Pathogenic Psychrotolerant Sporeformers: An Emerging Challenge for Low-Temperature Storage of Minimally Processed Foods

Abstract

Sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. Beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. In these products, the sporeforming pathogens of concern are Bacillus cereus, Bacillus weihenstephanensis, and Clostridium botulinum type E. This review article examines the foods, conditions, and organisms responsible for the food safety issue caused by the germination and outgrowth of psychrotolerant sporeforming pathogens in minimally processed refrigerated foods.

Introduction

Bacterial spores

N o other life form is as difficult to eliminate as a bacterial endospore. Spores have evolved into resilient survival packages withstanding just about any stress agent or conditions imaginable over extremely long periods of time. Normally the success of any sterilization procedure is judged by its ability to inactivate the last remaining spore. In foods, ingestion of spores themselves is generally of no concern unless the spores are capable of germinating in the digestive tract. Otherwise, intact resting spores harbor no measurable pathogenic activity. This makes the understanding of conditions for spore germination and outgrowth in foods during storage and consumption of fundamental importance. This is of particular importance due to increased demand for minimally processed foods and refrigerated processed foods of extended durability, where psychrotolerant sporeformers can be a problem. These types of foods include those that are able to be refrigerated for extended periods of time without spoilage and are not heated prior to consumption. If these foods are contaminated with psychrotolerant sporeformers, these spores may have the ability to germinate and even grow at refrigeration temperatures.

Spores of Bacillus and Clostridium species are of considerable concern in the food industry, due to their common occurrence, robustness, and in some cases the toxins that can be synthesized upon germination and outgrowth (Setlow, 2003; Coleman et al., 2007; De Vries et al., 2004). Spore formation is triggered by nutrient depletion, causing a vegetative cell to be transformed into a dormant spore; however, the spore is still responsive to specific agents in the environment. When the environment becomes favorable again with available nutrients, spores can revert back to their active state.

The process of germination is triggered by germinant compounds that bind to receptors in the inner membrane of the spore (Fig. 1). These nutrient germinants include certain amino acids, sugars, and purine nucleosides (Setlow, 2003). There are also several non-nutrient germinants, including dodecyclamine (DPA), sublethal heat treatment, high pressure, specific peptidoglycan fragments, and bryostatin, an activator of serine/threonine protein kinases (Wei et al., 2010).

FIG. 1.

Events in germination triggered by nutrients. DPA, dodecyclamine. (Adapted from Black et al., 2007).

Spores can survive for long periods of time in food products, particularly in foods where nutrient content is low or nonexistent (Coleman et al., 2007). When these spores germinate, foodborne illness can occur (Setlow, 2003). It would be ideal to be able to trigger the germination of all spores present in the food product prior or during any preservation treatment in order to eliminate them since spores are much more susceptible to inactivation after they have germinated (Ghosh and Setlow, 2009b). Although this strategy seems simple, germination rates vary, and a small percentage of spores germinate extremely slowly or not at all after exposure to germinants (Ghosh and Setlow, 2009a; Wei et al., 2010). Such spores are known as superdormant spores. Tyndallization, whereby low-acid foods are heated and cooled several times over a period of days to germinate and inactivate spores, is based on this concept and is no longer used due to risks presented with superdormant spores.

Superdormant spores

Superdormant spores are spores that either fail to germinate or germinate very slowly (Ghosh and Setlow, 2009a). Superdormancy has been recognized as a characteristic in Bacillus species, and has also been found to occur in Clostridium difficile spores where it is more likely to occur as spores age (Rodriguez-Palacios and Lejeune, 2011). The study of superdormant spores has been facilitated by a simple isolation method developed by Ghosh and Setlow (2009a) for B. subtilis, B. megaterium, and B. cereus. This method, called buoyant density centrifugation, separates dormant spores from germinating spores and debris by density adjustment of the centrifuging fluid with multiple cycles of heat shock and germination.

It appears that the physiological state for superdormancy is similar for all Bacillus species (Ghosh and Setlow, 2009a) and more than likely similar with regard to Clostridium species. Recent studies provide evidence that superdormancy is a result of a reduced level of germinant receptors in the inner membrane (Ghosh and Setlow, 2009b; Wei et al., 2010; Zhang et al., 2010).

Sublethal heat treatment prior to germination reduces the yield of superdormant spores; however, superdormant spores still show a higher temperature optimum for heat activation than the remainder of the spore population (Ghosh and Setlow, 2009b). It also appears that superdormant spores have greater wet-heat resistances and lower core water contents (Ghosh et al., 2009). Superdormant spores germinate poorly in the presence of nutrient germinants as compared to other germinants such as dodecylamine or calcium dipicolinic acid. This is not surprising, since germination by dodecylamine or calcium dipicolinic acid does not require nutrient binding by receptors, nor does it require prior heat activation (Ghosh and Setlow, 2009b; Wei et al., 2010).

A number of factors increase the rate of spore germination (Ghosh and Setlow, 2009b; Zhang et al., 2010). These include heat activation and an increased level of germinant receptors. Different germinant receptors within an individual spore are suspected to interact through aggregation. This could potentially amplify signals from large numbers of germinant receptors. The lack of these nutrient receptors may inhibit the amplification of this germination signal and may explain why higher yields of superdormant spores are observed with Bacillus strains that lack one or more germinant receptors (Ghosh and Setlow, 2009b). Heterogeneity in a spore population, resulting in varying rates of germination among individual spores, may also be due to adaptation of a particular bacterial species. Spores that germinate more slowly or at a reduced rate than the majority of the population are more likely to survive environmental changes where the majority of germinating spores are inactivated, thus increasing the likelihood of survival for the entire population (Ghosh and Setlow, 2009b).

Currently it is difficult to assess the food safety importance of superdormant spores in B. cereus, B. weihenstephanensis, and C. botulinum type E; however, one can assume that temperatures at or below 3°C used in chilled foods substantially reduce the frequency of spore germination. The phenomenon of superdormancy can reduce the rate even more. Therefore, a greater percentage of superdormancy in spore populations would help reduce the risk of foodborne disease from these bacteria in chilled foods.

Pathogenic Psychrotolerant Sporeformers

Bacillus cereus

B. cereus is a motile Gram-positive sporeforming bacterium that is a well-established foodborne pathogen (Chorin et al., 1997; Kotiranta et al., 2000; De Vries et al., 2004). It is found throughout nature but is most commonly isolated from soil and plants (Valero et al., 2003; Priest et al., 2004). Foodborne illnesses caused by B. cereus are directly related to the production of two toxin types: An emetic-type enterotoxin, and a group of several diarrheogenic-type enterotoxins (Chorin et al., 1997; De Vries et al., 2004). The emetic-type toxin, also known as cereulide, is a thermostable, cyclic peptide. The emetic-type toxin is not able to survive the acidic conditions of the host gastrointestinal environment and therefore is unable to contribute to diarrheal illness (Ceuppens et al., 2012). The enterotoxins responsible for the diarrheogenic symptoms are hemolysin, nonhemolytic enterotoxin, and cytotoxin (Ehling-Schulz et al., 2005; Lucking et al., 2009). The cell wall of vegetative B. cereus is also covered by proteins (called the S-layer) that play a role in cell adhesion and attribute to virulence of the organism (Kotiranta et al., 2000). Since the emetic toxin of B. cereus is heat stable, it can remain stable after cooking or heating. In a study reported by Rajkovic et al. (2008), cereulide toxin was demonstrated to remain stable at temperatures as high as 150°C at a pH as high as 10.6. The authors of this study also reported that highly alkaline conditions are needed to achieve cereulide inactivation, and when alkaline buffer was removed, toxin activity could be recovered. Cereulide production does not occur until a stationary-growth phase is achieved; therefore, relatively high counts of vegetative cells or spores able to germinate in foods are usually required for cereulide intoxication to occur (Thorsen et al., 2009).

Cereulide toxin is absorbed from the gut into the bloodstream and induces emetic-like symptoms including nausea and vomiting through stimulation of the vagus nerve (Jääskeläinen et al., 2003). Ingestion of approximately ≤8 μg/kg body weight of cereulide toxin within a food product is required to cause illness in humans (Jääskeläinen et al., 2003). Cereulide toxin affects the mitochondria by acting as a potassium ion channel–former (Mikkola, 1999) and causes apoptosis of human natural killer cells (Paananen et al., 2002). Mesophilic strains of B. cereus can only produce cereulide at temperatures above 10–15°C (Thorsen et al., 2009). This may explain why the toxin is mostly associated with foods that are improperly cooled and stored, such as rice and pasta dishes.

The diarrheogenic toxins are heat labile and can be destroyed by heat. These toxins are produced during the exponential-growth phase (Fermanian et al., 1994). Since spores of B. cereus are capable of surviving heat treatment and the acidic environment of the stomach, diarrheal-like symptoms can occur when spores of B. cereus are consumed in raw or minimally processed foods. Upon entering the small intestine, spores can germinate, outgrow, and multiply, enabling the production of the diarrheogenic toxin. According to an in vitro gastrointestinal transit study by Ceuppens et al. (2012), no vegetative cells were able to survive passage through the gastrointestinal system, but spores were able to very successfully survive the passage, indicating the importance of the physiological state of B. cereus cells in foods in order to cause foodborne illness. Depending on the amount of bacteria present in the food product, sometimes both sets of symptoms (emesis and diarrhea) can develop, causing what is known as “two-bucket disease.” Symptoms from either B. cereus toxin should resolve within 24–48 h of onset; however, in extreme cases and in immunocompromised individuals, emetic intoxication can lead to liver failure and ultimately death.

The foods most frequently recognized with B. cereus intoxication are milk, vegetables, rice, potatoes, grains, cereals (including batters, mixes, and breadings), spices, and various sauces (Doona and Feeherry, 2007). B. cereus is not nutritionally fastidious, which is why the bacterium can replicate in soil and the low-nutrient foods including rice and pasta (Kotiranta et al., 2000). The reservoir for B. cereus is the soil, where transmission of the organism can occur through various vectors (Abee et al., 2011).

It is estimated that there are approximately 63,000 illnesses and 20 hospitalizations caused by B. cereus every year in the United States (Scallan et al., 2011). Foodborne illness caused by B. cereus is often underreported; however, recalls, illnesses, and even deaths caused by the organism have been documented within the past few years. According to The Sydney Morning Herald, an 81-year-old man died on January 12, 2007 after eating contaminated asparagus sauce prepared at a local restaurant. The article reported that the sauce was left at room temperature for more than 4 h after being refrigerated, and was found to contain 9.8 log₁₀ colony-forming units/g of B. cereus. Naranjo et al. (2011) described the presumed cause of death of a healthy 20-year-old male in Brussels, Belgium following the ingestion of pasta contaminated with emetic strains of B. cereus. High levels of cereulide (14.8 μg/g) were found in the leftover spaghetti. B. cereus counts of 9.5×10⁷ colony-forming units/g were found in the pasta but not in the tomato sauce. The authors emphasized the need for adequate refrigeration of prepared foods because the emetic toxin is preformed in the food and not inactivated by heat treatment. Toxin production is closely linked to temperature (Finlay et al., 2000) and not strictly correlated with bacterial counts.

The Bacillus cereus group comprises seven species: B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, B. weihenstephanensis, and B. cytotoxicus. The members of the B. cereus group are nearly impossible to distinguish phenotypically from each other (Kotiranta et al., 2000; Guinebretière et al., 2008). It has even been suggested through genetic evidence that members of the B. cereus group may actually represent one single species (Priest et al., 2004; Ehling-Schulz et al., 2005; Auger et al., 2009). Because of the inability to separate these species genetically, they have been distributed between seven distinct phylogenic groups (Guinebretière et al., 2008; 2010). It has been found that there is a clear correlation between these phylogenic groups and adaptation to temperature, pH, and water activity, which can be used to predict the risk of a particular species to cause foodborne illness (Carlin et al., 2013). Several epidemiological studies have been performed comparing the genetic sequences of a variety environmental and food B. cereus isolates. The authors of these studies reported that psychrotolerant B. cereus isolates were more genetically similar to other psychrotolerant species, including B. mycoides, than to mesophilic B. cereus species, which were identified to be more genetically similar to B. thuringiensis (Schraft et al., 1996; Daffonchio et al., 2000; Sorokin et al., 2006; Guinebretière et al., 2008). Therefore, it was suggested that the taxonomy of the B. cereus group be revised. In 1998, a new species named Bacillus weihenstephanensis was proposed to accommodate the psychrotolerant strains of B. cereus (Lechner et al., 1998).

Bacillus weihenstephanensis

B. weihenstephanensis is differentiated from B. cereus by its ability to grow aerobically at 7°C in liquid culture and the absence of ability to grow at 43°C (Lechner et al., 1998). Genetically, B. weihenstephensis can be differentiated by the presence of the 16S rDNA signature sequence ¹⁰⁰³TCTAGAGATAGA and the signature sequence of the major cold-shock gene cspA, ⁴ACAGTT (Lechner et al., 1998). Other research shows that not all strains of psychrotolerant B. cereus can be classified as B. weihenstephanensis and that there is an intermediate form between the two species (Stenfors et al., 2001). These intermediate forms produce both mesophilic and psychrotolerant genetic (polymerase chain reaction) products; for example, B. cereus strains identified as mesophilic by polymerase chain reaction demonstrated the ability to grow at 6°C, and other strains containing the cspA signature sequence were able to grow at 43°C (Stenfors et al., 2001). According to Guinebretière et al. (2008), B. weihenstephanensis belongs to B. cereus phylogenic group VI, which was shown to have a minimal growth temperature of 5°C. Specific guidelines need to be established for researchers to be able to distinguish mesophilic and psychrotolerant species of B. cereus and B. weihenstephanensis.

According to Guinebretière et al. (2010), only those species included in B. cereus phylogenic groups III, IV, and VII have been implicated as the cause of foodborne illness. These species include B. cereus, B. thuringiensis, and B. anthracis, which have a minimum growth temperature of ≥10°C. To assess whether B. weihenstephanensis may serve as a hazard in refrigerated food products, it must first be determined whether this species can produce toxin, and if so, at what temperatures cereulide toxin can be produced. This is especially important since this toxin is heat stable and cannot be destroyed during cooking and food processing. In a study where 93 strains of B. weihenstephanensis were screened for the presence of genes responsible for toxin production including cesB cytK-1 and cytK-2, none of these strains were found to contain any of these genes (Guinebretière et al., 2010). B. weihenstephanensis strain isolated from whole liquid egg product was able to produce toxin in the food at 6, 8, and 10°C, but not at 4°C; however, the isolate did not contain the cesB gene, which encodes for cereulide production (Baron et al., 2007). Environmental isolates of B. weihenstephanensis were able to produce the emetic toxin at temperatures as low as 8°C in food; however, toxin was not produced at levels great enough to cause illness (Thorsen et al., 2006). Strains of B. weihenstephanensis have been found to contain the gene responsible for cereulide production (cesB) (Thorsen et al., 2006; 2009), although B. weihenstephanensis has not been demonstrated to be able to produce cereulide at recommended refrigeration temperatures of 4°C or below. Temperature abuse can often occur during food shipping, distribution, and storage. Because B. weihenstephanensis has demonstrated the ability to grow at 6°C and produce toxin at 8°C, this bacteria should be identified as a potential hazard for refrigerated foods that are commonly subject to temperature abuse.

Because of their potential to grow in refrigerated food products, their ability to produce toxins, and their implications in foodborne outbreaks, psychrotolerant species of B. cereus, including B. weihenstephanensis, have been of concern in the food industry (Baron et al., 2007). B. weihenstephanensis is a known causative agent of spoilage in white liquid egg products but can also cause spoilage in pasteurized milk. The B. weihenstephanensis strain isolated from the spoiled whole liquid egg product also demonstrated the ability to adhere to surfaces and form biofilms. These films can form on processing equipment commonly used in egg-breaking facilities including stainless steel, model hydrophilic materials (glass), and model hydrophobic materials (polytetrafluoroethylene) (Baron et al., 2007).

Clostridium botulinum type E

C. botulinum is a Gram-positive, anaerobic bacterium that produces the most potent natural neurotoxin known. It is classified as a category A terrorism agent (CDC, 2003; Yule et al., 2006; Peck, 2010). There are seven types of C. botulinum that make up the proteolytic and nonproteolytic groups, which are distinguished on the basis of antigenically distinct toxins (A–G). Four types cause disease in humans. These types are A, B, E, and F (Telzak et al., 1990; Peck et al., 2010). Foodborne intoxication is caused by consumption of food containing amounts as small as 30–100 ng of preformed botulinal neurotoxin (Peck et al., 2010). Each year in the United States, there are approximately 55 cases, 42 hospitalizations, and 9 deaths that occur due to foodborne botulism (Scallan et al., 2011).

Type E neurotoxin is produced by the nonproteolytic, psychrotrophic form of C. botulinum, which has the ability to secrete neurotoxin at temperatures as low as 3°C (Peck, 2010). It has been demonstrated that environmental factors and pretreatments can affect lag time duration and variability of nonproteolytic C. botulinum more than germination rates (Stringer et al., 2009). Lowering incubation temperature has a proportionally greater affect on outgrowth and doubling time of nonproteolytic C. botulinum rather than germination rates of spores (Stringer et al., 2009), which indicates that historical treatment and growth conditions of nonproteolytic spores may help determine the risk of C. botulinum germination and outgrowth at refrigeration temperatures. Proteolytic varieties of C. botulinum are mesophiles producing neurotoxins A, B, or F and demonstrate little genetic similarity to the nonproteolytic types (Sebaihia et al., 2007; Peck, 2010). Because nonproteolytic strains of C. botulinum are psychotrophs, they are able to derive their energy through the degradation of sugars and produce neurotoxins at reduced temperatures (Peck, 2010). The nonproteolytic strains of C. botulinum are reportedly the main hazard associated with minimally heated refrigerated foods (Peck et al., 2010). Botulism causes flaccid paralysis, respiratory failure, and ultimately death, depending on the amount of toxin exposure (Telzak et al., 1990).

C. botulinum type E is the most prevalent form of botulism associated with marine life. Home-canned, raw or fermented, dried, and vacuum-packaged seafoods are most commonly associated with outbreaks (Telzak et al., 1989; Peck, 2010). Probably the botulism outbreak most relevant for comparison to contemporary minimally processed refrigerated foods was the 2006 outbreak associated with temperature abuse of pasteurized organic carrot juice. The carrot juice was low-acid (pH 6.0), low-sugar, and low-salt. It was flash-pasteurized, clean-filled, and sealed in 1-L bottles prior to refrigerated storage. Pasteurization will not inactivate spores of C. botulinum or other sporeforming bacteria. After purchase, it appears consumers did not properly refrigerate the juice, allowing for eventual production of neurotoxin type A in the product. Toxin-positive bottles of carrot juice gave no indication of clostridial growth. The juice smelled normal and no gas was produced (CDC, 2006).

Challenges for the Food Industry

Minimally processed foods

The increase in demand by consumers for convenient food products of premium sensory quality, including ready-to-eat, cooked, or chilled foods, and minimally processed foods, has led to the development of food products known as refrigerated processed foods of extended durability (Nissen et al., 2002). These products are normally low-acid foods refrigerated at close to freezing to maintain wholesomeness and safety (i.e., a national cold chain). They are often globally sourced. Fluid products usually are pasteurized. Produce and other raw foods commonly rely on surface cleaning/washing, modified or controlled atmosphere packaging, and other traditional hurdle approaches to ensure integrity and shelf-life.

Low-temperature storage

For mesophilic sporeforming species, temperatures below 15°C are generally thought to prevent spores from germinating. This is why in a laboratory setting spore crops are suspended in water and stored under refrigeration with the assumption that the spore crop concentration will remain stable until use. Low temperatures and limited nutrients prevent germination; however, in the case of psychrotolerant sporeforming species, temperatures at or above 6°C (Lechner et al., 1998) may allow for spore germination (albeit slowly) with outgrowth and perhaps permit cell multiplication in nutrient-rich environments. This possibility is why psychrotolerant sporeforming species, such as B. weihenstephanensis, which can potentially germinate and grow in refrigerated foods stored above optimum refrigeration temperatures of 4°C, are of possible concern. Such would be the case with minimally processed foods not heated or significantly heat processed prior to eating.

Influence of sporulation temperature on rates of germination and outgrowth

Spores populations of mesophilic B. cereus isolates were shown to germinate more rapidly in 10 mM of l-alanine when sporulated at 37°C than populations sporulated at 20°C (Raso et al., 1998); however, Gounina-Allouane et al. (2008) found the opposite with psychrotolerant B. cereus spores sporulated at 15 and 20°C. These spores were more easily germinated than spores sporulated at 37°C. Several previous studies demonstrated similar patterns with other sporeforming species. Spores of psychrotolerant strains of B. subtilis and psychrotolerant C. botulinum type E were more easily germinated with nutrients when sporulation occurred at lower temperatures (Cortezzo and Setlow, 2005; Evans et al., 1997). These results may indicate that spores sporulated at temperatures close to 37°C may have lower rates of germination and have higher percentages of superdormant spores. Heat activation of spores prior to germination increases spore germination and reduces overall yields of superdormant spores (Ghosh and Setlow, 2009a; Garcia et al., 2010; Wei et al., 2010); however, the mechanism by which heat activation affects germination is not well understood. Further investigation on the relationship of sporulation temperature to spore germination and superdormancy is needed. Understanding these mechanisms may also lead to further understanding of spore germination and outgrowth in refrigerated food products. In addition to affecting the rate of spore germination, sporulation temperature has been shown to affect spore size. In a study by Garcia et al. (2010), spores of B. weihenstephanensis sporulated at 12, 20, and 30°C were sized by scanning electron microscopy. Spores sporulated at 12 and 30°C were of similar size at approximately 1.5 μm, while spores sporulated at 20°C were slightly larger at approximately 1.8 μm. It was suggested that sporulation temperature may also affect the degree by which spores are germinated or inactivated through food-processing methods. The authors observed that as much as 99% of B. weihenstephanensis spore populations sporulated at 30°C germinated using 150 MPa of pressure, whereas only 50 and 15% germinated when sporulated at 12 and 20°C, respectively (Garcia et al., 2010).

Research Needs and Conclusions

The botulism outbreak involving Bolthouse™ carrot juice demonstrated the somewhat tenuous nature of nonsterile, low-acid products that rely on strict maintenance of the cold chain until the product is fully consumed. In this instance, anaerobic conditions and elevated temperatures allowed one or more spores of C. botulinum to germinate, replicate, and synthesize neurotoxin. Given the innate abilities of B. cereus, B. weihenstephanensis, and C. botulinum type E to conduct metabolic activities at lower temperatures than other bacteria, and the inherent resistance of their spores to inactivation, a high level of vigilance for these pathogens in nonsterile refrigerated foods carries merit. History has witnessed many emerging pathogens over the last 40 years become established issues in our food manufacturing and distribution systems. With continued demands for foods with minimal processing and reduced use of preservatives, there is clearly a need for additional hurdles that will protect these foods against temperature abuse during storage, distribution, and while in the hands of the consumer. These hurdles must meet the criteria of minimal processing and be perceived as generally regarded as safe preservatives.

Footnotes

Disclosure Statement

No competing financial interests exist.

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