Survival and Outgrowth of Clostridium botulinum Type E Spores in Smoked Fish

Chub injected in the loin muscle with 10(6)Clostridium botulinum type E spores were smoked to an internal temperature of 180 F (82.2 C) for 30 min, sealed in plastic bags, and incubated at room temperature (20 to 25 C) for 7 days. Viable type E spores were found in practically all such fish. Toxin formation by the survivors in the smoked fish was dependent on the brine concentration of the smoked fish. A brine concentration of 3% or higher, as measured in the loin muscle, inhibited toxin formation. Six different type E strains gave similar results. Only a few hundred of the million spores in the inoculum survived the smoking. Moisture in the atmosphere during smoking did not reduce the incidence of fish with type E survivors.     

Effect of Sodium Nitrite, Sodium Chloride, and Sodium Nitrate on Germination and Outgrowth of Anaerobic Spores

The effects of meat-curing agents on germination and outgrowth of putrefactive anaerobe 3679h (PA 3679h) spores were studied in microcultures. Nitrite concentrations up to 0.06% at pH 6.0 or between 0.8 and 1% at pH 7.0 allowed emergence and elongation of vegetative cells but blocked cell division. The newly emerged cells then lysed. With more than 0.06% nitrite at pH 6.0 or more than 0.8 to 1% at pH 7.0, the spores lost refractility and swelled, but vegetative cells did not emerge. Even as much as 4% nitrite failed to prevent germination (complete loss of refractility) and swelling of the spores. Sodium chloride concentrations above 6% prevented complete germination (i.e., the spores retained a refractile core). In the presence of 3 to 6% sodium chloride, most of the spores germinated and produced vegetative cells, but cell division was often blocked. Sodium nitrate had no apparent effect on germination and outgrowth at concentrations up to 2%.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

The Resistance of Spores of Clostridium botulinum Type E to Heat and Radiation

Spores of Clostridium botulinum type E have been screened for resistance to heating in aqueous suspension at 60, 70 and 80°. D ¹⁷⁶ ranged from <0·33 min to 1·25 min. z values of eight strains were between 9·0 and 10·7, and for one strain was 7·4.
Radiation survivor curves have been plotted for nine strains, and a further nine strains screened. The shoulder extends to about 0·4 megarad, whereafter the curve becomes exponential within a D value from 0·065 to 0·16 megarad.     

Clostridium botulinum Type E Toxin: Effect of pH and Method of Purification on Molecular Weight

The toxin of Clostridium botulinum type E was isolated from intact cells and from toxic culture filtrates by column chromatography at three pH values, 4.5, 5.3, and 6.0. At pH 6.0 and 5.3, the isolated toxin was in a form with a molecular weight (MW) of 86,000. This toxin was homogeneous on polyacrylamide gel electrophoresis and gel filtration and had an optical density ratio, 280 nm/260 nm, greater than 2.0. It did not dissociate at higher pH levels, but was dissociated into nontoxic components of approximately 12,000 MW when reduced and alkylated in the presence of 6 M guanidine hydrochloride. At pH 4.5, smaller amounts of an impure toxic moiety with a MW of 12,000 were found. After storage for 6 months, the 86,000-MW moiety had lost 60% of its lethality. Gel filtration revealed that the bulk of the toxicity was associated with a component having a MW of 150,000. Toxic components with MW of 12,000 and over 200,000 were also found. The toxin appears to polymerize or aggregate when in a pure form, so that most, if not all, of the MW previously reported for the toxin may belong to different polymers of a monomer with a MW of 12,000 or less. Treatment of the 86,000-MW toxin with trypsin resulted in an 18- to 128-fold increase in lethality, but no detectable change in MW.     

Heterogeneity of Times Required for Germination and Outgrowth from Single Spores of Nonproteolytic Clostridium botulinum

Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (t_germ), time to emergence (t_emerg), and times to reach the lengths of one (t_C1) and two (t_C2) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (t_germ), emergence (t_emerg − t_germ), cell maturation (t_C1 − t_emerg), and doubling (t_C2 − t_C1) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.     

Serological Studies of Clostridium botulinum Type E and Related Organisms II. Serology of Spores

Pure spore antigens for the immunization of rabbits were prepared by enzymic digestion of vegetative components and separation of the cleaned spores in polyethylene glycol. Spore antisera were prepared to strains representative of toxigenic Clostridium botulinum type E; nontoxigenic boticin E-producing variants; nontoxigenic nonproducers of boticin E; nontoxigenic "atypical" strains, which differ somewhat from C. botulinum type E in their physiology; C. botulinum types A and B; and C. bifermentans. They were tested against these and additional strains representative of the above groups, other types of C. botulinum, and other Clostridium species. There was no evidence of agglutination of flagellar or somatic antigens of vegetative cells by these antisera. Agglutination and agglutinin absorption tests showed common antigens among toxigenic type E strains and nontoxigenic variants, both producers and nonproducers of boticin E. Some nontoxigenic "atypical" strains varied in their ability to be agglutinated by type E antisera, and others did not agglutinate at all. Of those atypical strains that were not agglutinated, one was agglutinated by C. bifermentans antiserum. Antisera prepared against C. botulinum types A and B and C. bifermentans did not agglutinate the spores of type E or its variants nor share antigens common to each other. Similarly, antisera to type E, its nontoxigenic variants, and nontoxigenic atypical strains did not agglutinate other C. botulinum types or any other Clostridium species investigated.     

Interrelationship of Heat and Relative Humidity in the Destruction of Clostridium botulinum Type E Spores on Whitefish Chubs

Heat destruction of types B and E Clostridium botulinum spores on whitefish chubs was observed to be dependent upon the relative humidity (RH) in the chamber in which fish were heated. Experimental conditions were designed to simulate those attainable in commercial fish-smoking plants. Low numbers of type E spores were destroyed with regularity, within 30 min, on fish which were held at an internal temperature of 77 C (170.6 F) in an atmosphere of at least 70% RH. However, an internal temperature of 82 C (179.6 F) and a minimum RH of 70% were required to destroy several hundred thousand type E spores. Quantitative estimates of spore destruction were arrived at with a modified most probable number procedure. Type E spore populations were reduced by 2 to 4 logarithms at 77 C (170.6 F), by 5 to 6 logarithms at 82 C (179.6 F), and by more than 6 logarithms at 88 C (190.4 F) when fish were heated in an atmosphere of 70% RH. A 5 to 6 logarithm reduction of spores was also observed when fish inoculated with type B spores were processed at 82 C (179.6 F) in an atmosphere of 70% RH.     

Effect of Lysozyme on the Recovery of Heated Clostridium botulinum Spores

Lysozyme in the recovery medium increased the recovery of heated spores, thereby raising the measured heat resistance of type E Clostridium botulinum spores about 1,800-fold and type A spores up to 3-fold.     

Effect of Nutrients on Physiological Properties of Clostridium botulinum Type E

Eight strains of Clostridium botulinum type E out of twelve tested showed good growth and normal cell morphology in a synthetic medium containing choline. Growth and toxin production by a representative strain was not influenced by repeated subculturing. In the chemically defined medium, acetylcholine, N,N-dimethylethanolamine, and lecithin could replace choline to get normal cell division and cell morphology of C. botulinum type E. Choline could not be replaced by ethanolamine, N-methylethanolamine, or betaine. A toxigenic strain of C. botulinum type E showed proteolytic, lipolytic, and lecithinase activity in complex media but not in a synthetic medium. On prolonged incubation in the high temperature range of growth, the toxicity of the culture filtrate decreased in a complex, but not in a synthetic medium. The implications of these findings are discussed.     

Outgrowth and sporulation studies on Clostridium botulinum type E: influence of isoleucine.

A defined medium (CDM) is described which supported growth and sporulation of type E strains of Clostridium botulinum, but not sporulation of other serotypes of C. botulinum or C. sporogenes. As compared to growth in complex medium, spore outgrowth was delayed and both the growth rate and the cell yield was reduced. However, efficiency of sporulation of the type E MSpt strain in a chemically defined medium (CDM) was the same as that in complex medium and, in fact, sporulation was nearly synchronous and completed within 3 h of the first appearance of phase-bright endospores, compared with completion in 9 h in TPGY. Growth studies with CDM, from which single amino acids were omitted, showed that isoleucine was essential for outgrowth of heat-activated spores of the MSp+ strain, whereas valine was required for that of the Ts-25 mutant. Radioactive isoleucine was incorporated by germinating MSp+ spores at an earlier stage and at a more rapid rate than labelled methionine or mixed amino acids. Uptake studies showed that isoleucine accumulated in a prominent acid-soluble pool during outgrowth, a period when its incorporation into protein was not evident. The results suggest that the isoleucine may be required for a purpose other than protein synthesis during outgrowth.     

Antagonistic Effect on Clostridium botulinum Type E by Organisms Resembling It

A bacteriocin-like substance, active against strains of Clostridium botulinum type E, is produced by certain nontoxic organisms whose biochemical properties and morphological characteristics are similar to type E. The substance, for which the name "boticin E" is proposed, is bacteriolytic for vegetative cells and bacteriostatic for spores of type E. Its spectrum of activity is somewhat strain-specific. Of the clostridial species tested, only C. botulinum type E and, to a lesser extent, C. perfringens and C. acetobutylicum, but not C. botulinum types A, B, or F, are sensitive. Irreversibly resistant variants originating from both vegetative cells and spores of certain strains were obtained. The active substance is heat-stable and dialyzable, and is not inactivated by chloroform but is digested by trypsin. Ethyl alcohol and acetone precipitates are fully active, whereas trichloroacetic acid precipitates are only partially active. Other nontoxic organisms producing similar antagonistic substances are discussed.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic Clostridium botulinum Bacteria

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (t_germ), one mature cell (t_C1), and two mature cells (t_C2) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the t_germ, the outgrowth time (t_C1 − t_germ), and the first doubling time (t_C2 − t_C1). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (t_C1) and outgrowth (t_C1 − t_germ) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.Clostridium botulinum is a group of four physiologically and phylogenetically distinct anaerobic spore-forming bacteria (known as groups I, II, III, and IV) that produce the highly toxic botulinum neurotoxin (12). The severity of the intoxication, botulism, ensures considerable effort is directed at preventing the growth of this pathogen in food. Nonproteolytic (group II) C. botulinum is one of the two groups most frequently associated with food-borne botulism. It forms heat-resistant spores and can germinate, grow, and produce toxin at 3°C (8); thus, nonproteolytic C. botulinum is a particular concern in mild heat-treated chilled foods (16, 17).Spores formed by pathogens such as C. botulinum are a significant food safety issue since they are able to resist many of the processes, such as cooking, used to kill vegetative cells. Understanding the transformation from a dormant spore to active vegetative cells is an important part of quantifying the risk associated with such organisms. Considerable effort has been targeted at measuring and relating the kinetic responses of populations of C. botulinum to environmental conditions and such data have been used to create predictive models, for example, ComBase (www.combase.cc). Such approaches have made a considerable contribution to ensuring food safety but problems with using population based predictions may arise when an initial inoculum is very small or additional information beyond point values is required. Spores typically contaminate foods at low concentrations so that growth of C. botulinum, when it occurs, is likely to initiate from just a few spores. In these circumstances the distribution of times to growth in packs will reflect the heterogeneity of times to growth from the contaminating individual spores. There is an intrinsic variability between individual spores within a population, and the relationship between population lag and individual lag is complex. Consequently, individual lag times cannot be predicted from population measurements (3). Knowledge of the underlying distribution would allow greater refinement of risk assessments.The lag period between a spore being exposed to conditions suitable for growth and the start of exponential growth will reflect the combined times of germination, emergence, elongation, and first cell division. Currently, very little is known about the variability and duration of these stages and any relationships between them. Measuring the kinetics of spore germination is usually achieved by measuring a population to identify time to percent completion. Such germination curves represent the summation of responses by individual spores. Some authors have measured the biovariability associated with individual spores, but most studies have examined only germination (4-7, 11, 22) and not subsequent outgrowth. More recently, we have used phase-contrast microscopy and image analysis to follow individual spores of nonproteolytic C. botulinum from dormancy, through germination and emergence, to cell division (21, 23). These experiments showed there is very little, or no, relationship between the time spent in each stage by individual spores. We have now extended this work to determine distributions of times for different stages in lag phase as affected by heat treatment and incubation temperature.     

Minimal Growth Temperature, Sodium Chloride Tolerance, pH Sensitivity, and Toxin Production of Marine and Terrestrial Strains of Clostridium botulinum Type C

Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 × 10⁶ viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 × 10⁶ unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin.     

Effect of Plating Medium on Heat Activation Requirement of Clostridium botulinum Spores

Clostridium botulinum 62A and ATCC 25763 spores required heat activation for maximum colony formation when plated on reinforced clostridial agar (BBL Microbiology Systems) but not when plated on botulinum assay medium. Spores from strains B-aphis and 53B did not exhibit heat activation when plated on either medium.