Jeffrey L. Kornacki

Development of thermal surrogate microorganisms in ground beef for in-plant critical control point validation studies.

In search of a suitable surrogate microorganism for in-plant critical control point validation, we compared the rates of thermal inactivation of three bacteria, Enterococcus faecium B2354, Pediococcus parvulus HP, and Pediococcus acidilactici LP, to those of Listeria monocytogenes and Salmonella. Ground beef samples containing 4 and 12% fat were inoculated with E. faecium, L. monocytogenes, and Salmonella Senftenberg 775W and heated at 58, 62, 65, or 68 degrees C. The decimal reduction times (D-values) for E. faecium B2354 in 4 and 12% fat ground beef were 4.4 to 17.7 and 3.6 to 14.6 times greater, respectively, than those for L. monocytogenes or Salmonella Senftenberg 775W at all temperatures tested, with the greatest differences in D-values occurring at 58 and 62 degrees C. Higher fat content protected bacteria from thermal inactivation in general, especially at temperatures lower than 68 degrees C. The heat resistance in a broth medium at 62degrees C of two food-grade bacteria, P. parvulus HP and P. acidilactici LP, was compared with that of the three strains under study. The D-values of P. parvulus HP and P. acidilactici LP were lower than those of E. faecium B2354 but 4.1 and 2.5 times greater, respectively, than those of Salmonella Senftenberg 775W, the most resistant pathogen. These results indicate that thermal treatments of ground beef at 58 to 68 degrees C that kill E. faecium B2354 will also kill Salmonella and L. monocytogenes, and the two Pediococcus isolates may serve as alternate surrogates for validation studies when a less heat-resistant surrogate is desired. However, additional studies in ground beef are needed with the Pediococcus strains in the desired temperature range intended for validation purposes.

Foodborne Illness Caused by Escherichia coli: A Review

Enteropathogenic Escherichia coli (EEC) can be defined as any strain of E. coli that has the potential to cause diarrheal illness. Four major categories of EEC exist. Classical enteropathogenic E. coli (EPEC) commonly refers to serogroups of E. coli historically associated with outbreaks of diarrhea in young children and infants. Facultatively enteropathogenic E. coli (FEEC) are non-EPEC serogroups associated with sporadic diarrhea, and include many serogroups associated with the normal intestinal flora. Enterotoxigenic E. coli (ETEC) is commonly isolated from outbreaks of travelers diarrhea, and includes those strains which produce a heat-stable enterotoxin (ST) only, a heat-labile enterotoxin (LT) only and those which produce both ST and LT. These organisms adhere to and colonize the epithelial cell surfaces of the proximal small intestine. This colonization is mediated by specific types of fimbriae which are host-specific. Toxigenicity is plasmid-related. Enteroinvasive E. coli (EIEC) exert their pathogenic effect through an invasive infection of the gastrointestinal tract. Many techniques currently exist to determine the presence of enterotoxins produced by a particular strain of E. coli . These include bioassay, tissue culture and in vitro immunological techniques. Of the newer in vitro immunological methods, the staphylococcal coagglutination technique to detect LT seems to have potential for routine use in diagnostic microbiology laboratories. Since large numbers (106 - 109) of EEC are necessary for diarrhea, an unsanitary environment is needed for transmission of illness. Presence of EEC varies geographically; however, E. coli diarrhea is not likely to occur in the more hygenic areas of the world, except in occasional common-source outbreaks where the organism has time to replicate in food or water. The following foods have been implicated in documented E. coli diarrheal outbreaks worldwide: meat and meat products, fish, poultry, milk and dairy products, vegetables, baked products, rice formulations, coffee substitutes and water.

The Microbiological Safety of Low Water Activity Foods and Spices

Low-water activity (lowa w ) foods (those with a w < 0.70), which were once thought to be microbiologically safe, have, in recent years, been shown to be contaminated with foodborne pathogens, most notably and frequently Salmonella spp., leading to numerous food product recalls and foodborne illness outbreaks. Lowa w food products can no longer be considered inherently safe, simply because Salmonella will not grow in such products. Therefore, diligence must be applied to ensure that safe food practices are employed for lowa w foods. Areas of concern include the sourcing of major and minor ingredients, unsanitary drying or storage conditions, contaminated processing equipment or improper maintenance, faulty sanitary design of manufacturing or processing equipment, sick or infected employees, cross-contamination of ready-to-eat foods, improper sanitation procedures, improper testing methods, inappropriate sampling plans, failure to act on foodborne pathogen-positive samples, and failure to validate and verify antimicrobial intervention treatments. Other areas in need of attention include failure to implement approved Hazard Analysis and Critical Control Points (HACCP) plans in manufacturing facilities, improper supplier or importer standards or failure to monitor or audit suppliers for hygiene and pathogen control, a faulty assumption that a given lowa w food or food product is innately safe from foodborne pathogen contamination, or, fi nally, overt criminal negligence on the part of a manufacturer or supplier involving one or more of the items mentioned above. Examples of lowa w food products that have previously been considered inherently safe from foodborne pathogens are raw fl our (responsible for a 2008 outbreak sickening 67 people and hospitalizing 12) and two peanut butter or paste outbreaks in 2007–2009, which sickened over 1,400 people in 48 US states and Canada. It is conceivable that lowa w food products not yet considered at risk for foodborne pathogens may emerge. Salmonella spp., of all common foodborne pathogens, will continue to pose the greatest threat in these foods, due to its uncanny ability to survive desiccation in foods and live for years in the environment of food processing facilities. J. B. Gurtler (*) USDA Research Scientist , 1029 Square Dr. , Phoenixville , PA 19460 , USA e-mail: gurtljb@gmail.com M. P. Doyle University of Georgia , Center for Food Safety , 1109 Experinat St. Griffi n , GA 30223 , USA J. L. Kornacki Kornacki Microbilogy Solutions , 9 Woodgien Ct. , Madison , WI 53716 , USA

A Solid Agar Overlay Method for Recovery of Heat-Injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58 degrees C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.

Fate of Nonpathogenic and Enteropathogenic Escherichia coli During the Manufacture of Colby-like Cheese

Pasteurized whole milk was artificially contaminated with 100 to 1000 Escherichia coli /ml and was used to manufacture Colby-like cheese. Some cheeses were made so their composition differed from that of normal Colby cheese. Cheeses were cut in half and stored at 3°C and 10°C. E. coli was enumerated by surface-plating of samples on Trypticase Soy Agar (TSA) with an overlay of Violet Red Bile Agar (VRB). E. coli increased by 100 to 1000-fold, depending on the strain, to about 1 × 106/g of curd, in most instances, by the end of the cook (3.5-3.9 h). After this point numbers of E. coli in cheeses generally decreased over a period of weeks. One strain of enteropathogenic E. coli (EEC) could not be detected after 4 weeks, and another (in all but one instance) after 6 weeks. However, EEC in one lot of cheese persisted at numbers in excess of 1 × 103/g after 12 weeks of refrigerated storage. EEC survived at low levels (<350/g) for many weeks in one instance. Cheeses of poor quality (high moisture and pH) were made to assess the effects of improper manufacture on survival of E. coli . In these cheeses, E. coli eventually reached numbers in excess of 1 × 108/g and persisted for many weeks at high numbers. Survival of E. coli in Colby-like cheese appeared to be influenced by pH, salt and temperature; pH seemed to have the greatest effect on survival of the bacterium.

Evaluation of Several Modifications of an Ecometric Technique for Assessment of Media Performance

Recovery of Listeria monocytogenes 101M, Jonesia denitrificans, salmonellae, and Pediococcus sp. NRRL B-2354 across nine media was evaluated with three modified versions of an ecometric method. Two approaches involved the use of broth cultures (10(8) to 10(9) CFU/ml) of individual strains and either large (10-microl) or small (1-microl) presterilized plastic loops. The third approach involved precultured slants and the inoculation of media with presterilized plastic inoculating needles (10(4) CFU per needle). Absolute growth indices (AGIs) were compared. No significant differences (P < 0.05) between methods were found when tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) was used for the recovery of L. monocytogenes, J. denitrificans, Pediococcus sp. NRRL B-2354, and Salmonella spp. However, the small loop-broth technique recovered significantly fewer Salmonella enterica Typhimurium DT104 and Salmonella Senftenberg 775W cells than the other two techniques did. The performance of each individual bacterial strain on each of nine media was assayed. The recovery of L. monocytogenes was excellent (AGI > 4.8) with TSAYE, PALCAM, modified Oxford medium (MOX), and Baird-Parker agar and slight with modified PRAB (AGI = 0.4) and deMan Rogosa Sharpe (MRS) agar (< 0.1), and the organism was not recovered with the remaining media (modified lysine iron agar [MLIA], xylose lysine desoxycholate [XLD] agar, and xylose lysine tergitol 4 [XLT4] agar). The recovery of J. denitrificans with TSAYE and MOX was excellent, significantly better than that achieved with PALCAM (AGI = 3.0), but the organism was not recovered with Baird-Parker agar or with the other media tested. The recovery of Pediococcus sp. NRRL B-2354 was excellent with TSAYE and modified PRAB medium > Baird-Parker agar > acidified MRS agar, but the organism was not recovered with any of the other media tested. The best recovery of S. enterica Typhimurium DT104 was achieved with TSAYE > MLIA > or = XLD agar > or = XLT4 agar > Baird-Parker > PALCAM, MOX, acidified MRS agar, modified PRAB, and MRS agar. The best recovery of Salmonella Senftenberg 775W was achieved with TSAYE, MLIA, and XLD agar > XLT4 agar, but the organism was not recovered with the other media evaluated.

Selected Pathogens of Concern to Industrial Food Processors: Infectious, Toxigenic, Toxico-Infectious, Selected Emerging Pathogenic Bacteria

This chapter, written by several contributing authors, is devoted to discussing selected microbes of contemporary importance. Microbes from three categories are described by the following: (1) infectious invasive agents like Salmonella, Listeria monocytogenes, and Campylobacter; (2) toxigenic pathogens such as Staphylococcus aureus, Bacillus cereus, and Clostridium botulinum; and (3) toxico-infectious agents like enterohemorrhagic Escherichia coli and Clostridium perfringens. In addition, emerging pathogens, like Cronobacter (Enterobacter) sakazakii, Arcobacter spp., and Mycobacterium avium subspecies paratuberculosis are also described.

Thermal inactivation of staphylococcus aureus in retentates from ultrafiltered milk

Cells of Staphylococcus aureus strains 196E, 481, and 425 were thermally stressed at 56°C for 10 min in milk and enumerated on Plate Count Agar (PCA), Mannitol Salt Agar (MSA), and PCA with an overlay of MSA. PCA recovered more S. aureus 196E and 481 than did PCA/MSA, which recovered more than MSA. PCA/MSA recovered slightly more S. aureus 425 than did PCA, which recovered more than MSA. At 58°C, in order of decreasing heat resistance, the four strains of S. aureus originally isolated from food were 425 > 100 and 481 > 196E. Their D-values were 26,14,13, and 3.0 min, respectively. S. aureus 425 was more heat resistant in the stationary than in the log phase when heated at 58°C in whole milk. Heat resistance at 58°C increased overall during the stationary growth phase, but was fairly stable when the culture was from 17 to 25 h or from 41 to 49 h old. S. aureus 425 exhibited no consistent differences in heat resistance in concentrated (4X by volume) and unconcentrated skim or whole milk. Adjustments of protein (3.5-4.0% to 12.6-16%), milkfat (0.28-1.12% to 10%), and lactose (ca. 4.5-5.0% to ca. 14.5-15%) contents of milk and 4X (volume concentration) UF milk retentates afforded no significant thermal protection to S. aureus 425. Diafiltration of 4X skim milk reduced thermal protection of S. aureus 425 in the retentate over that of unconcentrated skim milk of the same lot when tested at 63 and 74°C. S. aureus 425 had greatest D-values (min) in skim milk (0.36 ± 0.05) and permeate (0.30 ± 0.14) followed by permeate from diafiltration (0.28 ± 0.06) when tested at 63°C.

Heat-Inactivation of Streptococcus faecium var. casseliflavus in Skim Milk Cultures with Pseudomonas fluorescens

Thermal destruction of Streptococcus faecium var. casseliflavus (SFC) at 57°C in autoclaved skim milk was determined at several pH values, and when skim milk was inoculated with Pseudomonas fluorescens and incubated 3 d at 10°C plus 4 d at room temperature before SFC was added. The pH values between 6.4 and 6.6 had little effect on heat resistance of SFC. At pH 5.6, however, accelerated thermal destruction occurred in sterile skim milk as compared to skim milk at pH 6.5. (D-values were 4.8 min and 10.2 min, respectively). Presence of large populations (log10 bacterial count = 9.8 to 9.9/ml) of P. fluorescens had a protective effect on SFC (D = 7.6 ± .7 min in skim milk preincubated with P. fluorescens and 6.2 min in skim milk without P. fluorescens ).

The Microbiological Safety of Spices and Low-Water Activity Foods: Correcting Historic Misassumptions

Low-water activity (low-a w) foods (those with a w < 0.70), which were once thought to be microbiologically safe, have, in recent years, been shown to be contaminated with foodborne pathogens, most notably and frequently Salmonella spp., leading to numerous food product recalls and foodborne illness outbreaks. Low-a w food products can no longer be considered inherently safe, simply because Salmonella will not grow in such products. Therefore, diligence must be applied to ensure that safe food practices are employed for low-a w foods. Areas of concern include the sourcing of major and minor ingredients, unsanitary drying or storage conditions, contaminated processing equipment or improper maintenance, faulty sanitary design of manufacturing or processing equipment, sick or infected employees, cross-contamination of ready-to-eat foods, improper sanitation procedures, improper testing methods, inappropriate sampling plans, failure to act on foodborne pathogen-positive samples, and failure to validate and verify antimicrobial intervention treatments. Other areas in need of attention include failure to implement approved Hazard Analysis and Critical Control Points (HACCP) plans in manufacturing facilities, improper supplier or importer standards or failure to monitor or audit suppliers for hygiene and pathogen control, a faulty assumption that a given low-a w food or food product is innately safe from foodborne pathogen contamination, or, finally, overt criminal negligence on the part of a manufacturer or supplier involving one or more of the items mentioned above. Examples of low-a w food products that have previously been considered inherently safe from foodborne pathogens are raw flour (responsible for a 2008 outbreak sickening 67 people and hospitalizing 12) and two peanut butter or paste outbreaks in 2007–2009, which sickened over 1,400 people in 48 US states and Canada. It is conceivable that low-a w food products not yet considered at risk for foodborne pathogens may emerge. Salmonella spp., of all common foodborne pathogens, will continue to pose the greatest threat in these foods, due to its uncanny ability to survive desiccation in foods and live for years in the environment of food processing facilities.