Environmental Sponge Samples Research Articles

Intensive Environmental Sampling and Whole Genome Sequence-based Characterization of Listeria in Small- and Medium-sized Dairy Facilities Reveal Opportunities for Simplified and Size-appropriate Environmental Monitoring Strategies

Small- and medium-sized dairy processing facilities (SMDFs) may face unique challenges with respect to controlling Listeria in their processing environments, e.g., due to limited resources. The aim of this study was to implement and evaluate environmental monitoring programs (EMPs) for Listeria control in eight SMDFs in a ∼1-year longitudinal study; this included a comparison of pre-operation (i.e., after cleaning and sanitation and prior to production) and mid-operation (i.e., at least 4 h into production) sampling strategies. Among 2,072 environmental sponge samples collected across all facilities, 272 (13%) were positive for Listeria. Listeria prevalence among pre- and mid-operation samples (15% and 17%, respectively), was not significantly different. Whole genome sequencing (WGS) performed on select isolates to characterize Listeria persistence patterns revealed repeated isolation of closely related Listeria isolates (i.e., ≤20 high-quality single nucleotide polymorphism [hqSNP] differences) in 5/8 facilities over >6 months, suggesting Listeria persistence and/or reintroduction was relatively common among the SMDFs evaluated here. WGS furthermore showed that for 41 sites where samples collected pre- and mid-operation were positive for Listeria, Listeria isolates obtained were highly related (i.e., ≤10 hqSNP differences), suggesting that pre-operation sampling alone may be sufficient and more effective for detecting sites of Listeria persistence. Importantly, our data also showed that only 1/8 of facilities showed a significant decrease in Listeria prevalence over 1 year, indicating continued challenges with Listeria control in at least some SMDFs. We conclude that options for simplified Listeria EMPs (e.g., with a focus on pre-operation sampling, which allows for more rapid identification of likely persistence sites) may be valuable for improved Listeria control in SMDFs.

Detection of Selected Equine Respiratory Pathogens in Stall Samples Collected at a Multi-Week Equestrian Show during the Winter Months.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

Genetic Diversity of Listeria monocytogenes Isolated From Three Commercial Tree Fruit Packinghouses and Evidence of Persistent and Transient Contamination.

Whole genome analysis was performed on 501 isolates obtained from a previous survey which recovered 139 positive environmental sponge samples (i.e., up to 4 isolates per sample) from a total of 719 samples collected at 40 standardized sites in 3 commercial apple packinghouse facilities (i.e., P1, P2, and P3) over 3 successive seasons in a single production year. After excluding duplicated isolates, the data from 156 isolates revealed the clonal diversity of L. monocytogenes and allowed the detection of transient contamination, persistent contamination, and cross-area transmission events. Facility P2 with the poorest sanitary conditions had the least diversity (Shannon’s index of 0.38). P2 contained a Clonal Complex (CC) 554, serogroup IVb-v1 strain that persisted throughout the year and spread across the entire facility, a singleton Sequence Type (ST) 1003, lineage III strain that persisted through two seasons and spread across two areas of the facility, and 3 other clones from transient contaminations. P1 and P3, facilities with better sanitary conditions, had much higher diversity (i.e., 15 clones with a Shannon’s index of 2.49 and 10 clones with a Shannon’s index of 2.10, respectively) that were the result of transient contamination. Facilities P1 and P3 had the highest incidence (43.1%) of lineage III isolates, followed by lineage I (31.3%) and lineage II (25.5%) isolates. Only 1 isolate in the three facilities contained a premature stop codon in virulence gene inlA. Fourteen samples yielded 2–3 clones per sample, demonstrating the importance of choosing appropriate methodologies and selecting a sufficient number of isolates per sample for studying L. monocytogenes diversity. Only 1 isolate, belonging to CC5 and from facility P3, contained a known plasmid, and this was also the only isolate containing benzalkonium chloride tolerance genes. The persistent CC554 strain did not exhibit stronger sanitizer resistance than other isolates and did not contain any confirmed molecular determinants of L. monocytogenes stress resistance that were differentially present in other isolates, such as genes involved in sanitizer tolerance, heavy metal resistance, biofilm-forming, stress survival islet 1 (SSI-1), stress survival islet 2 (SSI-2) or Listeria genomic island (LGI2).

Occurrence and diversity of Listeria spp. in seafood processing plant environments

Listeria contamination in processing plant environments is a major issue for the seafood industry worldwide; faster and more reliable results are therefore desired for early detection and monitoring of environmental Listeria spp. This study aimed to gain a better understanding of the prevalence and diversity of Listeria spp., and to evaluate a rapid detection method, the 3M Molecular Detection Assay (MDA) Listeria, for its ability to detect Listeria spp. in environmental samples from seafood processing plants. Duplicate environmental sponge samples (n = 444) were collected from 152 different sites within three seafood processing plants, and analyzed for Listeria spp. by the MDA method (after 26 and 48 h of enrichment) and the U.S. Food and Drug Administration Bacteriological Analytical Manual method. Overall, detection of Listeria spp. by the two methods did not differ significantly (p > 0.05); 11 (4.9%) and 13 (5.9%) samples were positive for Listeria spp. by the MDA and FDA-BAM method, respectively. The sensitivity of the MDS was 87.0% (95% CI: 77.4–96.6%), specificity was 97.6% (95% CI: 95.5–99.7%), accuracy was 95.3%, and the positive predictive value was 89.4% (95% CI: 80.5–98.2%). Classification of 19 Listeria isolates by partial SigB sequencing analysis identified three allelic types. Twelve of these isolates were ATs 58 and 60 which were classified as Listeria monocytogenes lineage I and serotypes 1/2b, 3b, 4b, 4d, 4e, by multiplex-PCR serotyping. Six Listeria isolates were classified as Listeria innocua (AT31). Our data show that the 3M Molecular Detection Assay Listeria provides rapid and reliable results for detection and monitoring of Listeria spp., which are important for seafood processing plants. Effective Listeria monitoring programs will allow for improved development of Listeria control measures in order to minimize cross-contamination in finished products.

Detection of Listeria spp. using ACTERO listeria enrichment media.

ACTERO Listeria Enrichment Media (ACTERO Listeria) is a selective medium developed for a single-step recovery and enrichment of Listeria spp. from environmental samples. Robustness testing of the ACTERO Listeria medium demonstrated good performance when minor changes were introduced to the incubation temperature and time. All 54 Listeria strains tested, representing the most frequently isolated Listeria species from food (L. monocytogenes, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi), were successfully enriched in ACTERO Listeria. None of the 30 nontarget strains tested in the exclusivity study was recovered after incubation in ACTERO Listeria. Recovery of Listeria was consistent across three independently produced lots of the ACTERO Listeria, and the prepared medium was stable for 45 days when stored at 4 degrees C in the dark. Matrix studies performed with environmental sponge samples from plastic and stainless steel surfaces demonstrated similar recovery of Listeria spp. in a single-step enrichment using ACTERO Listeria from plastic, and significantly better recovery from stainless steel surfaces when compared to the U.S. Department of Agriculture-Food Safety and Inspection Service reference method. The results of this study prove that ACTERO Listeria Enrichment Media can be effectively used in replacement of the two-step enrichment suggested by the reference method without affecting the recovery of Listeria spp. from environmental samples.

Validation of the 3M Molecular Detection System for the Detection of Listeria in Meat, Seafood, Dairy, and Retail Environments

AbstractThere is a continued need to develop improved rapid methods for detection of foodborne pathogens. The aim of this project was to evaluate the 3M Molecular Detection System (3M MDS), which uses isothermal DNA amplification, and the 3M Molecular Detection Assay Listeria using environmental samples obtained from retail delicatessens and meat, seafood, and dairy processing plants. Environmental sponge samples were tested for Listeria with the 3M MDS after 22 and 48 h of enrichment in 3M Modified Listeria Recovery Broth (3M mLRB); enrichments were also used for cultural detection of Listeria spp. Among 391 samples tested for Listeria, 74 were positive by both the 3M MDS and the cultural method, 310 were negative by both methods, 2 were positive by the 3M MDS and negative by the cultural method, and one sample was negative by the 3M MDS and positive by the cultural method. Four samples were removed from the sample set, prior to statistical analyses, due to potential cross-contamination during testing. Listeria isolates from positive samples represented L. monocytogenes, L. innocua, L. welshimeri, and L. seeligeri. Overall, the 3M MDS and culture-based detection after enrichment in 3M mLRB did not differ significantly (P > 0.05) with regard to the number of positive samples, when chi-square analyses were performed for (i) number of positive samples after 22 h, (ii) number of positive samples after 48 h, and (iii) number of positive samples after 22 and/or 48 h of enrichment in 3M mLRB. Among 288 sampling sites that were tested with duplicate sponges, 67 each tested positive with the 3M MDS and the traditional U.S. Food and Drug Administration Bacteriological Analytical Manual method, further supporting that the 3M MDS performs equivalently to traditional methods when used with environmental sponge samples.

Isolation and Characterization of Methicillin-Resistant Staphylococcus aureus from Pork Farms and Visiting Veterinary Students

In the last decade livestock-associated methicillin-resistant S. aureus (LA-MRSA) has become a public health concern in many parts of the world. Sequence type 398 (ST398) has been the most commonly reported type of LA-MRSA. While many studies have focused on long-term exposure experienced by swine workers, this study focuses on short-term exposures experienced by veterinary students conducting diagnostic investigations. The objectives were to assess the rate of MRSA acquisition and longevity of carriage in students exposed to pork farms and characterize the recovered MRSA isolates. Student nasal swabs were collected immediately before and after farm visits. Pig nasal swabs and environmental sponge samples were also collected. MRSA isolates were identified biochemically and molecularly including spa typing and antimicrobial susceptibility testing. Thirty (30) veterinary students were enrolled and 40 pork farms were visited. MRSA was detected in 30% of the pork farms and in 22% of the students following an exposure to a MRSA-positive pork farm. All students found to be MRSA-positive initially following farm visit were negative for MRSA within 24 hours post visit. Most common spa types recovered were t002 (79%), t034 (16%) and t548 (4%). Spa types found in pork farms closely matched those recovered from students with few exceptions. Resistance levels to antimicrobials varied, but resistance was most commonly seen for spectinomycin, tetracyclines and neomycin. Non-ST398 MRSA isolates were more likely to be resistant to florfenicol and neomycin as well as more likely to be multidrug resistant compared to ST398 MRSA isolates. These findings indicate that MRSA can be recovered from persons visiting contaminated farms. However, the duration of carriage was very brief and most likely represents contamination of nasal passages rather than biological colonization. The most common spa types found in this study were associated with ST5 and expands the range of livestock-associated MRSA types.

Naturally colonized beef cattle populations fed combinations of yeast culture and an ionophore in finishing diets containing dried distiller's grains with solubles had similar fecal shedding of Escherichia coli O157:H7.

Beef steers (n = 252) were used to evaluate the effects of dietary supplement on fecal shedding of Escherichia coli O157:H7. Seven pens of 9 steers (63 steers per treatment) were fed diets supplemented with or without yeast culture (YC) or monensin (MON) and their combination (YC × MON). YC and MON were offered at 2.8 g/kg and 33 mg/kg of dry matter intake, respectively. Environmental sponge samples (from each pen floor, feed bunk, and water trough) were collected on day 0. Rectal fecal grab samples were collected on days 0, 28, 56, 84, 110, and 125. Samples were collected and pooled by pen and analyzed for presumptive E. coli O157:H7 colonies, which were confirmed by a multiplex PCR assay and characterized by pulsed-field gel electrophoresis (PFGE) typing. On day 0, E. coli O157:H7 was detected in 7.0% of feed bunk samples and 14.3% of pen floor samples but in none of the water trough samples. The 71.4% prevalence of E. coli O157:H7 in fecal samples on day 0 decreased significantly (P < 0.05) over time. E. coli O157:H7 fecal shedding was not associated with dietary treatment (P > 0.05); however, in cattle fed YC and YC × MON fecal shedding was 0% by day 28. Eight Xba I PFGE subtypes were identified, and a predominant subtype and three closely related subtypes (differing by three or fewer bands) accounted for 78.7% of environmental and fecal isolates characterized. Results from this study indicate that feeding YC to cattle may numerically decrease but not eliminate fecal shedding of E. coli O157:H7 at the onset of treatment and that certain E. coli O157 subtypes found in the feedlot environment may persist in feedlot cattle.

Molecular ecology of Listeria monocytogenes and other Listeria species in small and very small ready-to-eat meat processing plants.

A longitudinal study was conducted to track Listeria contamination patterns in ready-to-eat meats from six small or very small meat processing plants located in three states over 1 year. A total of 688 environmental sponge samples were collected from nonfood contact surfaces during bimonthly visits to each plant. Overall, L. monocytogenes was isolated from 42 (6.1%) environmental samples, and its prevalence ranged from 1.7 to 10.8% across different plants. Listeria spp., other than L. monocytogenes, were isolated from 9.5% of samples overall, with the prevalence ranging from 1.5 to 18.3% across different plants. The prevalence of L. monocytogenes correlated well with that of other Listeria spp. for some but not all plants. One L. monocytogenes isolate representing each positive sample was characterized by molecular serotyping, EcoRI ribotyping, and pulsed-field gel electrophoresis typing. Seven sample sites tested positive for L. monocytogenes on more than one occasion, and the same ribotype was detected more than once at five of these sites. Partial sigB sequencing was used to speciate other Listeria spp. isolates and assign an allelic type to each isolate. Other Listeria spp. were isolated more than once from 14 sample sites, and the same sigB allelic type was recovered at least twice from seven of these sites. One plant was colonized by an atypical hemolytic L. innocua strain. Our findings indicate that small and very small meat processing plants that produce ready-to-eat meat products are characterized by a varied prevalence of Listeria, inconsistent correlation between contamination by L. monocytogenes and other Listeria spp., and a unique Listeria molecular ecology.

Tracking of Listeria monocytogenes in Smoked Fish Processing Plants

Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments. Each of the four plants was sampled monthly for approximately 1 year. At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots. In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources. A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested. Presumptive Listeria spp. were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples. L. monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%). Among the environmental samples, L. monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces. Listeria spp. were isolated from environmental samples in each of the four plants, whereas L. monocytogenes was not found in any of the environmental samples from one plant. Overall, the L. monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples. Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L. monocytogenes isolates. For each of the three plants with L. monocytogenes–positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period. In one plant, a specific L. monocytogenes ribotype represented 44% of the L. monocytogenes–positive environmental samples and was also responsible for one of the two finished product positives found in this plant. In another plant, a specific L. monocytogenes ribotype persisted in the raw fish handling area. However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas. Molecular subtyping methods can help identify plant-specific L. monocytogenes contamination routes and thus provide the knowledge needed to implement improved L. monocytogenes control strategies.

Detection of Listeria in Crawfish Processing Plants and in Raw, Whole Crawfish and Processed Crawfish (Procambarus spp.)

The foodborne pathogen Listeria monocytogenes represents a major concern to the food industry and particularly to producers of ready-to-eat (RTE) foods because of the severity of human listeriosis infections and because of the ubiquitous nature of this organism. Although several studies on the prevalence and sources of L. monocytogenes in various RTE seafoods have been conducted, limited information is available on the presence and potential sources of this organism in RTE crawfish products. We thus monitored the presence of L. monocytogenes and other Listeria spp. in the processing environment, in raw, whole crawfish, and in cooked crawfish meat from two processing plants. Samples were collected from the two plants throughout one crawfish season (April to June 2001) at 5 and 8 separate visits, respectively. At each visit, 6 raw, whole crawfish, 6 finished product samples (crawfish meat), and 14 mid- or end-of-processing environmental sponge samples were collected and tested for L. monocytogenes and Listeria spp. Of the 337 samples tested, 31 contained Listeria spp. Although Listeria innocua was the predominant Listeria spp. found (20 samples), four samples were positive for L. monocytogenes. L. monocytogenes was detected in three raw material samples and in one environmental sample. Listeria spp. were found in 29.5% of raw, whole crawfish (n = 78) and in 4.4% of environmental samples (n = 181) but in none of the finished product samples. Among the environmental samples, Listeria spp. were found in 15.4% of the drains (n = 39) and in 5.1% of the employee contact surfaces (gloves and aprons) (n = 39) but in none of the samples from food contact surfaces. Even though a high prevalence of Listeria spp. was detected on raw materials, it appears that the heat treatment during the processing of crawfish and the practices preventing postprocessing recontamination can significantly reduce Listeria contamination of RTE crawfish meat.

Prevalence of Listeria monocytogenes during Production and Postharvest Processing of Cabbage

From November 1999 to May 2000, analyses of 425 cabbage, 205 water, and 225 environmental sponge samples from four cabbage farms with packing sheds and from two packing sheds in the Rio Grande Valley and Uvalde, Tex., were conducted to determine whether Listeria monocytogenes was present. Samples were tested by the Food and Drug Administration method for the isolation of Listeria spp., and confirmed isolates were DNA fingerprinted by repetitive-element sequence-based polymerase chain reaction (rep-PCR). L. monocytogenes was isolated from 3% (26 of 855) of the samples. Twenty of these isolates were obtained from cabbage (7 isolates from farms and 13 from packing sheds). Three isolates were from water samples (two from farms and one from a packing shed), and three were from environmental sponge samples of packing shed surfaces. Rep-PCR–generated fingerprints of 21 of the isolates revealed 18 distinctive banding patterns. Four isolates from environmental sponge samples of conveyor belts and from cabbage samples shared identical banding patterns, suggesting common sources of contamination. These identical environmental isolates suggest that contact with packing shed surfaces may be a source of contamination of cabbage. However, the cabbage samples could have arrived contaminated, since they were not washed.

Evaluation of Universal Preenrichment Broth for Growth of Heat-Injured Pathogens

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis, and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2°C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37°C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (>103 CFU/ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37°C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation—but not at 6 h—sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.