 J. Food Prot., Vol. 67, No. 8 LISTERIA MONOCYTOGENES IN MANURE 1681 when inoculated at low numbers, grew modestly (ca. 1.5 log CFU/g) in 16 days in sandy soil with no added manure. In a study of Escherichia coli O7:H7, growth of the pathogen occurred at and C in 0 manure-amended autoclaved soil samples (10). Lazarovits (12) determined that manure application to soil reduced populations of plant pathogens but increased the overall populations of soil microorganisms by up to 1,000-fold. These results suggest that the initial levels of competing microbes and available nutrients can influence the fate of microbial pathogens in manure-amended soil. Other foodborne pathogens such as E. coli O7:H7 and Salmonella can survive in soil for several months following manure application (2, 7, 10, 11, 13). Factors influencing survival of these pathogens in manure-amended soil include temperature, types of manure and soil, manure/soil ratio, and level of competitive bacterial flora. Natvig et al. (13) suggested applying manure during cool spring conditions ( 10 C) to reduce the potential for contamination of vegetables by Salmonella enterica serovar Typhimurium. However, L. monocytogenes survival in manure-amended soil was higher at low temperatures than at high temperatures. Therefore, this recommendation may not apply to L. monocytogenes in soil containing manure. The survival of L. monocytogenes in manure-amended soil for up to 7 weeks can facilitate the spread of this pathogen to the soil, water, animals, and fresh produce. This information is useful for identifying manure application practices that will reduce the risk of L. monocytogenes transmission from contaminated animal waste in soil. Further study is needed to determine the influence of vegetation on the fate of L. monocytogenes in manure applied to soil as a plant fertilizer. ACKNOWLEDGMENTS This study was funded in part by a grant from the U.S. Department of Agriculture (USDA) Initiative for Future Agriculture and Food Systems and by a special grant from the USDA-CSREES for the Alliance for Food Protection. We thank Dr. Gascho (University of Georgia, Tifton campus) for assistance in obtaining soil samples. REFERENCES 1. Al-Ghazali, M. R., and S. K. Al-Azawi Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq. J. Appl. Bacteriol. 60: Baloda, S. B., L. Christensen, and S. Trajcevska Persistence of a Salmonella enterica serovar Typhimurium DT12 clone in a piggery and in agricultural soil amended with Salmonella-contaminated slurry. Appl. Environ. Microbiol. 67: Brackett, R. E Incidence, contributing factors, and control of bacterial pathogens in produce. Postharvest Biol. Technol. : Dandie, C. E., S. M. Thomas, and N. C. 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