Listeria monocytogenes: Strain Heterogeneity, Methods, and Challenges of Subtyping.

Listeria monocytogenes is a food-borne bacterial pathogen that is associated with 20% to 30% case fatality rate. L. monocytogenes is a genetically heterogeneous species, with a small fraction of strains (serotypes 1/2a, 1/2b, 4b) implicated in human listeriosis. Monitoring and source tracking of L. monocytogenes involve the use of subtyping methods, with the performance of genetic-based methods found to be superior to phenotypic-based ones. Various methods have been used to subtype L. monocytogenes isolates, with the pulsed-field gel electrophoresis (PFGE) being the gold standard. Although PFGE has had a massive impact on food safety through the establishment of the PulseNet, there is no doubt that whole genome sequence (WGS) typing is accurate, has a discriminatory power superior to any known method, and allows genome-wide differences between strains to be quantified through the comparison of nucleotide sequences. This review focuses on the different techniques that have been used to type L. monocytogenes strains, their performance challenges, and the tremendous impact WGS typing could have on the food safety landscape.

Comparing the behavior of multidrug-resistant and pansusceptible Salmonella during the production and aging of a Gouda cheese manufactured from raw milk.

Outbreaks of salmonellosis have been linked to the consumption of cheese, and emerging multidrug-resistant (MDR) strains of Salmonella may be more virulent and more tolerant than less resistant strains to stresses encountered in food production, which may enhance the survival of these resistant strains in cheese. This study was conducted to compare the behavior of MDR and pansusceptible Salmonella strains during the manufacture and aging of Gouda cheese and compare pathogen recovery via several rapid and traditional methods. Cheeses were manufactured from raw milk inoculated with a six-strain cocktail of either MDR or susceptible Salmonella Newport and Salmonella Typhimurium at initial levels of <20 CFU/ml. Samples of milk, whey, curd, and finished cheese were analyzed using eight enrichment and detection protocols. Overall, changes in pathogen levels observed throughout manufacture and aging did not differ significantly between MDR and susceptible Salmonella strains. Salmonella counts increased significantly during manufacture to a mean of 734 CFU/g on day 1 followed by a significant decrease over 60 days of aging to <1 CFU/g. Although levels fell and stayed below the direct plating detection limit of $ 5 CFU/g after 54 days on average, viable cells remained detectable after enrichment for an average of 210 ± 40 days. The International Organization for Standardization methods with and without PCR detection provided the most accurate results, and the remaining methods, notably those with selective primary incubation, produced results that disagreed significantly with the true result. Overall, our findings suggest that MDR Salmonella strains may not pose a greater threat to cheese safety than do non-MDR Salmonella strains.

Rapid differentiation of Listeria monocytogenes epidemic clones III and IV and their intact compared with heat-killed populations using Fourier transform infrared spectroscopy and chemometrics.

UNLABELLED: The objectives of this study were to determine if Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis (chemometrics) could be used to rapidly differentiate epidemic clones (ECs) of Listeria monocytogenes, as well as their intact compared with heat-killed populations. FT-IR spectra were collected from dried thin smears on infrared slides prepared from aliquots of 10 µL of each L. monocytogenes ECs (ECIII: J1-101 and R2-499; ECIV: J1-129 and J1-220), and also from intact and heat-killed cell populations of each EC strain using 250 scans at a resolution of 4 cm(-1) in the mid-infrared region in a reflectance mode. Chemometric analysis of spectra involved the application of the multivariate discriminant method for canonical variate analysis (CVA) and linear discriminant analysis (LDA). CVA of the spectra in the wavelength region 4000 to 600 cm(-1) separated the EC strains while LDA resulted in a 100% accurate classification of all spectra in the data set. Further, CVA separated intact and heat-killed cells of each EC strain and there was 100% accuracy in the classification of all spectra when LDA was applied. FT-IR spectral wavenumbers 1650 to 1390 cm(-1) were used to separate heat-killed and intact populations of L. monocytogenes. The FT-IR spectroscopy method allowed discrimination between strains that belong to the same EC. FT-IR is a highly discriminatory and reproducible method that can be used for the rapid subtyping of L. monocytogenes, as well as for the detection of live compared with dead populations of the organism. PRACTICAL APPLICATION: Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis can be used for L. monocytogenes source tracking and for clinical case isolate comparison during epidemiological investigations since the method is capable of differentiating epidemic clones and it uses a library of well-characterized strains. The FT-IR method is potentially less expensive and more rapid compared to genetic subtyping methods, and can be used for L. monocytogenes strain typing by food industries and public health agencies to enable faster response and intervention to listeriosis outbreaks. FT-IR can also be applied for routine monitoring of the pathogen in food processing plants and for investigating postprocessing contamination because it is capable of differentiating heat-killed and viable L. monocytogenes populations.

From Pasteur to Probiotics: A Historical Overview of Cheese and Microbes.

Cheese is a food which has been produced for centuries. While cheese was originally developed as a product which extended the shelf life of milk, over time distinct cheese varieties arose, being shaped by geographic, climate, cultural, and economic factors. Global demand for artisan cheeses is creating new economic opportunities. Consumers seeking distinctive products with regional flavor, or terroir, are becoming connoisseurs of hand-crafted cheeses with distinctive tastes and character. These demands have spurred new inquiry into microorganisms used as starter cultures and adjunct cultures, as well as the microbiological consortia of finished cheeses. Such demands have also created new concerns for food safety and international trade. New bacterial pathogens such as Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium DT104 have emerged in the food supply, causing a reevaluation of the efficacy of traditional cheesemaking procedures to control these pathogens. Similarly, pathogens such as Listeria monocytogenes pose problems to susceptible human populations, and cheese can be a vehicle of transmission for this deadly pathogen. With changes in sanitary requirements due to the globalization of the food industry, governments around the world are increasingly requiring assurances of cheese safety. While many governments recognize the safety of traditional artisan cheeses manufactured from raw milk, others are demanding pasteurization of all milk intended for cheesemaking to provide assurance of microbiological safety. In response, new technologies are being proposed to increase cheese safety, but these technologies fundamentally alter the traditional artisan practices and may not enhance microbiological safety. A reevaluation of the safety of traditional artisan practices, validation thereof, and communication of the scientific principles which promote safety will be necessary to enable the continued production of traditional artisan cheeses in global commerce. This also affords the opportunity to more fully explore the microbial diversity and microbial ecology of the great cheeses of the world.

Characterization of Staphylococcus aureus strains isolated from raw milk utilized in small-scale artisan cheese production.

Staphylococcus aureus is an important agent of bacterial mastitis in milking animals and of foodborne intoxication in humans. The purpose of this study was to examine the genetic and phenotypic diversity, enterotoxigenicity, and antimicrobial resistance of S. aureus strains isolated from raw milk used for the production of artisan cheese in Vermont. Cross-tabulations revealed that the 16 ribotypes identified among the 90 milk isolates examined were typically associated with a specific animal species and that more than half of these ribotypes were unique to individual farms. In general, specific EcoRI ribotypes were commonly associated with specific phenotypical characteristics, including staphylococcal enterotoxin production or the lack thereof. Limited antimicrobial resistance was observed among the isolates, with resistance to ampicillin (12.51%) or penicillin (17.04%) most common. Two isolates of the same ribotype obtained from the same farm were resistant to oxacillin with 2% NaCl. More than half (52.22%) of isolates produced toxin, and 31 of the 32 isolates solely produced staphylococcal enterotoxin type C. Although these data demonstrate that S. aureus strains found in raw milk intended for artisan cheese manufacture are capable of enterotoxin production, staphylococcal enterotoxin C is not typically linked to foodborne illness. Because S. aureus is a common contaminant of cheese, an understanding of the ecology of this pathogen and of the antimicrobial susceptibility and toxigenicity of various strains will ultimately contribute to the development of control practices needed to enhance the safety of artisan and farmstead cheese production.

Behavior of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses manufactured from raw milk.

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.

Detection, isolation, and incidence of Listeria spp. in small-scale artisan cheese processing facilities: a methods comparison.

Environmental sampling, focused on environmental sites with a high probability of contamination, was conducted in eight artisan cheese processing facilities. Samples (n = 236) from 86 food contact surfaces and 150 non-food contact surfaces were examined for the presence of Listeria spp. by comparing the efficacy of three different primary enrichment media used in five detection and isolation methods. University of Vermont broth was the most sensitive primary enrichment medium for the detection of Listeria spp., including Listeria monocytogenes. These results, however, did not differ significantly from those obtained with Listeria repair broth or Oxoid 24 Listeria enrichment broth. When full methods were considered, the use of dual enrichment methods identified the most Listeria spp.-positive samples, whereas the BAX System PCR Assay for the Genus Listeria 24E provided the most rapid results (approximately 30 h). Cultural results from the direct plating of secondary enrichment broths were generally in agreement with PCR results when compared within methods. Despite minor differences in efficacy, all five methods were in agreement with one another. Overall, 24 (10.7%) of the 236 environmental samples were positive for Listeria spp., all of which were collected from non-food contact surfaces. Nine of these sites were also positive in previous sampling events, suggesting that these sites serve as Listeria niches and that certain ribotypes are particularly persistent, inhabiting environments and specific sites for over 2 years. Overall, our results suggest that the extent of Listeria spp. contamination, notably L. monocytogenes, in small-scale artisan cheese processing environments is low.

Enhanced detection of Listeria spp. in farmstead cheese processing environments through dual primary enrichment, PCR, and molecular subtyping.

The incidence and ecology of Listeria spp. in farmstead cheese processing environments were assessed through environmental sampling conducted in nine different plants over a 10-week period. Environmental samples (n = 705) were examined for the presence of Listeria spp. by using three detection/isolation protocols. The use of dual enrichment methods, which allowed for the recovery of injured Listeria spp. (mUSDA), identified more Listeria species-positive samples with higher sensitivity than the standard USDA method. The addition of PCR to the mUSDA method identified the most Listeria monocytogenes-positive samples, achieving greater sensitivity of detection while substantially reducing time. Overall, 7.5% of samples were positive for Listeria spp., yielding 710 isolates, 253 of which were subtyped by automated ribotyping to examine strain diversity within and between plants over time. The isolation of specific ribotypes did not appear to be affected by the enrichment protocol used. Fifteen (2.1%) samples yielded L. monocytogenes isolates differentiated almost equally into ribotypes of lineages I and II. Of most concern was the persistent and widespread contamination of a plant with L. monocytogenes DUP-1042B, a ribotype previously associated with multiple outbreaks of listeriosis. Our results suggest that the extent of contamination by Listeria spp., notably L. monocytogenes, in farmstead cheese plants is comparatively low, especially for those with on-site farms. The results of this study also identified points of control for use in designing more effective Listeria spp. control and monitoring programs with a focus on ribotypes of epidemiological significance.

60-day aging requirement does not ensure safety of surface-mold-ripened soft cheeses manufactured from raw or pasteurized milk when Listeria monocytogenes is introduced as a postprocessing contaminant.

Because of renewed interest in specialty cheeses, artisan and farmstead producers are manufacturing surface-mold-ripened soft cheeses from raw milk, using the 60-day holding standard (21 CFR 133.182) to achieve safety. This study compared the growth potential of Listeria monocytogenes on cheeses manufactured from raw or pasteurized milk and held for > 60 days at 4 degrees C. Final cheeses were within federal standards of identity for soft ripened cheese, with low moisture targets to facilitate the holding period. Wheels were surface inoculated with a five-strain cocktail of L. monocytogenes at approximately 0.2 CFU/ cm2 (low level) or 2 CFU/cm2 (high level), ripened, wrapped, and held at 4 degrees C. Listeria populations began to increase by day 28 for all treatments after initial population declines. From the low initial inoculation level, populations in raw and pasteurized milk cheese reached maximums of 2.96 +/- 2.79 and 2.33 +/- 2.10 log CFU/g, respectively, after 60 days of holding. Similar growth was observed in cheese inoculated at high levels, where populations reached 4.55 +/- 4.33 and 5.29 +/- 5.11 log CFU/g for raw and pasteurized milk cheeses, respectively. No significant differences (P < 0.05) were observed in pH development, growth rate, or population levels between cheeses made from the different milk types. Independent of the milk type, cheeses held for 60 days supported growth from very low initial levels of L. monocytogenes introduced as a postprocess contaminant. The safety of cheeses of this type must be achieved through control strategies other than aging, and thus revision of current federal regulations is warranted.

Low incidence of foodborne pathogens of concern in raw milk utilized for farmstead cheese production.

Overall milk quality and prevalence of four target pathogens in raw milk destined for farmstead cheesemaking was examined. Raw milk samples were collected weekly from June to September 2006 from 11 farmstead cheese operations manufacturing raw milk cheese from cow's, goat's, and sheep's milk. Samples were screened for Listeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli O157:H7 both quantitatively (direct plating) and qualitatively (PCR). Overall, 96.8% of samples had standard plate counts of < 100,000 CFU/ml, 42.7% of which were < 1,000 CFU/ml. Although no federal standards exist for coliforms in raw milk, 61% of samples tested conformed to pasteurized milk standards under the U.S. Pasteurized Milk Ordinance (PMO) at < 10 CFU/ml. All cow and sheep milk samples and 93.8% of goat milk samples were within the limits dictated by the PMO for somatic cell counts. Of the 11 farms, 8 (73%) produced samples that were positive for S. aureus, which was detected in 34.6% (46 of 133) of milk samples. L. monocytogenes was isolated from three milk samples (2.3%), two of which were from the same farm. E. coli O157:H7 was recovered from one sample of goat's milk for an overall incidence of 0.75%. Salmonella was not recovered from any of the 133 samples. The findings of this study suggest that most raw milk intended for farmstead cheesemaking is of high microbiological quality with a low incidence of pathogens. These data will help inform risk assessments associated with the microbiological safety of farmstead cheeses, particularly those manufactured from raw milk.

Comprehensive survey of pasteurized fluid milk produced in the United States reveals a low prevalence of Listeria monocytogenes.

A comprehensive survey was undertaken to generate contemporary data on the prevalence of Listeria monocytogenes in pasteurized fluid milk produced in the United States. Samples (5,519) near the sell-by expiration date were purchased at retail outlets over a 5-week period and analyzed for presence of L. monocytogenes. Products consisted of whole milk, nonfat milk, and chocolate milk packaged in gallon, half gallon, quart, pint, and half-pint containers. Samples were collected from both large and small retail stores in urban and suburban locations in four FoodNet cities (Baltimore, Md., Atlanta, Ga., St. Paul/ Minneapolis, Minn., and San Francisco, Calif.). Samples were prescreened for L. monocytogenes by the AOAC-approved rapid Vitek immunodiagnostic assay system, enzyme-linked fluorescent assay method. Positive prescreening samples were cultured according to the Bacteriological Analytical Manual, enumerated for L. monocytogenes with a nine-tube most-probable-number (MPN) procedure, and confirmed by biochemical characterization. The frequency of isolation of L. monocytogenes in these products was 0% (0 of 1,897) in whole milk, 0.05% (1 of 1,846) in nonfat milk, 0% (0 of 1,669) in chocolate milk, and 0% (0 of 107) in other (reduced fat and low fat) milk samples. Overall, L. monocytogenes was confirmed in only 0.018% of pasteurized milk samples (1 of 5,519). Enumeration of the single confirmed positive nonfat milk sample revealed low-level contamination (<0.3 MPN/g), even when sampled 5 days past the expiration of the sell-by date. The results confirm the low frequency of contamination of pasteurized fluid milk products by L. monocytogenes for products sold in the United States and reaffirm the reduction of contamination frequency of fluid milk by L. monocytogenes when compared with earlier estimates from the U.S. Food and Drug Administration Dairy Safety Initiatives Program.

Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury.

Detection of L. monocytogenes is often limited by the performance of the enrichment media used to support bacterial growth to detectable levels. Because Listeria may exist at extremely low levels in foods, sample enrichment protocols must amplify these low initial populations to detectable limits. Listeria may also exist in an injured state in food products as a result of processing treatments such as heating, freezing, exposure to acids, or exposure to sanitizing compounds. Selective agents in enrichment media normally used for recovery of Listeria may inhibit repair and detection of sublethally injured Listeria, which may go on to repair, grow, and regain pathogenicity. Simple modifications to existing regulatory protocols, such as those that use more than one enrichment broth, raise sensitivity of detection to 90%. This review shows the efficacy of repair/enrichment strategies, which increase sensitivity of detection to 97.5-98.8% compared with 65-70% by standard regulatory protocols. Ribotype analysis of isolates obtained from meat samples reveals a complex microbial ecology, with striking differences in both number and distribution of distinct genetic types of Listeria, depending upon whether samples are enriched in selective or repair/enrichment media. In studies on enrichment of dairy environmental samples in University of Vermont medium and Listeria repair broth (UVM and LRB), combining these 2 primary enrichment media into a single tube of Fraser broth for dual secondary enrichment yielded a significantly higher percentage (p < 0.05) of Listeria-positive samples than did use of either LRB or UVM alone. Refinement of conventional Listeria recovery methods should consider the importance of the enrichment step, the nutritional needs of specific genetic types, and the physiological condition of Listeria isolates in foods.

Diversity of Listeria Ribotypes Recovered from Dairy Cattle, Silage, and Dairy Processing Environments.

Listeria strains isolated over the past 10 years from farms and dairy processing environments were subjected to strain-specific ribotyping using the automated Riboprinter microbial characterization system, alpha version (E. I. du Pont de Nemours & Co., Inc.). A total of 388 Listeria isolates from 20 different dairy processing facilities were examined along with 44 silage, 14 raw milk bulk tank, and 29 dairy cattle (26 udder quarter milk, 1 brain, 1 liver, and 1 aborted fetus) isolates. These 475 isolates included 93 L. monocytogenes , 362 L. innocua , 11 L. welshimeri , 6 L. seeligeri , 2 L. grayi , and 1 L. ivanovii strains. Thirty-seven different Listeria ribotypes (RTs) comprising 16 L. monocytogenes (including five known clinical RTs responsible for foodborne listeriosis), 12 L. innocua , 5 L. welshimeri , 2 L. seeligeri , 1 L. ivanovii , and 1 L. grayi were identified. Greatest diversity was seen among isolates from dairy processing facilities with 14 of 16 (87.5%) of the L. monocytogenes RTs (including five clinical RTs) and 19 of 21 (90.5%) of the non- L. monocytogenes RTs detected. Sixty-five of the 93 L. monocytogenes isolates belonged to a group of five clinical RTs. These five clinical RTs included one RT unique to dairy processing environments, two RTs common to dairy processing environments and silage, and one RT common to dairy processing environments, silage, and dairy cattle with the last RT appearing in dairy processing environments, silage, raw milk bulk tanks, and dairy cattle. These findings, which support the link between on-farm sources of Listeria contamination (dairy cattle, raw milk, silage) and subsequent contamination of dairy processing environments, stress the importance of farm-based HACCP programs for controling listeriae.

Increased Detection of Acid-Injured Escherichia coli O157:H7 in Autoclaved Apple Cider by Using Nonselective Repair on Trypticase Soy Agar †.

Three different acid-resistant strains of Escherichia coli O157:H7 were inoculated individually and as a cocktail into sterile apple cider (pH 3.2) at a level of approximately 105 cells per ml and incubated at 2°C. Samples were plated on Trypticase soy agar (TSA), violet red bile agar (VRBA), sorbitol MacConkey agar (SMA), and Petrifilm E. coli count plates (Petrifilm) at 24-h intervals. Repair of acid-injured cells was assessed by surface plating cider samples on TSA and allowing a 2-h room-temperature incubation period followed by overlaying with double-strength VRBA or SMA. Since SMA is a surface plate medium, the repair procedure was modified by overlaying SMA with Trypticase soy broth after 2 h of room-temperature incubation. Populations of all three strains and the cocktail of strains decreased rapidly in apple cider and approached undetectable levels within 72 h. At 24 and 48 h, 98.4% and >99% of the E. coli populations were injured, respectively. Repair procedures significantly (α = 0.05) increased detection of E. coli O157:H7. After 72 h E. coli O157:H7 was not detected by using SMA and Petrifilm; however, it was detected using repair procedures. Although detection levels were increased with resuscitation procedures, the levels detected were still lower than those obtained using nonselective TSA. This research confirms the need for special recovery steps when analyzing acidic food products suspected of containing E. coli O157:H7.

Comparison of Methods for Determining Coliform and Escherichia coli Levels in Apple Cider †.

The main objective of this research was to determine the easiest and most reliable media for enumerating coliform bacteria and Escherichia coli levels in apple cider. During the autumn of 1994 a total of 59 apple cider samples were collected directly from 12 cider producers and were assessed for bacterial levels and pH. Plate count agar was used to determine heterotrophic bacteria levels. Coliform levels were determined using three different media: violet red bile agar (VRBA), Petrifilm High Sensitivity Coliform Count Plates (PHSCCP), and Trypticase soy agar with a VRBA overlay (TSA/VRBA) for attempted recovery of coliforms injured by the low pH of the apple cider. Eosin methylene blue agar (EMBA) and Petrifilm E. coli Count Plates were used to screen cider samples for E. coli . Apple cider had an average pH of 3.34 ± 0.08. Heterotrophic bacterial levels ranged from 2.30 to 7.11 log CFU/ml. All cider samples contained coliform bacteria with levels varying greatly; on the different media, we found the following: on VRBA, <1.00 to 4.37 log CFU/ml; on TSA/VRBA, 1.20 to 4.40 log CFU/ml; and on PHSCCP, < 1.00 to 4.56 log CFU/ml. Coliform levels were most easily determined in apple cider by using PHSCCP. However TSA/VRBA proved to be more reliable; coliform detection was significantly (P < 0.05) increased. EMBA was ineffective for screening apple cider for E. coli , with the low pH of the cider producing many false-positive results. E. coli was only recovered by using Petrifilm E. coli Count Plates with one of the 59 samples positive for E. coli (non-O157:H7) at a level of 10 CFU/ml.

Enhanced Recovery of Listeria from Dairy-Plant Processing Environments through Combined Use of Repair Enrichment and Selective Enrichment/Detection Procedures.

The efficacy of using a repair step to increase sensitivity of recovery of injured Listeria from environmental sources in dairy processing plants was investigated. The USDA-FSIS Listeria isolation protocol using UVM-modified Listeria enrichment broth medium University of Vermont (UVM) for primary enrichment was the standard method chosen for comparison. UVM broth was used in conjunction with rapid methods (Organon Teknika and Gene-Trak™), following manufacturer's guidelines. Listeria Repair Broth (LRB) was used as the repair enrichment medium in modified protocols of the standard and rapid procedures. LRB employs a nonselective period (2-5 hours) for repair of injured Listeria prior to selective-agent addition. Of 80 environmental sites positive by any method, UVM and LRB showed similar recovery rates (87.5% and 88.8%, respectively). Thus LRB provided little advantage over current procedures for use in contaminated sites. UVM was superior when used in conjunction with either rapid method. The USDA and modified USDA (mUSDA) procedures gave identical recovery rates (93%), but 10 additional positive sites were attributed to the use of two enrichment broths. The culture method combined with either rapid method from each broth increased the sensitivity to 97.5-98.8% when data from UVM and LRB was combined. False negative rates in the USDA method (7.1%) were attributed to the lack of color change in Fraser secondary broth. Fraser broth also yielded many false positive results (overall 66.2%) making this broth of limited value as a screening tool for highly contaminated samples. In order to optimize methodology for detection of Listeria , suppression of background flora and the recovery of potentially injured Listeria in the processing environment must be addressed. Overgrowth occurring during the nonselective enrichment period was suspected of causing suboptimal sensitivity in LRB; however, the combination of UVM and LRB showed promising recovery rates. Ceftazidime was evaluated against 68 background isolates that survived throughout the various enrichment and detection methods. Inhibition of 57 of the contaminants indicates a potential role for ceftazidime in the LRB selective-agent regime for sites with high microbial background.

Environmental Surveillance of Dairy Processing Plants for the Presence of Yersinia Species.

Thirty dairy processing plants were surveyed to determine if the psychrotrophic nature of Yersinia influenced its environmental niche within the plants. Three hundred forty-seven samples obtained from 30 dairy processing plants were tested for the presence of Yersinia spp. Ten (33.3%) plants had one or more positive sites. A total of 20 of the 347 (5.8%) sites tested were positive for one or more species of Yersinia . Yersinia enterocolitica was present at 85% (17/20) of the positive sites. It was identified by itself on 15 occasions and twice with other species of Yersinia . Other species identified included Yersinia ruckeri in 3 of the 20 positive samples (15%); Yersinia kristensenii and Yersinia frederiksenii both in 1 of the 20 (5%) positive samples. Ten of the 20 (50%) positive samples were obtained from coolers and entrances to freezers. Yersinia -positive cooler/freezer sites were obtained from 9 of the 10 positive plants. The remaining 10 Yersinia -positive sites were from raw milk receiving areas (4/10) and drains around machinery (6/10), including an ice maker, a case washer, two holding tanks, one filler machine and a packaging area. These results reinforce both the psychrotrophic nature of Yersinia and the evidence that raw milk can be a primary source of the organism. Our findings show that sanitation schemes need to be developed for coolers and freezers to prohibit the colonization and proliferation of Yersinia within these areas.

Increased Incidence of Listeria Species in Dairy Processing Plants Having Adjacent Farm Facilities.

The processing environments of 30 dairy facilities were surveyed for the presence of Listeria species. Two different primary enrichment media - University of Vermont Modified Listeria Enrichment Broth and Listeria Repair Broth - were employed to increase the probability of identifying positive samples. Samples were also tested using both an enzyme-linked immunosorbent-based (ELISA-based) assay and a gene probe assay. A total of 346 sponge samples were evaluated for the presence of Listeria . Listeria spp. were identified via one or more of the assays 122 (35.3%) times. Fifty-five of the positive samples (37.2%) contained Listeria monocytogenes and 93 (62.8%) contained Listeria innocua . Of the 30 plants tested, 9 had a dairy farm contiguous to the processing facilities. Our results show that these plants are more likely to be contaminated (9/9) than those plants without on site dairy farms (17/21). Analysis of the Listeria spp. results indicated that contamination was significantly higher (α = 0.1) at those plants with an on-site dairy farm (x = 50.1 %) than those plants without an on-site dairy farm (x = 33.5%). Plants producing dairy ingredients, frozen milk products or fluid milk were all shown to have significantly higher incidence rates than expected. Conversely, plants producing cultured dairy foods, or a combination of cultured dairy foods and fluid milk were found to have significantly lower incidence rates than expected. There was no statistically significant difference in contamination by area within the plants.

Effect of Incubation Temperature on Repair of Heat-Injured Listeria in Milk.

Heat-injured Listeria species were examined for their ability to repair in pasteurized whole and 2% (fat) bovine milk. Listeria monocytogenes F5069 (serotype 4B) and F5027 (serotype 1/2a) and Listeria innocua CWD139 were heated at 55°C. After 20 min, 99% of the surviving population was injured as determined by their inability to grow in the presence of 4% NaCl. Bacterial cells were immediately suspended in sterile milk at a concentration of 102 to 103 per ml and incubated at 4, 10, 26 and 37°C. For all of the Listeria tested, repair at 4°C was initiated between days 8 and 10 and was complete between days 16 and 19; at 10°C, repair began immediately and was complete in 4 d; at 26 and 37°C, repair was complete by 13 and 9 h, respectively. The kinetics of repair were similar in whole and 2% (fat) milk. The relationship between the time required for repair and increasing temperature was nonlinear and indicated that repair of heat-injured Listeria in milk is highly sensitive to minor increases in temperature. Current Listeria detection techniques are not adequate for the detection of injured organisms. The public health consequences associated with failure to detect injured L. monocytogenes which subsequently repair in milk may be significant.

Destruction, Injury, and Repair of Listeria Species Exposed to Sanitizing Compounds.

The efficacy of four commonly used dairy plant sanitizers against Listeria coupled with an examination of ability of these sanitizers to induce injury was investigated. Listeria monocytogenes F 5069, F 5027, and Listeria innocua CWD 350 were tested against different concentrations of sanitizers (for periods of 30 s and 2 min) which included two quaternary ammonium compounds (QAC), an acid anionic sanitizer, and a chlorine-containing sanitizer. According to the Sanitizer Test of the Association of Analytical Chemists, all sanitizers showed efficacy of >99.999% against all test organisms, regardless of the type of sanitizer, its concentration, or exposure time. Two enrichment broths consisting of the University of Vermont (UVM), which is currently used for selective enrichment of Listeria , and Listeria repair broth (LRB), which is a nonselective highly nutritious medium, were used to determine the ability of standard enrichment procedures to recover injured Listeria . Use of these enrichment procedures suggests that sanitizers may induce injury rather than death as indicated by recovery of the organisms in LRB versus failure of growth in UVM, or through variation in the lag phase and viable cell count between both broths. The extent of cell death, injury, and repair was found to be affected by the type and concentration of sanitizer, exposure time, bacterial strain, and the enrichment procedure. QAC were the most effective while the acid anionic sanitizer was the least effective. The lethal effect of the sanitizer was found to increase by increasing its concentration or exposure time. L. monocytogenes F 5027 was the most resistant of the strains tested to QAC and the acid anionic sanitizer, while L. innocua was more resistant to the chlorine sanitizer. In all instances LRB proved to be superior to UVM in promoting repair and growth of sanitizer-injured cells.