PRODUCTION AND PROCESSING
Safer Supply Chains
The volume and international breadth of human illnesses associated with alfalfa and radish sprouts justifi ed extensive modifi cations to pathogen control programs within the sprout industry. In 1998, the FDA issued the Guide to Minimize Microbial Con-tamination of Fresh Fruits and Vegetables. As a result, vast improvements have been made to the conven-tional germination, cultivation, harvesting, and pro-cessing of sprouts. Among the most signifi cant of the improvements was the screening of water used during hydroponic cultivation for E. coli (http://www.cfsan.
fda.gov/~dms/prodguid.html). Seed decontamination technologies include the application of acidifi ed sodium chlorite, quaternary ammonium compounds, ozone gas, and irradiation, all of which are capable of reducing levels of E. coli O157:H7 contamination
removal of all visible soil, feces, ingesta, and milk contaminants from the surface of nonintact beef (FSIS, 1993). Knives are invaluable food processing tools but may also be contaminated with pathogens (Bell, 1997). According to the section of the Code of Federal Regulations (9 CFR 415.4, Code of Federal Regulations, 2001) describing sanitary operations,
“all [direct and indirect] food-contact surfaces including…utensils and equipment, must be sanitized as frequently as necessary to prevent the creation of insanitary conditions and the adulteration of prod-uct.” The dual-knife system has been a great asset to the meat packing industry. In an effort to reduce the incidence of cross-contamination events between carcasses, knives are rotated between consecutive carcasses (one knife is applied to a carcass, while the other knife is submerged in sanitizer) (Scanga, 2005).
Other carcass-spot decontamination technologies include steam pasteurization or steam vacuum units, which are used to remove visible contamination or to systematically treat the area most likely to become contaminated during hide removal (Sofos and Smith, 1998; Sofos et al., 1999a, 1999b). While zero toler-ance implies that all such matter is removed, attempts to remove contamination from the entire surface of every carcass using spot decontamination strategies is not feasible, especially with the chain speeds used in many domestic commercial facilities. Inappropri-ate terminology aside, spot decontamination efforts effectively reduce microbial counts and limit the spread of particulate matter over carcass surfaces during subsequent carcass washing steps (Sofos et al., 1999a, 1999b).
Inactivation
Decontamination with scientifi cally validated decontamination fl uids may be valuable in minimiz-ing the incidence of diarrheagenic E. coli, specifi cally STEC contamination, on surfaces of seeds, produce, eggs, nuts, and fresh or processed meat products (Beuchat, 1998; Sapers et al., 1999; Sofos et al., 2006;
Warriner et al., 2005). In lieu of pasteurization, such treatments may be used to decontaminate the outer surfaces of fruits destined for juice production and only when accompanied by rigid microbiological
“hold and test” programs; unpasteurized juice must be labeled as such (21 CFR 120.24, Code of Federal Regulations, 2002). Available decontamination fl uids include chlorine and chlorine derivatives, organic acids, iodophors, trisodium phosphate, peroxides and peroxyacid solutions, ozone and electrolyzed oxidizing water, and protein compounds (Acuff, 2005; Beuchat, 1998; Cords et al, 2005; Sapers et al., 1999; Sofos, 2005; Sofos et al., 1999a, 1999b;
as workers can serve as original vectors of enterically derived contamination or transfer existing contami-nation to unsoiled areas or products. In-plant con-tamination issues may be reduced by simply reviewing the basic fundamentals related to personal hygiene and hand washing, product and equipment handling, and appropriate behavior during illness (FSIS, 1999).
Employee education and training courses are typi-cally included in the prerequisite program portion of a HACCP system and can be customized to fi t the needs of an individual operation. Once proper behav-iors are introduced and improper practices have been corrected, supervisors must provide access to and/or enforce the use of appropriate restroom and eating facilities and good hygiene practices. The advent of mechanical devices as processing aids, which limit worker contact with food products, has also reduced cross-contamination events during process-ing (Scanga, 2005).
When present, microorganisms can contaminate previously sterile inner tissue through cuts or tears in outer skin, rinds, or hides (CDC, 1997; Sofos, 1994, 2005; Sofos et al., 1999c). Therefore, it is imperative that injurious events to protective outer surfaces are minimized, raw produce with surface defects are rejected, and the levels of contamination on outer sur-faces are minimized before inner tissues are exposed.
The outer surfaces/trimmings from carcasses are gen-erally intended for ground meat production, even though the outer surface typically encounters a higher level of contamination than any other carcass region (Gill, 2005). Although cooking (72C or 160F) raw ground beef before consumption should inactivate pathogens of concern, improper cooking is very likely to occur and should be anticipated. Thus, it is not surprising that the majority of fresh-meat-related out-breaks and recalls involve ground meat (Rangel et al., 2005). Carcasses are sterile before hide removal, at which time contamination may be transferred from the hide to the carcass (Sofos, 1994; Gill, 2005).
Advances in mechanical hide removal should reduce the incidence of hide-to-carcass transfer of pathogenic E. coli and also reduce the number of carcasses which fall from overhead rails and onto the ground and must then be diverted to areas reserved for trimming and additional FSIS inspection, as well as the incidence of hide and carcass defects (Scanga, 2005). In addition, the number of potential harbors for pathogenic con-tamination may be reduced by covering tears in the subcutaneous fat layer with plastic fi lm prior to wash-ing carcasses (Simpson et al., 2006). The evisceration process also has the potential to generate high levels/
incidence of contamination on carcasses (Gill, 2005).
Knife trimming has been implemented to address the “zero tolerance” policy, which requires the
process, as well the components (e.g., pulp and fat content), pH, and level of target microorganisms associated with a product (NACMCF, 2006).
Low doses of irradiation are highly lethal for most microorganisms, specifi cally E. coli, and many improvements to irradiation technologies have been developed in recent years (http://www.wisc.edu/fri/
briefs/foodirrd.htm). Irradiation programs have been scientifi cally validated and are available to inactivate E. coli O157:H7 associated with juices, seeds, sprouts, spices, and meat products (Bari et al., 2004; Foley et al., 2004; Murano et al. 1998; NACMCF, 2006).
With adherence to the guidelines for optimization of temperature, packaging conditions, dose, and depth of penetration, the administration of 3 kGy suc-cessfully inactivates the pathogen, with little impact on the fl avor, texture, or color of treated ground beef samples (Arthur et al., 2005; Murano et al., 1998;
Vickers and Wang, 2002). Nonmeat ingredients also appear to increase overall effi cacy of irradiation treat-ments (Bricher, 2003; Kamat et al., 1997; Lacroix et al., 2004; Smith and Pillai, 2004; http://www.wisc.
edu/fri/briefs/foodirrd.htm). The generation of free radicals is also responsible for the generation of hydrogen peroxide and quality defects in irradiated produce, which include mushiness, alterations in fl a-vor profi les, and rapid oxidation (Kuby, 1997; Lewis et al., 2002). Other nonthermal processes include high hydrostatic pressure, shock waves, ultrasonica-tion, and pulsed ultraviolet light or electric fi eld treat-ments (Guan and Hoover, 2005; NACMCF, 2006).
Inhibition
Postlethality contamination events pose a signifi -cant risk to human health, and tools are available to inhibit pathogen growth in both processed and unprocessed products. Antimicrobial agents within processed food product formulations and sprays applied to fi nal products or in packaging material may be used to control postlethal pathogen growth.
However, outbreaks of E. coli infections are still asso-ciated with fresh, minimally processed, and/or ready-to-eat food products, and the use of antimicrobial ingredients to control diarrheagenic E. coli is limited.
Dry fermented sausages are the exception; the use of various lactic acid bacteria cultures, which produce inhibitory compounds (e.g., acids, bacteriocins, diacetyl, and ethanol) are added to raw sausage for-mulations to ensure proper fermentation and prod-ucts of acceptable quality and safety (Lahti et al., 2001; Kang and Fung, 1999; Jay, 1982).
The rate and extent of product chilling, as affected by packaging, stocking density, air fl ow, rela-tive humidity, temperature, and time, can infl uence Warriner et al., 2005). It is extremely important that
these solutions be applied under appropriate condi-tions, as sublethal exposures may only enhance the stress tolerance of diarrheagenic E. coli (Davidson and Harrison, 2002; Duffy et al., 2000; Samelis and Sofos, 2003; Sofos and Smith, 1998). Lipids, pro-teins, other organic compounds, water hardness, con-tact time, and pressure during application infl uence the killing power of many decontamination fl uids (Arrit et al., 2002; Cutter et al., 1997; Pordesimo et al., 2002; Russell and Axtell, 2005; Sofos et al., 1999, Sofos and Smith, 1998; Sofos et al., 2006). In general, many decontamination treatments tend to be more effective when used sequentially or when com-bined with other antimicrobial factors, such as heat (Bacon et al., 2003; Hardin et al., 1995; Leistner and Gould, 2002; Sofos et al., 1999a, 1999b, 2006).
Spray applications may be more convenient than immersion treatments (Beuchat, 1998), although all surfaces of a product may not be exposed or ade-quately decontaminated (Warriner et al., 2005).
Appropriate cooking and handling recommen-dations are available for many raw or partially cooked food products, and adherence to such recommenda-tions should eliminate the risk associated with raw foods. However, possible pathogen contamination must be addressed during processing of products intended for immediate consumption (i.e., ready to eat). In 1998, it became mandatory for all noncitrus juices entering interstate commerce to be pasteurized or be clearly labeled as an unpasteurized product, or as an alternative to pasteurization, fruit juice manu-facturers may adopt HACCP programs which include alternative scientifi cally validated processes which are capable of reducing 5 log cycles of E. coli O157:H7 (21 CFR 120.24). Pasteurization is recommended (21 CFR 120.24, Code of Federal Regulations, 2002), and a thermal pasteurization guide for milk, Grade
“A” Pasteurized Milk Ordinance, is available at http://www.cfsan.fda.gov/~ear/pmo01toc.html. The successful pasteurization of milk and other dairy products can be determined by screening for the pres-ence of postpasteurization phosphatase activity (http://www.cfsan.fda.gov/~ebam/bam-27.html).
Alkaline phosphatase is an LT enzyme naturally found in raw milk and is inactivated at temperatures just above those required to inactivate most milk-borne microorganisms, including diarrheagenic E. coli (http://www.hc-sc.gc.ca/fn-an/alt_formats/hpfb-dgpsa/pdf/res-rech/mfo25-eng.pdf). For these reasons, alkaline phosphatase inactivation is used to verify the successful application of pasteurization treatments (15 min at 71.7 or 30 min at 62.8C) (http://www.
cfsan.fda.gov/~ebam/bam-27.html). In general, the effi cacy of pasteurization treatments depends on the
many tests can be used for more than one of these objectives, it is important to employ the most appro-priate method regarding the type of information needed, sample composition, and the expected level of contamination.
Enrichment and Isolation
Enrichment protocols usually precede most con-ventional and rapid detection methods. Although results of microbiological analyses may be delayed as a result, enrichment protocols are critical when attempting to isolate very low levels of pathogenic E. coli from samples with or without excessive levels of background fl ora (fresh produce and fermented foods), as well as in the recovery of injured cells (Hep-burn et al., 2002; Sanderson et al., 1995). Selective E. coli enrichment broths include brain heart infu-sion, tryptic soy broth, and gram-negative or E. coli broth supplemented with novobiocin (Hajna, 1955;
Hussein et al., 2008). Additional compounds added to selective media to suppress the growth of back-ground fl ora include antimicrobials to which E. coli are particularly resistant, such as cefi xime, cefsulo-din, potassium tellurite, and vancomycin (Hussein et al., 2008). Hussein et al. (2008) found that brain heart infusion supplemented with potassium tellurite, novobiocin, vancomycin, and cefi xime was more suc-cessful than other selective enrichment broths in the suppression of background fl ora during enrichment of STEC recovered from cattle feces.
In general, detection of microorganisms in food product samples can be problematic due to low levels of contamination, a nonhomogeneous distribution of cells throughout the product/sample, or interactions between food components and reagents used in detec-tion protocols (Gill, 2005; Smith et al., 2000). Practi-cal detection methods must be sensitive, specifi c, repeatable, and economical and also generate results expressed in practical units in a timely manner. To date, conventional culture methods remain the gold standard in pathogen detection, and numerous selec-tive and differential media, which require as little as 18 h for the detection of diarrheagenic E. coli, are available (Zadik et al., 1993; http://www.cfsan.fda.
gov/~ebam/bam-a1.html). Popular solid media used to isolate generic biotype I E. coli, EPEC, ETEC, and EAEC include violet red bile agar, Levine’s eosine methylene blue agar, MacConkey agar (MAC), and 3M PetriFilm rehydratable fi lms (http://www.cfsan.
fda.gov/~ebam/bam-a1.html). MAC and Hektoen enteric agar are most useful when isolating EIEC (Doyle and Padhye, 1989).
Sorbitol is added to MAC (SMAC) to differenti-ate E. coli O157 from non-O157 strains, which the fate of microorganisms during storage. As oxygen
and other gases in the environment affect microbial growth, modifi ed atmosphere packaging is used for microbial inhibition. Such technologies manipulate individual levels of the main components of air (O2, N2, and CO2) and include common vacuum packag-ing (Phillips, 1996). The performance of E. coli O157:H7 under modifi ed atmospheres is not well understood and may be similar to its performance under aerobic conditions (Hao and Brackett, 1993).
Product quality and safety are almost certainly compromised after encounters of abusive tempera-tures for sporadic/extended periods of time during holding, transport, or distribution (Koutsoumanis and Taoukis, 2005). Diarrheagenic E. coli can sur-vive but will not grow at recommended refrigeration temperatures (4C or 40F), but it is capable of growth at temperatures that barely exceed (7 to 9C or 45F) the recommendations (ICMSF, 1996). Thus, to effec-tively inhibit the proliferation of these pathogens dur-ing cold storage of produce and meat products, temperature must be lowered as quickly as possible and maintained for the duration of product holding (Hao and Brackett, 1993; Simpson et al., 2006).
Chilling food products too rapidly or storing them at 4C may result in quality defects (e.g., cold-shortening in rapidly chilled beef, lamb, and pork or freeze-damaged fresh produce) (Lee et al., 2004).
Temperature monitoring/recording systems, equipped with audible default alarms, can be used to effectively maintain the correct temperature during each phase of cold storage.
METHODS FOR ISOLATION, DETECTION,