Foodborne Listeriosis 601 Table 25–4 Summary of Some Findings on the Thermal Destruction of L monocytogenes Strains Tested/State Scott A, free suspension Scott A, intracellular Scott A, free suspension F5069, intracellular F5069, free suspension Scott A, free suspension Ten strains Chicken/meat isolate Number of Cells (ml) Heating Menstrum ∼ 105 ∼ 105 ∼ 105 ∼ 105 ∼ 105 ∼ 106 ∼ 106 ∼ 105 ∼ 108 ∼ 108 ∼ 107 ∼ 107 ∼ 105 ∼ 105 Sterile skim milk Sterile skim milk Sterile skim milk Whole raw milk Whole raw milk Sterile whole milk Sterile whole milk Ice cream mix pH 7.2, phosphate buffer pH 5.9, meat slurry Liquid whole egg Irradiated ground meats Beef Minced chicken Heating D z Temperature Value Value (◦ C) (sec) (◦ C) Reference 71.7 71.7 71.7 71.7 71.7 71.7 71.7 79.4 70.0 70.0 72.0 62.0 70.0 70.0 1.7 2.0 0.9 1.9 1.6 5.0 3.1 2.6 9.0 13.8 36.0 61.0 6.5 6.5 6.3 6.0 6.1 8.0 7.3 7.0 — — 7.1 4.92 7.2 6.7 109 11 15 15 14 14 8 41 36 88 88 been reported on its thermal destruction in dairy products D values have been determined on many strains of L monocytogenes in whole and skim milk, cream, ice cream, and various meat products As this organism is an intracellular pathogen, several studies were undertaken to determine its relative heat resistance inside and outside phagocytes Overall, standard pasteurization protocols for milk are adequate for destroying L monocytogenes at levels of 105 –106 /ml, whether freely suspended or in intracellular state Some of the specific findings are presented below For a more extensive review, see Doyle et al.29 Dairy Products A summary of thermal D and z values for some L monocytogenes strains is presented in Table 25–4 The D values indicate that the high-temperature, short-time (HTST) protocol for milk (71.7◦ C for 15 seconds) is adequate to reduce normally existing numbers of this organism below detectable levels The vat or low-temperature, long-time (LTLT) pasteurization protocol (62.8◦ C for 30 minutes) is even more destructive (see Chapter 17) Employing the Scott A strain (serovar 4b from the Massachusetts outbreak), D values ranged from 0.9 to 2.0 seconds with z values of 6.0–6.5◦ C The F5069 strain (serovar 4b) appeared to be a bit more heat resistant than Scott A from these results, although Scott A was the most heat resistant of three other strains evaluated, not including F5069.11 The thermal resistance of L monocytogenes is not affected by its intracellular position With Scott A freely suspended in whole raw milk at mean levels of 2.6 × 105 cfu/ml and heating at 71.7◦ C for 15 seconds, no survivors could be found after five heating trials.82 In seven heating trials with Scott A engulfed in vitro by bovine phagocytes, no survivors could be detected with a mean number of × 104 cfu/ml Further, these investigators experimentally infected cows with Scott A and were still unable to find survivors following 11 pasteurization trials at 71.7◦ C for 15 seconds with numbers of Scott A that ranged from 1.4 × 103 to 9.5 × 103 cfu/ml I90I employing five strains of L monocytogenes in 602 Modern Food Microbiology whole milk, skim milk, and 11% nonfat milk solids, Donnelly and Briggs27 found that composition did not affect heat destruction and that at 62.7◦ C, the D values were 60 seconds or less The five strains employed included serotypes 1, 3, and When milk that was naturally contaminated with a serotype strain at around 104 /ml was subjected to an HTST protocol at temperatures ranging from 60◦ to 78◦ C, no viable cells could be detected at processing temperatures of 69◦ C or above.37 In their review of the early studies on the thermal resistance of L monocytogenes in milk, Mackey and Bratchell89 concluded that normal pasteurization procedures will inactivate this organism but that the margin of safety is greater for the vat protocol (LTLT) than the HTST protocol Their mathematical model predicted a 39 D reduction for vat and a 5.2 D reduction for HTST Nondairy Products For liquid whole egg and meat products, D values are generally higher than for milk, a fact not unpredicted, considering the effect of proteins and lipids on the thermal resistance of microorganisms (discussed in Chapter 17) For one strain of L monocytogenes isolated from a chicken product, D values at 70◦ C were 6.6–8.4 seconds; they were essentially the same in beef and two poultry meats.88 In one study, viable cells could be recovered by enrichments from eight of nine samples following heating in ground beef to 70◦ C.8 In a study of blue crabmeat, strain Scott A at levels of about 107 had a D value of 2.61 minutes with a z of 8.4◦ C, indicating that the crabmeat pasteurization protocol of 30 minutes at 85◦ C was adequate to render the product safe from this organism.55 Processing frankfurters to an internal temperature of 160◦ F (71.1◦ C) has been shown to effect at least a 3-log cycle reduction of strain Scott A.133 The cooking of meat products to an internal temperature of 70◦ C for minutes will destroy L monocytogenes.43,83,89 In liquid whole egg (LWE) exposed to 60◦ C for 3.5 minutes, the calculated D value for strain Scott A was 2.1 minutes.5 However, the same strain in LWE + 10% NaCl heated at 63◦ C for 3.5 minutes had a D of 13.7 minutes, whereas LWE + 10% sucrose gave a D of 1.9 minutes under the same conditions The 10% NaCl lowered the aw from 0.98 to 0.915, which could account in part for the higher D value Higher D values were found for seven serovars incubated at 4◦ C for days followed by 37◦ C incubation for days.119 In saline, D60 values were 0.72–3.1 and D62 were 0.30–1.3 minutes In the sausage-type meat employed by Farber,36 the D value at 62◦ C was 61 seconds, but when cure ingredients were added, the D value increased to 7.1 minutes, indicating some heat-protective effects of the cure compounds, which consisted of nitrite, dextrose, lactose, corn syrup, and 3% (w/v) NaCl An approximate doubling in D value in ground beef containing 30% fat, 3.5% NaCl, 200 ppm nitrite, and 300 ppm nitrate was found by Mackey et al.88 who attributed the increased heat resistance to the 3.5% NaCl The destruction of strain Scott A by microwave cooking was investigated by Lund et al.,86 where more than 107 cells/g were placed in chicken stuffing and 106 –107 /g on chicken skin By use of a home-type microwave unit, the adequacy of heating to an internal temperature of 70◦ C for minute was shown to give a 6-log reduction in numbers The thermal destruction of L monocytogenes is similar to that of most other bacteria relative to pH of suspending menstrum where resistance is higher at pH values closer to 7.0 than values in the acid range This was demonstrated in cabbage juice, where D values were higher at a pH of 5.6 than at 4.6.7 In a study of rainbow trout from retail markets in east Tennessee, 51% of the 74 samples were positive for L monocytogenes.31 The log10 means for aerobic plate counts (APC) and coliforms were 6.2 and 3.2, respectively, and the higher percentage of L monocytogenes was associated with samples that had the highest APC and coliform numbers See Chapters 4, and for numbers of listeriae in a variety of foods Foodborne Listeriosis 603 Effect of Sublethal Heating on Thermotolerance It is unclear whether sublethal heating of L monocytogenes cells renders them more resistant to subsequent thermal treatments Some investigators have reported no effect,11,13 and others have reported increased resistance.35,38,79 In one study, the heat shocking of strain Scott A at 48◦ C for 20 minutes resulted in a 2.3-fold increase in D values at 55◦ C.79 In another study employing Scott A in broth and ultrahigh temperature (UHT)-treated milk, an increase in heat resistance was observed following exposure to 48◦ C for 60 minutes and subsequent exposure to 60◦ C.38 Finally, in a study employing 10 strains at a level of about 107 /g in a sausage mix and heat shocking at 48◦ C for 30 or 60 minutes, no significant increase in thermotolerance was observed at 62◦ or 64◦ C, but those shocked for 120 minutes did show an average 2.4-fold increase in D values at 64◦ C.35 In this study, the thermotolerance was maintained for at least 24 hours when the cells were stored at 4◦ C If sublethal heating does lead to greater thermoresistance, it would not pose a problem for milk that contains fewer than 10 cells/ml assuming that a twofold to threefold increase in D value occurs VIRULENCE PROPERTIES Of listerial species, L monocytogenes is the pathogen of concern for humans Although L ivanovii can multiply in the mouse model, it does so to a much less degree than L monocytogenes, and up to 106 cells caused no infection in the mouse.59 L innocua, L welshimeri, and L seeligeri are nonpathogens, although the last produces a hemolysin The most significant virulence factor associated with L monocytogenes is listeriolysin O (LLO) Listeriolysin O and Ivanolysin O In general, the pathogenic/virulent strains of L monocytogenes produce beta-hemolysis on blood agar and acid from rhamnose but not from xylose Strains whose hemolysis can be enhanced with either the prepurified exo-substance or by direct use of the culture are potentially pathogenic.117 Regarding hemolysis, the evidence is overwhelming that all virulent strains of this species produce a specific substance that is responsible for beta-hemolysis on erythrocytes and the destruction of phagocytic cells that engulf them The substance and perfringolysin O (PFO) have been shown to be highly homologous to streptolysin O (SLO) and pneumolysin O (PLO) It has been purified and shown to have a molecular weight of 60,000 Da and to consist of 504 amino acids.44,95 It is produced mainly during the exponential growth phase, with maximum levels after 8–10 hours of growth.43 Less LLO is synthesized at 26◦ C than at 37◦ C with high glucose, and synthesis was found to be best with 0.2% glucose at 37◦ C.24 Sorbate at a level of 2% inhibited LLO synthesis at 35◦ C under aerobic or anaerobic conditions.72 LLO has been detected in all strains of L monocytogenes, including some that were nonhemolytic, but not in L welshimeri or L grayi The gene that encodes its production is chromosomal and it is designated hly Its role in virulence is discussed below L ivanovii and L seeligeri produce thiol-dependent exotoxins that are similar but not identical to LLO Large quantities are produced by L ivanovii but only small quantities by L seeligeri.43 The L ivanovii thiol-activated cylolysin is ivanolysin O (ILO) Antiserum raised to the L ivanovii product cross-reacts with that from L monocytogenes and SLO.73 ILO-deficient mutants have been shown to be avirulent in mice and chick embryos.1 604 Modern Food Microbiology Purified LLO has been shown to share in common with SLO and PLO the following properties: activated by SH-compounds such as cysteine, inhibited by low quantities of cholesterol, and common antigenic sites as evidenced by immunological cross-reactivity Unlike SLO, LLO is active at a pH of 5.5 but not at pH 7.0, suggesting the possibility of its activity in macrophage phagosomes (phagolysosomes) Its LD50 for mice is about 0.8 µg, and it induces an inflammatory response when injected intradermally.44 It appears that LLO and the other poreforming toxins evolved from a single progenitor gene Intracellular Invasion When L monocytogenes is contracted via the oral route, it apparently colonizes the intestinal tract by mechanisms that are poorly understood From the intestinal tract, the organism invades tissues, including the placenta in pregnant women, and enters the blood stream, from which it reaches other susceptible body cells As an intracellular pathogen, it must first enter susceptible cells, and then it must possess means of replicating within these cells In the case of phagocytes, entry occurs in two steps: directly into phagosomes and from the phagosomes into the phagocyte’s cytoplasm Entry or uptake into nonphagocytic cells is different In nonphagocytic cell lines, uptake requires surface-bound proteins of the bacterium designated In1A and In1B.78 The former has a molecular weight of 88 kDa and the latter 65 kDa They are involved in aiding the entry of L monocytogenes cells into host cells The In1A protein, i.e., internalin, and its mammalian surface receptor is E-cadherin It is required for entry into cultured epithelial cells, whereas In1B is required for invasion of cultured mouse hepatocytes.32 Another invasion-associated protein of Listeria is p60, a 60-kDa protein encoded by the iap gene It is secreted by all species of Listeria Another surface protein, ActA (90 kDa), is required for actin polymerization and it allows for intracytoplasmic movement of cells.61 Ami (ca 90 kDa) is located on the surface of L monocytogenes and it is a bacteriolysin From a study of 150 food isolates, Ami was present in 149 while 283 of 300 human isolates contained this protein.61 All of the positive strains contained LLO, InlB, and ActA L monocytogenes survives inside macrophages by escaping from phagolysosomal membranes into the cytoplasm (cytosol), and this process is facilitated in part by LLO Once inside the cytosol, the surface protein ActA (encoded by actA) aids in the formation of actin tails that propel the organism toward the cytoplasmic membrane At the membrane, double membrane vacuoles form With LLO and the two bacterial phospholipases, the phosphatidylinositol-specific phospholipase C (encoded by plcA) and the broad-range phospholipase C (encoded by plcB), the bacteria are freed and the process is repeated upon entry of bacteria into adjacent host cells The latter occurs following the pushing out of the membrane to form a filopodium (a projection), which is absorbed by an adjacent cell and the invasion process is repeated Thus, the spread of L monocytogenes from cell to cell occurs without the bacterium having to leave the inner parts of host cells For more information, see reference 97 and Chapter 22 Monocytosis-Producing Activity An interesting yet incompletely understood part of the L monocytogenes cell is a lipid-containing component of the cell envelope that shares at least one property with the lipopolysaccharide (LPS) that is typical of Gram-negative bacteria In Gram-negative bacteria, LPS is located in the outer membrane, but listeriae and other Gram-positive bacteria not possess outer membranes The L monocytogenes substance is lipoteichoic acid (LTA) It was shown several decades ago that phenol–water extracts of L monocytogenes cells induce the production of monocytes, and it was because of this monocytosisproducing activity (MPA) factor that the organism was given the species name monocytogenes This Foodborne Listeriosis 605 LTA fraction accounts for about 6% of the dry weight of cells and is associated with the plasma membrane It has a molecular weight of about 1,000 Da, contains no amino acids or carbohydrates, and stimulates only mononuclear cells.42 It possesses low tissue toxicity and is serologically inactive121 but it kills macrophages in vitro.42 It has been shown to share the following properties with LPS: it is pyrogenic and lethal in rabbits, produces a localized Schwartzman reaction, contains acylated hydroxy fatty acids, produces a positive reaction with the Limulus amoebocyte lysate (LAL) reagent, contains 2-keto3-deoxyoctonic acid (KDO), and contains heptose Regarding its LAL reactivity, µg/ml was required to produce a positive reaction115 whereas the same can be achieved with picogram quantities of LPS Sphingomyelinase L ivanovii is known to be infectious for sheep, in which it causes abortions, and to be a prolific producer of hemolysin on sheep erythrocytes It has been shown to possess an LLO-like hemolysin (ILO), sphingomyelinase, and lecithinase.73 Sphingomyelinase has a molecular weight of 27,000 Da.127 Whereas the LLO-like agent is responsible for the inner complete zone of hemolysis on sheep erythrocytes, the halo of incomplete hemolysis that is enhanced by Rhodococcus equi appears to be caused by the two enzymes noted In one study, a mutant defective in sphingomyelinase and another protein exhibited lower virulence than wild-type strains.1 ANIMAL MODELS AND INFECTIOUS DOSE The first animal model employed to test the virulence of L monocytogenes was the administration of a suspension of cells into the eye of a rabbit or guinea pig (Anton’s test), where 106 cells produced conjunctivitis.2 Chicken embryos have been studied by a large number of investigators Inocula of 100 cells of L monocytogenes into the allantoic sac of 10-day-old embryos led to death within 2–5 days, and the LD50 was less than × 102 cells for virulent strains L ivanovii is also lethal by this method Injections of 100–30,000 cells/egg into the chorioallantoic membrane of 10-day-old chick embryos resulted in death within 72 hours compared to about days for mice.123 Although Anton’s test and chick embryos may be used to assess the relative virulence of strains of listeriae, the mouse is the model of choice for the additional information that it gives relative to cellular immunity Not only the mouse is the most widely used laboratory animal for virulence studies of listeriae, but also it is widely used in studies of T cell immunity in general This model is employed by use of normal, baby, juvenile, or adult mice, as well as a variety of specially bred strains such as athymic (T cell-deficient) nude mice Listerial cells have been administered intraperitoneally (IP), intravenously (IV), and intragastrically (IG) When normal adult mice are used, all smooth and hemolytic strains of L monocytogenes at levels of 103 –104 /mouse multiply in the spleen.59 With many strains, inocula of 105 –106 are lethal to normal adult mice, although numbers as high as × 109 have been found necessary to produce an LD50 A low of 50 cells for 15-g mice has been reported (see below) Whereas the IP route of injection is often used for mice, IG administration is employed to assess gastrointestinal behavior of listeriae The administration of L monocytogenes to 15-g mice by the IG route produced more rapid infection and more deaths in the first days of the 6-day test than by IP.105 By this method, the approximate 50% lethal dose (ALD50 ) ranged from 50 to 4.4 × 105 cells for 15 food and clinical isolates of L monocytogenes.105 Six- to eight-week-old mice were given IP and oral challenges of a serovar 4b strain by one group to study their effect under normal and compromised states Cells were suspended in 11% nonfat milk solids and administered to four groups of mice: normal, hydrocortisone treated, pregnant, and cimetidine treated Minimum numbers of cells ... embryos.1 604 Modern Food Microbiology Puri? ?ed LLO has been shown to share in common with SLO and PLO the following properties: activated by SH-compounds such as cysteine, inhibited by low quantities... is E-cadherin It is required for entry into cultured epithelial cells, whereas In1B is required for invasion of cultured mouse hepatocytes.32 Another invasion-associated protein of Listeria is... normal and compromised states Cells were suspended in 11% nonfat milk solids and administered to four groups of mice: normal, hydrocortisone treated, pregnant, and cimetidine treated Minimum numbers