Fluorescent Protein–Marked Escherichia coli Biotype I Strains as Surrogates for Enteric Pathogens in Validation of Beef Carcass Interventions

2009 ◽  
Vol 72 (2) ◽  
pp. 295-303 ◽  
Author(s):  
ELISA CABRERA-DIAZ ◽  
TIFFANY M. MOSELEY ◽  
LISA M. LUCIA ◽  
JAMES S. DICKSON ◽  
ALEJANDRO CASTILLO ◽  
...  

The efficacy of antimicrobial interventions implemented in slaughter establishments to reduce enteric pathogens on beef carcasses should optimally be validated under commercial operation conditions. This study was conducted to identify surrogate organisms for enteric pathogens that could be used to validate beef carcass interventions. The growth, resistance, and attachment properties of nonpathogenic fluorescent protein–marked Escherichia coli strains were compared with those of E. coli O157: H7 and Salmonella strains. Growth curves were obtained based on growth in tryptic soy broth at 37°C. In general, growth parameters were not different among potential surrogates and target pathogens (P > 0.05). Thermal resistance was compared in phosphate-buffered saline (pH 7.4) at 55, 60, and 65°C, and D-values of potential surrogates were not different (P > 0.05) or were higher (P < 0.05) than those of the target pathogens. Acid resistance was tested in phosphate-buffered saline acidified with l-lactic acid at pH 2.5, 3.0, and 3.5, and log reductions (CFU per milliliter) were not different (P > 0.05) among potential surrogates and E. coli O157:H7 strains; however, some Salmonella serotypes were less acid resistant than were surrogates (P < 0.05). The cell surface hydrophobicity was different (P < 0.05) among surrogates and some E. coli O157:H7 strains, but the strength of attachment to beef carcasses was not different (P > 0.05) among all microorganisms. Log reductions (CFU per square centimeter) after application of hot water washes and 2% l-lactic acid sprays on beef carcasses were not different (P > 0.05) among surrogates and pathogens. The nonpathogenic E. coli strains evaluated in this study could be used as surrogates for E. coli O157:H7 and Salmonella to validate hot water and lactic acid interventions on beef carcasses.

2015 ◽  
Vol 78 (12) ◽  
pp. 2136-2142 ◽  
Author(s):  
BRITTNEY R. SCOTT ◽  
XIANG YANG ◽  
IFIGENIA GEORNARAS ◽  
ROBERT J. DELMORE ◽  
DALE R. WOERNER ◽  
...  

Studies were conducted to (i) determine whether inoculants of nonpathogenic Escherichia coli biotype I effectively served as surrogates for E. coli O157:H7, non-O157 Shiga toxin–producing E. coli, and Salmonella when prerigor beef carcass tissue was treated with a commercially available blend of lactic acid and citric acid (LCA) at a range of industry conditions of concentration, temperature, and pressure; (ii) determine the antimicrobial efficacy of LCA; and (iii) investigate the use of surrogates to validate a hot water and LCA sequential treatment as a carcass spray intervention in a commercial beef harvest plant. In an initial laboratory study, beef brisket tissue samples were left uninoculated or were inoculated (~6 log CFU/cm2) on the adipose side with E. coli O157:H7 (5-strain mixture), non-O157 Shiga toxin–producing E. coli (12-strain mixture), Salmonella (6-strain mixture), or nonpathogenic E. coli (5-strain mixture). Samples were left untreated (control) or were treated with LCA, in a spray cabinet, at one of eight combinations of solution concentration (1.9 and 2.5%), solution temperature (43 and 60°C), and application pressure (15 and 30 lb/in2). In a second study, the E. coli surrogates were inoculated (~6 log CFU/cm2) on beef carcasses in a commercial facility to validate the use of a hot water treatment (92.2 to 92.8°C, 13 to 15 lb/in2) followed by an LCA treatment (1.9%, 50 to 51.7°C, 13 to 15 lb/in2, 10 s). In the in vitro study, surrogate and pathogen bacteria did not differ in their response to the tested LCA treatments. Treatment with LCA reduced (P < 0.05) inoculated populations by 0.9 to 1.5 log CFU/cm2, irrespective of inoculum type. The hot water and LCA sequential treatments evaluated in the commercial facility reduced (P < 0.05) the inoculated nonpathogenic E. coli surrogates on carcasses by 3.7 log CFU/cm2. This study therefore provides the meat industry with data for this sequential multiple hurdle system for the operation parameters described.


2010 ◽  
Vol 73 (10) ◽  
pp. 1864-1874 ◽  
Author(s):  
STEVEN C. INGHAM ◽  
RYAN J. ALGINO ◽  
BARBARA H. INGHAM ◽  
RONALD F. SCHELL

We compared the survival of potential pathogen surrogates—meat-hygiene indicators (non–Escherichia coli coliforms), biotype I E. coli, and lactic acid bacteria starter cultures—with survival of an E. coli O157:H7 (ECO157:H7) inoculum in beef carcass intervention trials. Survival of one lactic acid bacteria starter culture (Bactoferm LHP Dry [Pediococcus acidilactici and Pediococcus pentosaceus]), a five-isolate biotype I inoculum, and a five-isolate non–E. coli coliform inoculum, was compared with survival of a 12-isolate ECO157:H7 inoculum in interventions by using beef brisket (adipose and lean), cod fat membrane, or neck tissue. Treatments were grouped by abattoir size: small (6-day dry aging; 22°C acid treatment [2.5% acetic acid, 2% lactic acid, or Fresh Bloom], followed by 1-day dry aging; hot water) and large (warm acid treatment [5% acetic acid or 2% lactic acid] with or without a preceding hot water treatment). Reductions in pathogen and surrogate inocula were determined with excision sampling. A surrogate was considered a suitable replacement for ECO157:H7 if the intervention produced a reduction in surrogate levels that was not significantly greater (P ≥ 0.05) than that observed for ECO157:H7. All three surrogate inocula were suitable as ECO157 surrogates for dry aging and acid spray plus dry-aging treatments used by small abattoirs. No one inoculum was suitable as an ECO157 surrogate across all intervention treatments used by large abattoirs. Effects seen on neck tissue were representative of other tissues, and the low value of the neck supports its use as the location for evaluating treatment efficacy in in-plant trials. Results support using nonpathogenic surrogate organisms to validate beef carcass intervention efficacy.


2006 ◽  
Vol 69 (8) ◽  
pp. 1808-1813 ◽  
Author(s):  
JOSEPH M. BOSILEVAC ◽  
XIANGWU NOU ◽  
GENEVIEVE A. BARKOCY-GALLAGHER ◽  
TERRANCE M. ARTHUR ◽  
MOHAMMAD KOOHMARAIE

Lactic acid has become the most commonly used organic acid for treatment of postevisceration beef carcasses. Many processors have also implemented 2% lactic acid washes on preevisceration carcasses. We previously demonstrated that hot water washing and steam vacuuming are effective carcass interventions. Because of the effectiveness of hot water, we compared its use with that of lactic acid as a preevisceration wash in a commercial setting. A commercial hot water carcass wash cabinet applying 74°C (165°F) water for 5.5 s reduced both aerobic plate counts and Enterobacteriaceae counts by 2.7 log CFU/100 cm2 on preevisceration carcasses. A commercial lactic acid spray cabinet that applied 2% l-lactic acid at approximately 42°C (105 to 110°F) to preevisceration carcasses reduced aerobic plate counts by 1.6 log CFU/100 cm2 and Enterobacteriaceae counts by 1.0 log CFU/100 cm2. When the two cabinets were in use sequentially, i.e., hot water followed by lactic acid, aerobic plate counts were reduced by 2.2 log CFU/100 cm2 and Enterobacteriaceae counts were reduced by 2.5 log CFU/100 cm2. Hot water treatments reduced Escherichia coli O157:H7 prevalence by 81%, and lactic acid treatments reduced E. coli O157:H7 prevalence by 35%, but the two treatments in combination produced a 79% reduction in E. coli O157:H7, a result that was no better than that achieved with hot water alone. These results suggest that hot water would be more beneficial than lactic acid for decontamination of preevisceration beef carcasses.


2007 ◽  
Vol 70 (5) ◽  
pp. 1174-1180 ◽  
Author(s):  
C. E. HELLER ◽  
J. A. SCANGA ◽  
J. N. SOFOS ◽  
K. E. BELK ◽  
W. WARREN-SERNA ◽  
...  

The prevalence of Escherichia coli O157:H7 on beef subprimal cuts intended for mechanical tenderization was evaluated. This evaluation was followed by the assessment of five antimicrobial interventions at minimizing the risk of transferring E. coli O157:H7 to the interior of inoculated subprimal cuts during blade tenderization (BT) or moisture enhancement (ME). Prevalence of E. coli O157:H7 on 1,014 uninoculated beef subprimals collected from six packing facilities was 0.2%. Outside round pieces inoculated with E. coli O157:H7 at 104 CFU/100 cm2 were treated with (i) no intervention, (ii) surface trimming, (iii) hot water (82°C), (iv) warm 2.5% lactic acid (55°C), (v) warm 5.0% lactic acid (55°C), or (vi) 2% activated lactoferrin followed by warm 5.0% lactic acid (55°C) and then submitted to BT or ME. Prevalence (n = 196) of internalized (BT and ME) E. coli O157:H7 was 99%. Enumeration of E. coli O157:H7 (n = 192) revealed mean surface reductions of 0.93 to 1.10 log CFU/100 cm2 for all antimicrobial interventions. E. coli O157:H7 was detected on 3 of the 76 internal BT samples and 73 of the 76 internal ME samples. Internal ME samples with no intervention had significantly higher mean E. coli O157:H7 populations than did those internal samples treated with an intervention, but there were no significant differences in E. coli O157:H7 populations among internal BT samples. Results of this study demonstrate that the incidence of E. coli O157:H7 on the surface of beef subprimal cuts is low and that interventions applied before mechanical tenderization can effectively reduce the transfer of low concentrations of E. coli O157:H7 to the interior of beef subprimal cuts.


Author(s):  
Kourtney A. Daniels ◽  
Katherine Modrow ◽  
Wesley N. Osburn ◽  
Thomas Matt Taylor

Water use for antimicrobial intervention application for beef harvest has come under increased scrutiny in recent years in an effort to enhance water conservation during beef harvest and fabrication. This study was conducted to determine the efficacy of beef safety interventions for reducing surrogates of the Shiga toxin-producing Escherichia coli (STEC) on beef cuts while lowering intervention-purposed water use for a Small or Very Small beef establishment. Beef briskets, shoulder/clods, and rounds were inoculated with a gelatin-based slurry containing 6.8±0.3 log CFU/g non-pathogenic E. coli . After 30 min of attachment, inoculated cuts were treated by: conventional lactic acid spray (LA; 2.5%, 55°C), lactic acid spray delivered by an electrostatic spray handheld wand (ESS; 2.5%, 55°C), hot water spray (HW; 82°C), recycled hot water spray (RW; 82°C) wherein previously applied hot water was collected, thermally pasteurized to 82°C, or left untreated (CON). 100 mL of each treatment was sprayed onto marked surfaces of inoculated cuts, after which surviving surrogate E. coli were enumerated. LA and ESS treatments produced greater reductions (1.0-1.1 log CFU/300 cm 2 ) versus hot water interventions (0.3-0.5 log CFU/300 cm 2 ) ( p =<0.0001). Recycling of water reduced water losses by no less than 45% on RW-treated beef cuts. Low water beef safety interventions offer Small and Very Small inspected beef establishments opportunities to incrementally reduce water use during intervention application, but not necessarily without loss of pathogen reduction efficacy.


2018 ◽  
Vol 9 (2) ◽  
pp. 275-780 ◽  
Author(s):  
O. М. Berhilevych ◽  
V. V. Kasianchuk ◽  
O. M. Deriabin ◽  
M. D. Kukhtyn

Escherichia coli is part of the normal microflora of the intestinal tract of humans and warm-blooded animals, but its presence in raw material and food of animal origin is considered as fecal contamination and can be very dangerous for consumers. The determination of the number of E. coli in raw material and food is important because among them can be pathogenic strains. The most dangerous strains are considered enterohemorrhagic E. coli as a causative agent of severe bloody diarrhea and hemorrhagic uremic syndrome in humans through the production of Shiga-toxin, which is the main virulence factor, responsible for disease. The aim of this study was to identify the prevalence of Shiga toxin-producing strains of E. coli (STEC) from swabs of beef and swine carcass in slaughterhouses in Ukraine and characterize their genes, which are responsible for pathogenic properties. A total of 230 samples of swabs from beef (130) and swine (100) carcasses were obtained from 5 slaughterhouses in Ukraine between 2012 and 2015. Samples of swabs from carcasses were randomly selected at the final point of the process after the final washing of the carcass from the following areas: distal hind limb, abdomen (lateral and medial) from swine carcasses, brisket, flank and flank groin areas from beef carcasses. All samples were examined by culture-dependent method, after that each positive isolate of STEC was analyzed by multiplex PCR to detect the stx1, stx2, and eae genes. Out of 230 collected samples, seven (7.2%) were contaminated with STEC. The highest prevalence of STEC was found in swabs from beef carcasses (8.1%) in comparison to swabs from swine carcasses (5.7%). The stx1 gene was the predominant gene detected in all STEC positive samples. The eae gene was found in one of the examined isolates from beef carcass. Three isolates from swabs of beef carcass carried both stx1 and stx2 genes, one isolate showed association between stx1 and eae genes, one isolate was positive for stx1 gene only. In swabs from swine carcasses (2 isolates) stx1 and stx2 genes were presented simultaneously. The results of this study suggested that fresh raw meat could be a potential vehicle for transmission of the Shiga toxin-producing strain of E. coli to humans. This is the first report of STEC prevalence in beef and swine carcasses in Ukraine and these data will be valuable for microbiological risk assessment and help the appropriate services to develop strategies to mitigate health risk.


2018 ◽  
Vol 81 (5) ◽  
pp. 762-768
Author(s):  
JOSHUA D. HASTY ◽  
JOHN A. HENSON ◽  
GARY R. ACUFF ◽  
DENNIS E. BURSON ◽  
JOHN B. LUCHANSKY ◽  
...  

ABSTRACT Scalding of hide-on bob veal carcasses with or without standard scalding chemical agents typically used for hogs, followed by an 82.2°C hot water wash and lactic acid spray (applied at ambient temperature) before chilling, was evaluated to determine its effectiveness in reducing Shiga toxin–producing Escherichia coli surrogate populations. A five-strain cocktail of rifampin-resistant, nonpathogenic E. coli surrogates was used to inoculate hides of veal carcasses immediately after exsanguination (target inoculation level of 7.0 log CFU/100 cm2). For carcasses receiving no scalding treatments, spraying with 82.2°C water as a final wash resulted in a 4.5-log CFU/100 cm2 surrogate reduction, and an additional 1.2-log CFU/100 cm2 reduction was achieved by spraying with 4.5% lactic acid before chilling. Scalding hide-on carcasses in 60°C water (no chemicals added) for 4 min in a traditional hog scalding tank resulted in a 2.1-log CFU/100 cm2 reduction in surrogate levels, and a subsequent preevisceration 82.2°C water wash provided an additional 2.9-log CFU/100 cm2 reduction. Spraying a 4.5% solution of lactic acid onto scalded, hide-on carcasses (after the 82.2°C water wash) resulted in a minimal additional reduction of 0.4 log CFU/100 cm2. Incorporation of scalding chemicals into the scald water resulted in a 4.1-log CFU/100 cm2 reduction (1.9 log CFU/100 cm2 greater than scalding without chemicals) in the surrogate population, and the first 82.2°C wash provided an additional 2.5-log CFU/100 cm2 reduction. Application of antimicrobial interventions did not affect the carcass temperature decline during chilling, the pH decline, or the color characteristics of the ribeye or the flank of the bob veal carcasses.


2015 ◽  
Vol 78 (6) ◽  
pp. 1197-1202 ◽  
Author(s):  
THELMA F. CÁLIX-LARA ◽  
KATIE R. KIRSCH ◽  
MARGARET D. HARDIN ◽  
ALEJANDRO CASTILLO ◽  
STEPHEN B. SMITH ◽  
...  

Although studies have shown antimicrobial treatments consisting of hot water sprays alone or paired with lactic acid rinses are effective for reducing Escherichia coli O157:H7 loads on beef carcass surfaces, the mechanisms by which these interventions inactivate bacterial pathogens are still poorly understood. It was hypothesized that E. coli O157:H7 exposure to hot water in vitro at rising temperatures for longer time periods would result in increasing deterioration of bacterial outer membrane lipids, sensitizing the pathogen to subsequent lactic acid application. Cocktails of E. coli O157:H7 strains were subjected to hot water at 25 (control) 65, 75, or 85°C incrementally up to 60 s, after which surviving cells were enumerated by plating. Formation of lipid hydroperoxides from bacterial membranes and cytoplasmic accumulation of l-lactic acid was quantified spectrophotometrically. Inactivation of E. coli O157:H7 proceeded in a hot water exposure duration- and temperature-dependent manner, with populations being reduced to nondetectable numbers following heating of cells in 85°C water for 30 and 60 s (P < 0.05). Lipid hydroperoxide formation was not observed to be dependent upon increasing water temperature or exposure period. The data suggest that hot water application prior to organic acid application may function to increase the sensitivity of E. coli O157:H7 cells by degrading membrane lipids.


2004 ◽  
Vol 67 (10) ◽  
pp. 2099-2106 ◽  
Author(s):  
J. D. STOPFORTH ◽  
Y. YOON ◽  
K. E. BELK ◽  
J. A. SCANGA ◽  
P. A. KENDALL ◽  
...  

Samples (10 by 20 by 2.5 cm) of beef carcass tissue were inoculated (104 to 105 CFU/cm2) with Escherichia coli O157: H7 that was either non–acid habituated (prepared by incubating at 15°C for 48 h in inoculated filter-sterilized composite [1:1] of hot and cold water meat decontamination runoff fluids, pH 6.05) or acid habituated (prepared in inoculated water fluids mixed with filter-sterilized 2% lactic acid [LA] runoff fluids in a proportion of 1/99 [vol/vol], pH 4.12). The inoculated surfaces were exposed to conditions simulating carcass chilling (−3°C for 10 h followed by 38 h at 1°C). Treatments applied to samples (between 0 and 10 h) during chilling included the following: (i) no spraying (NT) or spraying (for 30 s every 30 min) with (ii) water, (iii) cetylpyridinium chloride (CPC; 0.1 or 0.5%), (iv) ammonium hydroxide (AH; 0.05%), (v) lactic acid (LA; 2%), (vi) acidified sodium chlorite (ASC; 0.12%), (vii) peroxyacetic acid (PAA; 0.02%), (viii) sodium hydroxide (SH; 0.01%), or (ix) sodium hypochlorite (SC; 0.005%) solutions of 4°C. Samples were taken at 0, 10, 24, 36, and 48 h of the chilling process to determine changes in E. coli O157:H7 populations. Phase 1 tested water, SH, PAA, LA, and 0.5% CPC on meat inoculated with non–acid-habituated pathogen populations, whereas phase 2 tested water, SC, AH, ASC, LA, and 0.1% CPC on meat inoculated with acid- and non–acid-habituated populations. Reductions in non–acid-habituated E. coli O157:H7 populations from phase 1 increased in the order NT = water = SH < PAA < LA < CPC. Reductions from phase 2 for acid-habituated cells increased in the order NT = water = SC < ASC = LA = AH < CPC, whereas on non–acid-habituated cells the order observed was NT = water = SC < AH = ASC < LA < CPC. Previous acid habituation of E. coli O157:H7 inocula rendered the cells more resistant to the effects of spray chilling, especially with acid; however, the trend of reduction remained spray chilling with water = non–spray chilling < spray chilling with chemical solutions.


Author(s):  
Chevise L. Thomas ◽  
Harshavardhan Thippareddi ◽  
Sanjay Kumar ◽  
Macc Rigdon ◽  
Robert W. Mckee ◽  
...  

Ruminants are natural reservoirs of Shiga toxin producing Escherichia coli (STEC), and the STEC can be easily transferred to carcasses during the conversion of animals to meat. Three experiments were conducted to validate the efficacy of lactic acid (4%; LA), peroxyacetic acid (300 ppm; PAA), and hot water (80˚C; HW) for their individual or combined abilities to reduce STEC surrogates on bob veal carcasses pre- and post-chill and through fabrication. In experiment 1, hot carcasses (n=9) were inoculated with a 5-strain cocktail (ca. 8 log CFU/mL) containing rifampicin-resistant surrogate Escherichia coli ( E. coli ; BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431) and then treated with HW, LA, or PAA. Carcasses were then chilled (0±1°C; 24 h), split in half, and each side was treated with either LA or PAA. In experiment 2, hot carcasses (n=3) were inoculated and chilled (24 h). After 24 h, the carcasses were split, and each side was treated with either LA or PAA. For experiment 3, carcasses (n=3) were chilled for 24 h, split, inoculated, and treated with either LA or PAA. After chilling, carcasses from all three experiments were fabricated to subprimals and the cut surfaces were sampled to determine the translocation. Experiment 1 showed that LA+LA was the most effective ( P ≤ 0.05) treatment for reducing surrogate E. coli on veal. In experiments 2 and 3, LA and PAA were similar ( P > 0.05) in their abilities to reduce E. coli on chilled veal carcasses. In experiments 1 and 2, all antimicrobial treatments resulted in undetectable levels (< 0.2 log CFU/cm 2 ) of surrogate E. coli on cut surfaces after fabrication, while low levels (1.7 and 1.0 log CFU/cm 2 for LA and PAA, respectively) were observed in experiment 3. Of the antimicrobial interventions utilized, lactic acid was more effective for reducing STEC surrogate populations on veal carcasses, pre- and/or post-chill.


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