Evaluation of Culture Methods To Identify Bovine Feces with High Concentrations of Escherichia coli O157

Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log₁₀ CFU/g. The preenrichment streak detected >3.0 log₁₀ CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log₁₀ CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.     

Elimination of Escherichia coli O157:H7 from Fermented Dry Sausages at an Organoleptically Acceptable Level of Microencapsulated Allyl Isothiocyanate

Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log₁₀ CFU/g Pediococcus pentosaceus and 6 log₁₀ CFU/g Staphylococcus carnosus) and a five-strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log₁₀ CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at ≤26°C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13°C for at least 25 days. The water activity (a_w), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a_w from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log₁₀ CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log₁₀ CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists.     

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log₁₀ 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log₁₀ 6.9 CFU/g).     

Fate of pGFP-Bearing Escherichia coli O157:H7 in Ground Beef at 2 and 10°C and Effects of Lactate, Diacetate, and Citrate

Although beef has been implicated in the largest outbreaks of Escherichia coli O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing E. coli O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10°C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2°C, the initial numbers of E. coli O157:H7 (3 and 5 log₁₀ CFU/g) decreased by ~1 log₁₀ CFU/g when spoilage was manifest (>7 log₁₀ CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10°C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.     

Reduction of Escherichia coli O157:H7 Populations in Cattle by Addition of Colicin E7-Producing E. coli to Feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10⁷ CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log₁₀ CFU/g was observed, with a maximum decrease of 1.8 log₁₀ CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10⁸ CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

Survival of Cold-Stressed Campylobacter jejuni on Ground Chicken and Chicken Skin during Frozen Storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4°C, freezing at −20°C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log₁₀ CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log₁₀ CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and −20°C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Control of Listeria spp. by Competitive-Exclusion Bacteria in Floor Drains of a Poultry Processing Plant

In previous studies workers determined that two lactic acid bacterium isolates, Lactococcus lactis subsp. lactis C-1-92 and Enterococcus durans 152 (competitive-exclusion bacteria [CE]), which were originally obtained from biofilms in floor drains, are bactericidal to Listeria monocytogenes or inhibit the growth of L. monocytogenes both in vitro and in biofilms at 4 to 37°C. We evaluated the efficacy of these isolates for reducing Listeria spp. contamination of floor drains of a plant in which fresh poultry is processed. Baseline assays revealed that the mean numbers of Listeria sp. cells in floor drains sampled on six different dates (at approximately biweekly intervals) were 7.5 log₁₀ CFU/100 cm² for drain 8, 4.9 log₁₀ CFU/100 cm² for drain 3, 4.4 log₁₀ CFU/100 cm² for drain 2, 4.1 log₁₀ CFU/100 cm² for drain 4, 3.7 log₁₀ CFU/100 cm² for drain 1, and 3.6 log₁₀ CFU/100 cm² for drain 6. The drains were then treated with 10⁷ CE/ml in an enzyme-foam-based cleaning agent four times in 1 week and twice a week for the following 3 weeks. In samples collected 1 week after CE treatments were applied Listeria sp. cells were not detectable (samples were negative as determined by selective enrichment culture) for drains 4 and 6 (reductions of 4.1 and 3.6 log₁₀ CFU/100 cm², respectively), and the mean numbers of Listeria sp. cells were 3.7 log₁₀ CFU/100 cm² for drain 8 (a reduction of 3.8 log₁₀ CFU/100 cm²), <1.7 log₁₀ CFU/100 cm² for drain 1 (detectable only by selective enrichment culture; a reduction of 3.3 log₁₀ CFU/100 cm²), and 2.6 log₁₀ CFU/100 cm² for drain 3 (a reduction of 2.3 log₁₀ CFU/100 cm²). However, the aerobic plate counts for samples collected from floor drains before, during, and after CE treatment remained approximately the same. The results indicate that application of the two CE can greatly reduce the number of Listeria sp. cells in floor drains at 3 to 26°C in a facility in which fresh poultry is processed.     

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log₁₀ and 6 log₁₀ CFU g⁻¹.Bovines are a reservoir for verotoxigenic Escherichia coli O157:H7 and Campylobacter jejuni, pathogenic microorganisms responsible for severe human gastrointestinal disease (5, 12). Qualitative and quantitative detection of these organisms in bovine feces is essential for evaluating risk to human health. Real-time PCR (quantitative PCR [qPCR]) assays have been developed to detect and quantify both E. coli O157:H7 and C. jejuni bacteria by using DNA directly extracted from animal feces (20, 22). Analysis of DNA extracted from bovine feces can generate a high level of correlation between the actual target cell density and the PCR signal (7, 8). However, the detection of E. coli O157:H7 and C. jejuni by direct DNA extraction is less sensitive and more variable than detection by procedures based on a preliminary enrichment step (e.g., laboratory culture) (7, 9, 16, 20). We explored the potential of lyophilization for improving overall detection by qPCR through increasing the amount of bovine fecal material available for DNA extraction.Four sets of five fresh bovine fecal samples were collected, and each sample was divided into four equal portions. Samples were seeded with either (i) E. coli O157:H7 (strain NZRM 3614) grown for 18 h at 37°C in tryptic soy broth (BD, Sparks, MD) or (ii) C. jejuni (strain NZRM 1958) grown for 48 h at 42°C in Exeter broth (11) to obtain the following concentrations: set 1, 0 CFU g⁻¹ (unseeded) and 3.5 log₁₀, 4.5 log₁₀, and 5.5 log₁₀ CFU of E. coli O157:H7 g⁻¹, and set 2, 0 CFU g⁻¹ (unseeded) to 5.2 log₁₀ CFU of E. coli O157:H7 g⁻¹. Set 3 and 4 concentrations varied from 0 CFU g⁻¹ (unseeded) to 6.4 log₁₀ C. jejuni CFU g⁻¹. DNA was either extracted directly from fresh samples or extracted from samples after lyophilization. Lyophilization involved mixing of prepared fecal samples in phosphate-buffered saline (145 mM NaCl, 59 mM Na₂HPO₄, 8 mM KH₂PO₄, pH 7.5) at a ratio of 1:10 (wt/vol), homogenization with a lab blender model 400 (Seward Medical, London, United Kingdom), cooling to −35°C, and concentration using a 1015GP lyophilizer (Cuddon Ltd., Blenheim, New Zealand). Total DNA was extracted from 0.2 g of a fresh or lyophilized fecal sample by using a QIAamp DNA stool minikit (Qiagen Inc., Mississauga, Canada). DNA was amplified using either a TaqMan E. coli O157:H7 detection kit (Applied Biosystems, Foster City, CA) or mapA primers and a corresponding probe (1). Amplification and fluorescence data were collected with optical-grade 96-well plates by using a TaqMan 7300 PCR system (Applied Biosystems). For each DNA sample, a mean threshold cycle (C_T) value for triplicate qPCR runs was calculated. When no C_T value was obtained, an arbitrary C_T value of 40 was assigned. All data were reported as equivalent concentrations in fresh feces. Significance levels were determined by one-way analysis of variance. The relationship between the log₁₀ numbers of CFU g⁻¹ fresh feces (viable-cell counts) and C_T values was analyzed using GenStat software (version 10.2.0.175; VSN International, Oxford, United Kingdom). Confidence intervals were obtained using the software program Flexi (21).Lyophilized samples were associated with significantly improved sensitivity (P < 0.001) at seeding levels of 4.5 and 5.5 log₁₀ E. coli O157:H7 CFU g⁻¹ (Table ​(Table1).1). At 3.5 log₁₀ CFU g⁻¹, the rate of E. coli O157:H7 detection was also higher, with all lyophilized samples producing a C_T value of <40 (Table ​(Table1).1). Individual C_T values for the three qPCR amplification runs were sufficiently similar to allow averaging (P > 0.05). Regression analysis of the averaged set 2 and 3 data (Fig. ​(Fig.1)1) demonstrated that the detection of both E. coli O157:H7 and C. jejuni was linear for seeding levels ranging from ca. 2 log₁₀ to 6 log₁₀ CFU g⁻¹ fresh feces. The range of concentrations used reflects the reported range of concentrations of these bacteria in feces (i.e., 0 to 6 log₁₀ CFU g⁻¹) as determined by conventional culture (3, 4, 18, 19). The high coefficients of correlation for the relationships between the log₁₀ numbers of CFU g⁻¹ feces and the C_T values indicated the specific amplification of the target DNA. The reproducibility of detection of E. coli O157:H7 was reduced at the lowest seeding concentration (i.e., 2.2 log₁₀ CFU g⁻¹ feces), with 75% of the samples giving a C_T value of <40. The limit for 100% successful detection after lyophilization was 2.9 log₁₀ E. coli O157:H7 CFU g⁻¹. The detection of C. jejuni by qPCR varied between sets. For set 3, 100% reproducibility occurred at 2.2 log₁₀ C. jejuni CFU g⁻¹. For set 4, satisfactory detection was obtained only after dilution of the DNA extract prior to qPCR. Despite this requirement for dilution, C. jejuni was still detected in 80% of the samples of set 4 seeded at a density of 2.2 log₁₀ C. jejuni CFU g⁻¹.Open in a separate windowFIG. 1.Ranges of quantification of E. coli O157:H7 (A) and C. jejuni (B) bacteria obtained from lyophilized fecal samples by real-time PCR. Each point represents the average C_T value for triplicate runs of one fecal sample at one seeding concentration. The hatched areas represent the 95% confidence intervals.

TABLE 1.

Difference in C_T values obtained for real-time PCR detection of E. coli O157:H7 in seeded fecal samples (n = 5) with and without lyophilization

Evidence of Association of Salmonellae with Tomato Plants Grown Hydroponically in Inoculated Nutrient Solution

The possibility of uptake of salmonellae by roots of hydroponically grown tomato plants was investigated. Within 1 day of exposure of plant roots to Hoagland nutrient solution containing 4.46 to 4.65 log₁₀ CFU of salmonellae/ml, the sizes of the pathogen populations were 3.01 CFU/g of hypocotyls and cotyledons and 3.40 log₁₀ CFU/g of stems for plants with intact root systems (control) and 2.55 log₁₀ CFU/g of hypocotyls and cotyledons for plants from which portions of the roots had been removed. A population of ≥3.38 log₁₀ CFU/g of hypocotyls-cotyledons, stems, and leaves of plants grown for 9 days was detected regardless of the root condition. Additional studies need to be done to unequivocally demonstrate that salmonellae can exist as endophytes in tomato plants grown under conditions that simulate commonly used agronomic practices.     

Prediction of the nutrients value and biochemical characteristics of swine slurry by measurement of EC – Electrical conductivity

Slurry samples, collected from 41 commercial swine farms in South Korea, were characterized in various physico-biochemical, macro and micronutrients, heavy metals and microbial parameters. Interestingly, significant variations were observed in all the parameters. However, positive relationships were noticed between EC and ammonia nitrogen (NH₃-N), total nitrogen (TN), total potassium (TK), specific gravity (SG), total solids (TS), volatile solids (VS), fixed solids (FS), total dissolved solids (TDS) at R² = 0.91, 0.74, 0.69, 0.60, 0.50, 0.48, 0.55, and 0.52, respectively. Whereas phosphorous and other nutrients shown poor correlation. Escherichia coli and Salmonella were counted at an average of 5.04 log₁₀ colony forming unit (CFU)/mL and 3.55 log₁₀ most probable number (MPN)/mL, respectively. Equations for predicting nutrients content in swine slurries are presented with EC, because it is an easily determinable parameter. The data obtained in this study could be used as a guideline for Good Management Practices in South Korean swine farms as well as other countries.     

Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log₁₀ CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log₁₀ were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log₁₀ CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Sorting through the Wealth of Options: Comparative Evaluation of Two Ultraviolet Disinfection Systems

Background

Environmental surfaces play an important role in the transmission of healthcare-associated pathogens. Because environmental cleaning is often suboptimal, there is a growing demand for safe, rapid, and automated disinfection technologies, which has lead to a wealth of novel disinfection options available on the market. Specifically, automated ultraviolet-C (UV-C) devices have grown in number due to the documented efficacy of UV-C for reducing healthcare-acquired pathogens in hospital rooms. Here, we assessed and compared the impact of pathogen concentration, organic load, distance, and radiant dose on the killing efficacy of two analogous UV-C devices.

Principal Findings

The devices performed equivalently for each impact factor assessed. Irradiation delivered for 41 minutes at 4 feet from the devices consistently reduced C. difficile spores by ∼ 3 log₁₀CFU/cm², MRSA by>4 log₁₀CFU/cm², and VRE by >5 log₁₀CFU/cm². Pathogen concentration did not significantly impact the killing efficacy of the devices. However, both a light and heavy organic load had a significant negative impacted on the killing efficacy of the devices. Additionally, increasing the distance to 10 feet from the devices reduced the killing efficacy to ≤3 log₁₀CFU/cm² for MRSA and VRE and <2 log₁₀CFU/cm² for C.difficile spores. Delivery of reduced timed doses of irradiation particularly impacted the ability of the devices to kill C. difficile spores. MRSA and VRE were reduced by >3 log₁₀CFU/cm² after only 10 minutes of irradiation, while C. difficile spores required 40 minutes of irradiation to achieve a similar reduction.

Conclusions

The UV-C devices were equally effective for killing C. difficile spores, MRSA, and VRE. While neither device would be recommended as a stand-alone disinfection procedure, either device would be a useful adjunctive measure to routine cleaning in healthcare facilities.     

Concurrent Quantitation of Total Campylobacter and Total Ciprofloxacin-Resistant Campylobacter Loads in Rinses from Retail Raw Chicken Carcasses from 2001 to 2003 by Direct Plating at 42°C

This is the first report on the use of a normally lethal dose of ciprofloxacin in a Campylobacter agar medium to kill all ciprofloxacin-sensitive Campylobacter spp. but allow the selective isolation and quantitation of naturally occurring presumptive ciprofloxacin-resistant Campylobacter CFU in rinses from retail raw chicken carcasses (RTCC). Thermophilic-group total Campylobacter CFU and total ciprofloxacin-resistant Campylobacter CFU (irrespective of species) were concurrently quantified in rinses from RTCC by direct plating of centrifuged pellets from 10 or 50 ml out of 400-ml rinse subsamples concurrently on Campylobacter agar and ciprofloxacin-containing Campylobacter agar at 42°C (detection limit = 0.90 log₁₀ CFU/carcass). For 2001, 2002, and 2003, countable Campylobacter CFU were recovered from 85%, 96%, and 57% of RTCC, while countable ciprofloxacin-resistant Campylobacter CFU were recovered from 60%, 59%, and 17.5% of RTCC, respectively. Total Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.52, 0.90 to 4.58, and 0.90 to 4.48 log₁₀ CFU/carcass in 2001, 2002, and 2003, respectively. Total ciprofloxacin-resistant Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.06, 0.90 to 3.95, and 0.90 to 3.04 log₁₀ CFU/carcass in 2001, 2002, and 2003, respectively. Overall, total Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, 4 to 5 log₁₀ CFU/carcass, respectively, were recovered from 16%, 32%, 26%, and 5% of RTCC tested over the 2-year sampling period. For the same period, total ciprofloxacin-resistant Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log₁₀ CFU/carcass, respectively, were recovered from 24%, 11%, 7%, and 0.2% of RTCC tested. There was a steady decline in total Campylobacter and total ciprofloxacin-resistant Campylobacter loads in RTCC rinses from 2001/2002 to 2003.     

Room temperature sterilization of surfaces and fabrics with a One Atmosphere Uniform Glow Discharge Plasma

We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log₁₀ CFU reduction of bacteria when 2 × 10² cells were seeded on filter paper. Results showed ≥3 log₁₀ CFU reduction when polypropylene samples seeded with E. coli (5 × 10⁴) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect ≥6 log₁₀ CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated ≥5 log₁₀ CFU reduction of commercially prepared Bacillus subtilis spores (1 × 10⁶); 7 min OAUGDP exposures were required to generate a ≥3 log₁₀ CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed. Received 06 June 1997/ Accepted in revised form 01 November 1997     

Baseline Data from a Belgium-Wide Survey of Campylobacter Species Contamination in Chicken Meat Preparations and Considerations for a Reliable Monitoring Program

From February to November 2007, chicken meat preparations (n = 656) were sampled at 11 processing companies across Belgium. All samples were tested for Campylobacter by enrichment culture and by direct plating according to standard methods. Almost half (48.02%) of the samples were positive for Campylobacter spp. The mean Campylobacter count was 1.68 log₁₀ CFU/g with a standard deviation of ± 0.64 log₁₀ CFU/g. The study revealed a statistically significant variation in Campylobacter contamination levels between companies; processors with a wider frequency distribution range of Campylobacter counts provided chicken meat preparations with higher Campylobacter incidences and concentrations. There was no significant difference between the counts of Campylobacter spp. in various preparation types. However, the Campylobacter counts and incidences in chicken wings were the highest and portioned-form products (legs, wings, and breasts) showed a higher probability of being Campylobacter positive compared to minced-form products (sausages, burgers, and minced meat). The proportion of Campylobacter-positive samples was significantly higher in July than in other months. Recovery of Campylobacter spp. recovery by direct plating was higher (41.0%) compared to detection after enrichment (24.2%). Statistical modeling of the survey data showed that the likelihood of obtaining a positive result by enrichment culture increases with an increase in the Campylobacter concentration in the sample. In the present study, we provide the first enumeration data on Campylobacter contamination in Belgian chicken meat preparations and address proposals for improving Campylobacter monitoring programs.     

Effect of Bacteriophage Application on Campylobacter jejuni Loads in Commercial Broiler Flocks

Campylobacteriosis is the most frequent food-borne human enteritis. The major source for infection with Campylobacter spp. is broiler meat. Risk assessments consider the reduction of Campylobacter in primary production to be most beneficial for human health. The aim of this study was to test the efficacy of a bacteriophage application under commercial conditions which had proved to be effective in previous noncommercial studies under controlled experimental conditions. A phage cocktail for Campylobacter reduction was tested on three commercial broiler farms each with a control and an experimental group. Colonization of Campylobacter was confirmed prior to phage application in fecal samples. Subsequently, a phage cocktail was applied via drinking water in the experimental group (log₁₀ 5.8 to 7.5 PFU/bird). One day after phage application, Campylobacter counts of one experimental group were reduced under the detection limit (<50 CFU/g, P = 0.0140) in fecal samples. At slaughter, a significant reduction of >log₁₀ 3.2 CFU/g cecal content compared to the control was still detected (P = 0.0011). No significant reduction was observed in the experimental groups of the other trials. However, a significant drop in cecal Campylobacter counts occurred in a phage-contaminated control. These results suggest that maximum reduction of Campylobacter at the slaughterhouse might be achieved by phage application 1 to 4 days prior to slaughter.     

Behavior of Bacillus anthracis Strains Sterne and Ames K0610 in Sterile Raw Ground Beef

The behavior of Bacillus anthracis Sterne spores in sterile raw ground beef was measured at storage temperatures of 2 to 70°C, encompassing both bacterial growth and death. B. anthracis Sterne was weakly inactivated (−0.003 to −0.014 log₁₀ CFU/h) at storage temperatures of 2 to 16°C and at temperatures greater than and equal to 45°C. Growth was observed from 17 to 44°C. At these intermediate temperatures, B. anthracis Sterne displayed growth patterns with lag, growth, and stationary phases. The lag phase duration decreased with increasing temperature and ranged from approximately 3 to 53 h. The growth rate increased with increasing temperature from 0.011 to 0.496 log₁₀ CFU/h. Maximum population densities (MPDs) ranged from 5.9 to 7.9 log₁₀ CFU/g. In addition, the fate of B. anthracis Ames K0610 was measured at 10, 15, 25, 30, 35, 40, and 70°C to compare its behavior with that of Sterne. There were no significant differences between the Ames and Sterne strains for both growth rate and lag time. However, the Ames strain displayed an MPD that was 1.0 to 1.6 times higher than that of the Sterne strain at 30, 35, and 40°C. Ames K0610 spores were rapidly inactivated at temperatures greater than or equal to 45°C. The inability of B. anthracis to grow between 2 and 16°C, a relatively low growth rate, and inactivation at elevated temperatures would likely reduce the risk for recommended ground-beef handling and preparation procedures.     

Enumeration of Escherichia coli Cells on Chicken Carcasses as a Potential Measure of Microbial Process Control in a Random Selection of Slaughter Establishments in the United States

To evaluate whether the number of Escherichia coli bacteria in carcass rinses from chicken slaughter establishments could be monitored for the purpose of microbial process control, we drew a random sample from 20 of 127 large USDA-inspected operations. In 2005, every 3 months, two sets of 10 carcass rinses, 100 ml each, were collected from establishments, netting 80 sample sets from the rehang and postchill stages. E. coli and Campylobacter numbers and Salmonella prevalence were measured. Mixed-effect models were used to estimate variance of mean log₁₀ E. coli cell numbers of 10-carcass rinse sample sets. Relationships between E. coli and Campylobacter and Salmonella were examined. For 10-carcass rinse sets, at both the rehang and postchill stages the mean log₁₀ E. coli CFU/ml fit the logistic distribution better than the normal distribution. The rehang overall mean log₁₀ E. coli was 3.3 CFU/ml, with a within-sample set standard deviation of 0.6 CFU/ml. The overall postchill mean log₁₀ E. coli was 0.8 CFU/ml, with 13 establishments having mean log₁₀ E. coli CFU/ml values of less than 1.0 and 7 having mean values of 1.2 or more. At the midpoint separating these establishments, a mean log₁₀ E. coli CFU/ml of 1.1, the within-sample set standard deviation was 0.5 CFU/ml, with smaller standard deviations as means increased. Postchill sample sets with mean log₁₀ E. coli counts less than or equal to 1.1 CFU/ml had lower overall prevalence of Salmonella and mean log₁₀ Campylobacter CFU/ml than sample sets with higher means. These findings regarding reductions in E. coli numbers provide insight relevant to microbial process control.Regulatory food microbiology standards are defined and enforced with the intent of protecting public health and maintaining consumer confidence in the safety of the food supply. Resource demands (22) and legal constraints (21) have hindered the U.S. Department of Agriculture (USDA) from enforcing its current Salmonella performance standard (3). For this reason, in 2004 the USDA requested guidance from its national scientific advisory committee on the possible use of E. coli numbers to monitor sanitary conditions during poultry slaughter (12). The committee acknowledged that, if valid, such a performance standard could facilitate inspection of slaughter processing establishments. The committee recommended studies to define how E. coli numbers vary in poultry carcass rinses during poultry processing by processing stage, time of year, and geographic region and with respect to food-borne pathogens.The widespread presence and high numbers of generic E. coli bacteria on poultry entering the slaughter establishment (2, 5, 14) are suitable characteristics for an indicator organism used to monitor microbial control processes. The ease and lower cost (5, 13) of E. coli enumeration also allow more observations than can be made when comparable resources are allocated for Campylobacter or Salmonella testing (15).Regulatory agencies and food manufacturers have recognized the potential utility of E. coli numbers as a measure of slaughter process control. For example, USDA''s hazard analysis and critical control point rule (3) specifies two criteria for evaluating process control: establishments are to maintain less than 100 CFU of E. coli per ml in 80% of poultry carcass rinses and never exceed 1,000 CFU/ml. Surveys have been performed to define precise E. coli performance criteria for poultry (5), to monitor microbial reduction during slaughter processing (6), and to validate interventions to reduce microbial numbers on poultry (20).If generic E. coli numbers on poultry carcasses fit a parametric distribution, with a predictable mean and standard deviation, then carcasses could be monitored using a statistical process control plan. For example, if E. coli numbers decrease by an acceptable amount during processing to a reasonable level, then the process could be considered to be under control. Or a plan could be designed to monitor for acceptable occurrences of small, medium, and large deviations above a target E. coli number (7). If relationships were found between E. coli and Campylobacter numbers during chicken slaughter as well as Salmonella prevalence, they would further support the use of E. coli numbers as a measure of process control.This study of a random sample of 20 large chicken slaughter operations located throughout the United States measured microbial numbers at two processing line locations. Once a quarter, 10 carcass rinse samples were collected from both the post-feather-pick (rehang) and postchill locations. Rinses were examined to estimate mean Salmonella prevalence and E. coli and Campylobacter numbers by location within establishments. The primary objective was to assess whether the reduction in E. coli numbers between the rehang and postchill stages or numbers at the postchill location might have utility as a measure of process control during chicken slaughter. A related objective was to estimate values of parameters that could be used to design statistical process control plans (7).     

Influences of short-term pre-slaughter dietary manipulation in sheep and goats on pH and microbial loads of gastrointestinal tract

Sheep (BW = 39.9 kg, n = 16) and goats (BW = 32.8 kg, n = 16) were used in a completely randomized design to determine the effect of short-term pre-slaughter diet and feed deprivation (FD) time on pH and microbial loads in the gastrointestinal tract (GIT) contents. In a 2 × 2 × 2 factorial treatment arrangement, the main effects of species, diet, and FD time prior to slaughter and their interactions were studied. Animals were fed either a hay or concentrate diet for 4 d and then feed deprived for either 12 or 24-h prior to slaughter. The pH of rumen and colon contents as well as weight of GIT was measured. The contents of rumen and rectum were also sampled for microbial analysis. The GIT of sheep (1.82 kg) was heavier (P < 0.05) than that of goats (1.46 kg). The 12-h FD group (1.74 kg) had a higher (P < 0.05) GIT weight than the 24-h FD group (1.53 kg). Hay-fed animals had higher (P < 0.05) rumen (7.08 vs. 6.43) and colon pH values (7.02 vs. 6.56) than those of the concentrate-fed animals. The 24-h FD group (3.39 ± 0.272 log₁₀CFU/g) contained more (P < 0.05) Escherichia coli in the rumen than did the 12-h FD (2.17 ± 0.272 log₁₀CFU/g) group. The concentrate-fed animals (3.49 ± 0.289 log₁₀CFU/g) had higher (P < 0.05) coliform counts in the rumen than the hay-fed animals (2.43 ± 0.289 log₁₀CFU/g). The 24-h FD group (3.42 ± 0.289 log₁₀CFU/g) had a higher (P < 0.05) concentration of coliform than did the 12-h FD group (2.50 ± 0.289 log₁₀CFU/g). The 24-h FD group (3.31 ± 0.259 log₁₀CFU/g) also had higher (P < 0.05) Enterobacteriaceae counts in the rumen than did in the 12-h FD group (2.47 ± 0.259 log₁₀CFU/g). Goats (5.71 ± 0.158 log₁₀CFU/g) had lower (P < 0.05) total plate counts in the rumen compared to sheep (6.27 ± 0.158 log₁₀CFU/g). The concentrate-fed animals had higher (P < 0.05) E. coli (6.44 vs. 4.01 ± 0.468 log₁₀CFU/g), total coliform (6.74 vs. 4.16 ± 0.469 log₁₀CFU/g), Enterobacteriaceae (6.93 vs. 3.83 ± 0.651 log₁₀CFU/g), and total plate counts (7.79 vs. 7.28 ± 0.170 log₁₀CFU/g) in the rectum than the hay-fed animals. The results indicate that microbial loads in the GIT of small ruminants may be reduced by either feeding hay for 4 d or depriving feed for 12-h prior to slaughter.     

Growth promotion of Prunus rootstocks by root treatment with specific bacterial strains

A collection of bacterial strains obtained from a wide-range origin was screened for ability to promote growth in two types of Prunus rootstocks in a commercial nursery. Only few strains promoted growth significantly and consistently, and a strong specificity for the rootstock cultivar was observed. Irrigation of plants with Pseudomonas fluorescens EPS282 and Pantoea agglomerans EPS427 significantly increased plant height and root weight of the plum Marianna 2624 and the peach–almond hybrid GF-677, respectively. Plant height showed a higher rate of growth in early stages of development (2.6–3.5 times the non-treated controls), but the effect decreased with plant age. However, in aged plants growth promotion was more significant on root weight (1.9 times the non-treated controls) than on plant height. The efficacy of growth promotion and the persistence of strains in the root environment were dependent on the bacterial inoculum concentration applied. Increases in root development were maximum at inoculum concentrations of up to 8 log₁₀ CFU ml^–1 (ca 10 log₁₀ CFU L^–1 of potting mix). Population levels at the optimum inoculum concentration were around 7 log₁₀ CFU g f.w.^–1 root material at early stages of development and decreased to 4 log₁₀ CFU g f.w.^–1 after several months of development. The best plant growth-promoting strains were very diverse in secondary metabolite production and antagonistic ability against several plant pathogens.