Bacterial spore count

Introduction

Bacterial spores are resistance structures of bacteria and, once they are formed, remain in a dormant state. Contrarily to vegetative cells, spores are optically refractile, do not have metabolic activity, do not multiply, and can resist to environmental conditions that would be lethal to vegetative cells, including freezing, drying, irradiation, preservatives, disinfectants, and high temperatures. Under favorable conditions, they may germinate and originate new vegetative cells.

1.1 The bacterial spore

Endospore is the name used when the structure is formed intracellularly, before released into the environment. Spores are formed at the end of the exponential growth phase and may be induced by many factors such as nutritional deprivation, growth temperature, environmental pH, aeration, presence and concentration of minerals, carbon, nitrogen, and phosphorous sources, and population density (Logan and De Vos, 2009b).

1.1.1  Sequence of  spore formation

Hilbert and Piggot (2004) reviewed the formation of heat-resistant spores from vegetative cells of  Bacillus subtilis, which takes about 7 h at 37°C. The authors described the basic sequence of morphological changes during sporulation, which is similar for  Bacillus and Clostridium. Stage 0 is the vegetative cell. Stage I is the formation of an axial filament of chromatin with two copies of the chromosome. Stage II is the asymmetric cell division and septation to form a larger cell (mother cell or sporangium) and a smaller cell (prespore or fore-spore). Stage III is the engulfment of the prespore by the mother cell. Stage IV is the formation of two peptidoglycan layers surrounding the prespore, the cortex and the primordial germ cell wall (which will form the peptidoglycan layer of a new cell after germination).

Stage V is the construction of the coat, a complex structure of proteins on the outside surface of the prespore. When the cortex and the coat are formed the prespore is dehydrated and acquire its phase-bright appearance. Stage VI is the maturation, when the spore acquires its refractivity and full resistance.

1.1.2  Spore ultrastructure

Driks (2004) described the structure of Bacillus spores, which is composed of several concentric layers. The interior layer is the core, where the chromosome is located. The core is filled with small acid-soluble proteins (called SASP) that saturate the DNA and maintain the genetic material in a stable crystalline state. The SASPs are synthesized only during sporulation and are degraded when the spore germination begins. They bind to DNA and change the conformational and chemical properties of the molecule, which become much less reactive with a variety of chemicals. Surrounding the core is a lipid membrane and then a specialized thick layer of peptidoglycan, the cortex, which differs from the regular peptidoglycan found in the cell wall. The cortex is responsible for the spore relatively low water activity. Surrounding the cortex is a complex multilayered protein structure called the coat, which serves as a barrier against entry of large toxic molecules and play a role in germination.  B. anthracis and certain other  Bacillus species possess an additional layer, the exosporium, which is separated from the coat by a substantial gap. Its shape varies from spore to spore and its function is unknown.

1.1.3  Mechanisms of  spore resistance

Nicholson et al. (2000) reviewed the mechanisms of spore resistance, which are not well understood. Genetics is extremely important for wet heat resistance: spores of thermophiles are more resistant than spores of mesophiles, which in turn are more resistant than spores of psychrophiles. The sporulation conditions also have a significant effect, particularly temperature, since sporulation at an elevated temperature results in spores with increased heat resistance. The core water content appears to be inversely related to the spore wet-heat resistance.

The spore coats are believed to prevent access of pepti-doglycan-lytic enzymes to the spore cortex and to pro-tect from hydrogen peroxide and UV radiation. The small acid-soluble proteins (SASPs) appear to be important in protecting DNA from heat and oxidative dam-age. Logan and De Vos (2009b) also made reference to pyridine-2-6-dicarboxylic acid (dipicolinic acid; DPA), a unique and quantitatively important spore compo-nent, which comprises 5–14% of the spore dry weight. Ca²⁺ and other divalent cations are chelated by it, but their precise role in spore resistance is still unclear.

 Spore germination: Logan and De Vos (2009b) described the mechanism of germination in the genus Bacillus, which involves three steps: activation, germination and out-growth. Activation may be achieved by heat treatment (using time and temperature appropriate to the microorganism) or by ageing at low temperature. Spores of many species do not require the activation step, but the methods for spore isolation in foods always use the heat treatment to destroy vegetative cells. Germination can be induced by exposure to nutrients as amino acids and sugars, by mixtures of these, by non-nutrients such dodecyl amine, by enzymes and by high hydrostatic pressure. For many species, L-alanine is an important germinant, while D-alanine can bind at the same site as L-alanine and acts as competitive inhibitor. When the dormancy is broken the cortex is rapidly hydrolyzed, SASPs are degraded, and refractility is lost. The germinated spore protoplast then outgrows: it visibly swells owing to water uptake, biosynthesis recommences and a new vegetative cell emerges from the broken spore coat.

Importance in foods: Due to their thermal resistance, spores are particularly deleterious in foods that have been subjected to commercial sterilization processes, in which the competing flora is eliminated by heat. Their presence must be controlled in the ingredients used in the manufacture of these products, since high counts increase the probability of survival and future or later germination in the processed product. The ingredients most commonly used in formulating commercially sterile products are raw milk, milk powder, seasonings, spices and herbs, starch, sugar, fruits, fruit juices, vegetables and cereals. Heat resistance: Heat resistance is evaluated based on the D (decimal reduction time) and z (temperature coefficient) parameters. The D value, also called the lethal ratio, is defined as the time necessary to reduce to 1/10 the population of a given microorganism, at a given temperature. The z value is defined as the variation in temperature necessary to bring about a ten-fold variation in the D value, or, in other words, to promote decimal reduction or increase in the D value. Commercial sterilization and the D and z parameters are dis-cussed in detail in the specific chapter on commercial sterility.

1.2   Taxonomy of   spore forming bacteria important in foods

Up to 1990, spore forming bacteria associated with foods were restricted to the genera Clostridium, Desulfotomaculum,  Bacillus and  Sporolactobacillus. With the advances in phylogenetic studies, however, several new genera have been created, either to classify new species or to re-classify species that exhibit sufficient genetic diversity to justify separation.

1.2.1   Aeribacillus Miñana-Galbis et al. 2010

Nomenclature update from Euzéby (2012): Aeribacillus is a new genus created to contain one species, Aeribacillus pallidus, originally described as  Bacillus pallidus by Scholz  et al. (1987) and later transferred to the genus Geobacillus by Banat et al. (2004). There are no reports on the involvement of Aeribacillus pallidus in the spoil-age of foods, but Scheldeman et al. (2006) found this species as one of the most frequent in raw milk, and on equipment and in the environment of dairy farms. In the survey conducted by these authors, the species was isolated only after incubation at 55°C, and was not detected at 37°C.

Genus and species description from Logan  et al. (2009) (as  Geobacillus pallidus) and Miñana-Galbis et al. (2010) (as  Aeribacillus pallidus): Aeribacilli are thermophilic, aerobic, Gram positive, motile rods, occurring singly, in pairs and in chains. The spores produced are central to terminal, ellipsoidal to cylindrical and swell slightly the sporangia. The colonies on solid media are flat to convex, circular or lobed, smooth and opaque, with 2–4 mm in diameter after four days at 55°C. The optimum growth temperature is between 60 and 65°C with a minimum of 37°C and a maxi-mum of 65–70°C. Alkali tolerant, grow at pH 8.0–8.5 and do not grow at pH 6.0. Grow in presence of up to 10% NaCl. Catalase and oxidase reactions are positive. Nitrate is not reduced, citrate is not used as sole carbon source. Acid without gas is produced from glucose and from a small number of other carbohydrates. Casein, gelatin and urea are not hydrolyzed and starch is weakly hydrolyzed.

1.2.2   Alicyclobacillus Wisotzkey et al. 1992 emend. Goto et al. 2003 emend. Karavaiko et al. 2005

Nomenclature update from Euzéby (2012): This genus was proposed by Wisotzkey  et al. (1992) to reclassify the thermophilic and acidophilic species of  Bacillus with a typical cell membrane composition, consisting mainly  ω-alicyclic fatty acids (Bacillus acidocaldarius, Bacillus acidoterrestris and  Bacillus cycloheptanicus). The description of the genus was amended by Goto et al. (2003) and later by Karavaiko et al. (2005), when species previously classified as Sulfobacillus were transferred to the genus and new species was discovered. With this alteration, not all species contain this type of fatty acids and not all species are thermophilic, as originally described.

According to the International Federation of Fruit Juice Producers (IFU, 2007)  Alicyclobacillus is one of food spoilage microorganisms of major significance to the fruit juice industry. The spoilage is characterized by the formation of off flavors and odors from metabolic compounds such as guaiacol and the halophenols. Alicyclobacillus spores survive juice pasteurization and also survive for long periods in raw materials (fruit concentrates, liquid sugar, syrups, tea, etc) although more dilute environments are required for growth.  Alicyclobacillus acidoterrestris is the most common spoilage species but other species may also occur. The guaiacol-producing species currently known are positive in the peroxidase test and grow at temperatures less than 65°C. Strains of alicyclobacilli growing at 65°C and above are unlikely to spoil juice products. The other species that have been isolated from acidic food products are  A. acidocaldarius, A. acidiphilus,  A. contaminans,  A. fastidiosus,  A. herbarius,  A. pomorum and  A. sacchari.

Genus description from Costa  et al., (2009): Alicyclobacilli are motile or non-motile rods with Gram-positive cell wall and positive or variable Gram stain reaction. Sporogenic, the spores are ovoid, terminal or subterminal, in swollen or not swollen sporangium. Aerobes with a strictly respiratory type of metabolism, but a few strain are able to growth anaerobically using nitrate or Fe3+ as terminal electron acceptor. The oxidase and catalase reactions may be positive or negative. All species are acidophilic with pH range for growth of 0.5 to 6.5 and optimum between pH 1.5 and 5.5. The species can be grouped into three categories in terms of their growth temperature range. One group includes the strict thermophilic species (A. acidocaldarius and others) which grows between 45°C and 70°C with an optimum of about 65°C. Another group includes the facultative thermophilic species (A. acidoterrestris and others) which grows between 20°C and 65°C with an optimum between 40°C and 55°C. The third group includes species which grows between 4°C and 55°C with an optimum between 35°C and 42°C.

 Alicyclobacillus acidoterrestris: Previously called Bacillus acidoterrestris, this species is reported by Evancho and Walls (2001) as a facultative thermophile that produces acid, but not gas from carbohydrates (typical “flat-sour”). Common in fresh fruits, fruit juices and fruit concentrates, the spoilage caused by  A. acidoterrestris results in off-flavor and off-odor (described as “medicinal” or “phenolic”) due to the production of guaiacol during growth. The spoilage does not occur in concentrated juices with a Brix above 30º, but the spores can survive the heat process given to fruit juices and remain viable (D95ºC is 1.64 min in apple juice at pH 3.6) (Evancho and Walls, 2001). Species description from Costa et al. (2009): The cells of A. acidoterrestris are Gram-positive, endospore-forming rods. The spores are oval, subterminal to terminal and the sporangia are not generally swollen. Colonies formed on solid media are non-pigmented. Nitrate is not reduced to nitrite and growth factors are not required. The temperature range for growth is 35°C to 55°C and the optimum varies from 42°C to 53°C. The pH range for growth is 2.2 to 5.8 with an optimum around 4.0. Oxidase negative and catalase weakly positive. No growth occurs in the presence of 5% NaCl. The species was described as aerobic by Wisotzkey  et al. 1992 and Costa  et al., 2009, but the Compendium (Evancho and Walls, 2001) reported the existence of strains facultative anaerobic.

Alicyclobacillus acidocaldarius: Previously denominated Bacillus acidocaldarius this species does not appear to be a frequent spoilage microorganism, once there are only few reports on its presence in acid foods (mango juice and concentrates sterilized by the UHT process) (Gouws  et al., 2005). Species description from Costa et al. (2009): The cells of A. acidocaldarius are gram positive, spore-forming rods that often occur in short chains. The sporangia are not swollen by endospores, which are ellipsoidal and terminal to subterminal. Colonies on solid media are not pigmented. Aerobic, the carbon and energy sources used for growth include hexoses, disaccharides, organic acids, and amino acids. Nitrate is not reduced to nitrite. Growth occurs with ammonia but not with nitrate as a sole nitrogen source. The pH range for growth is 2.0 to 6.0, with an optimum around 3.0–4.0. Strict thermophile, the temperature range for growth is 45 to 70°C, with an optimum around 60–65°C. No growth factors are required. The oxidase reaction is negative and catalase is weakly positive.

 Alicyclobacillus acidiphilus: A novel acidophilic facultative thermophile, this species was isolated from an acidic beverage that had the odor of guaiacol. Species description from Costa  et al. (2009): The cells of A. acidophilus are Gram-positive rods, motile, forming subterminal or terminal oval spores in swollen sporangia. Colonies on solid media are not pigmented. The pH range for growth is 2.5 to 5.5 with an optimum around 3.0. The temperature range for growth is 20 to 55°C with an optimum around 50°C. Catalase is positive and oxidase is negative. Growth factors are not required, nitrate is not reduced to nitrite. Aerobic, acids but no gas are formed from several sugars.

 Alicyclobacillus contaminans: A novel moderately thermophilic, acidophilic species isolated from soil and from orange juice. Species description from Costa et al. (2009): The cells of  A. contaminans are straight rods with rounded ends. Gram-positive, but old cultures stain Gram-variable. Motile, endospore-forming, endospores are ellipsoidal and subterminal with swollen sporangia. Colonies on BAM agar are non-pigmented (creamy white), circular, opaque, entire and umbonate with 3–5 mm in diameter after 48 h. The temperature range for growth is 35 to 60°C with an optimum about 50 to 55°C. The pH optimum is 4.0 to 4.5 and growth does not occur at pH 3.0 or 6.0. Growth occurs in the presence of 0 to 2% (w/v) NaCl but not 5% (w/v) NaCl. Oxidase and catalase reactions are negative, nitrate reduction is negative. Strictly aerobic, acid is produced from a number of sugars and sugar alcohols.

 Alicyclobacillus fastidiosus: A novel moderately thermophilic, acidophilic species isolated from soil and from apple juice. Species description from Costa et al. (2009): The cells of A. fastidiosus are non-motile, endospore-forming straight rods. Gram-positive but stain Gram-variable in old cultures. Endospores are ellipsoidal and subterminal with swollen sporangia. Colonies on BAM agar are non-pigmented (creamy white), circular, opaque, entire and flat with 3–4 mm in diameter after 48 h. The temperature range for growth is 20 to 55°C and the optimum is 40 to 45°C. Optimum pH is 4.0 to 4.5; growth does not occur at pH 2.0 or 5.5. Growth occurs in the presence of 0 to 2% (w/v) NaCl, but not at 5% (w/v) NaCl. Oxidase negative and catalase positive. Strictly aerobic, acid is produced from a number of sugars and sugar alcohols.

 Alicyclobacillus herbarius: A novel thermo-acidophilic bacterium isolated from herbal tea made from the dried flowers of hibiscus. Species description from Costa et al. (2009): The cells of A. herbarius are Gram-positive, motile, spore-forming rods. Endospores are oval and subterminal with swollen sporangia. Colonies on solid media are not pigmented. Temperature range for growth is 35 to 65°C and the optimum is 55 to 60°C. The pH optimum is 4.5 to 5.0; growth does not occur at pH 3.0 or 6.5. Growth factors are not required, oxidase is negative, catalase is positive and nitrate is reduced to nitrite. Strictly aerobic, acid is produced from several sugars.

 Alicyclobacillus pomorum. A novel thermo-acidophilic endospore-forming bacterium isolated from spoiled mixed fruit juice (orange, apple, mango, pineapple and raspberry). Species description from Costa et al. (2009): The cells of A. pomorum are motile, endospore-forming rods. Gram-positive but stain Gram-variable in old cultures. Endospores are oval and subterminal with swollen sporangia. Colonies on solid media are non-pigmented. The temperature range for growth is 30 to 60°C and the optimum is 45 to 50°C. Optimum pH is 4.5 to 5.0; growth does not occur at pH 2.5 or 6.5. Oxidase and catalase positive, nitrate is not reduced to nitrite. Strictly aerobic, acid is produced from several sugars.

 Alicyclobacillus sacchari. A novel moderately thermophilic, acidophilic species isolated from soil and from liquid sugar. Species description from Costa et al. (2009): The cells of A. sacchari are endospore-forming straight rods, Gram-positive but Gram-variable in old cultures. Endospores are ellipsoidal and subterminal with swollen sporangia. Colonies on BAM agar are non-pigmented (creamy white), circular, opaque, entire and umbonate. The temperature range for growth is 30 to 55°C and the optimum is 45 to 50°C. The pH optimum is 4.0 to 4.5; growth does not occur at pH 2.0 or 6.0. Growth occurs in the presence of 0 to 2% (w/v) NaCl, but not at 5%(w/v) NaCl. Oxidase, catalase and nitrate reduction are negative. Strictly aerobic, acid is produced from a number of sugars and sugar alcohols.

22.1.2.3   Aneurinibacillus Shida et al. 1996 emend. Heyndrickx et al. 1997

Nomenclature update from Euzéby (2012): This genus was proposed to reclassify strains previously classified as Bacillus aneurinolyticus and Bacillus migulanus, which characteristically decompose thiamine. A data survey conducted by Scheldeman  et al. (2006) revealed the presence of species of this genus in raw milk, and on the equipment and in the environment of dairy farms. Logan and De Vos (2009a) related the isolation of A. thermoaerophilus from beet sugar.

Genus description from Logan and De Vos (2009a): The cells of aneurinibacilli are motile Gram-positive rods forming ellipsoidal spores, one per cell, central, paracentral or subterminal in swollen or not swollen sporangia. Strictly aerobic but one species is microaerophilic. Growth occurs on routine media such as Nutrient Agar and Trypticase Soy Agar. Decompose thiamine. Catalase reaction is positive, weakly positive, or negative. Growth temperature ranges from 20 to 65°C. Growth pH ranges from 5.5 to 9.0. Growth occurs in the presence of 2 to 5% NaCl; some strains grow weakly at 7% NaCl. Few carbohydrates are assimilated and acid is produced weakly or not produced from them. Amino acids and some organic acids are used as carbon sources.

 Aneurinibacillus thermoaerophilus: Previously denominated  Bacillus thermoaerophilus, the original strains were isolated from the high-temperature stages of beet sugar extraction and refining. Species description from Logan and De Vos (2009a): Vegetative cells are Gram positive rods, motile. Central and paracentral spores are formed in swollen sporangia. Colonies on nutrient agar after 24 h at 55°C are creamy grayish, flat, irregular and tend to swarm across the surface of the agar. Growth temperatures range from 40°C to 60°C and the pH for growth varies from 7.0 to 8.0. Growth occurs in presence of 3% NaCl but not in presence of 5% NaCl. Catalase production is variable, nitrate is not reduced. Strict aerobic, a range of amino acids, carbohydrates, and organic acids is assimilated as carbon sources.

22.1.2.4   Anoxybacillus Pikuta et al. 2000 emend. Pikuta et al. 2003

Nomenclature update from Euzéby (2012): This genus was created to contain  Anoxybacillus pushchinensis, a novel species of anaerobic, alkaliphilic, moderately thermophilic bacteria isolated from manure, and to reclassify “Bacillus flavothermus” as Anoxybacillus flavithermus. Most species of the genus have been isolated from hot springs but the novel species Anoxybacillus contaminans was isolated from gelatin.

Genus description from Pikuta (2009): Anoxybacilli form rod-shaped and straight or slightly curved, cells, motile or nonmotile. Angular division and Y-shaped cells may occur, often arranged in pairs or in short chains. The Gram stain is positive or variable. Terminal, round, oval or cylindrical endospores are formed, only one per cell. Colonies on solid media vary with the species including yellow colonies, white colonies with yellowish center and cream colored colonies. Catalase reaction is variable. Moderately thermophilic, the growth occurs in the temperature range between 30–45°C to 60–72°C, with an optimum between 50°C and 62°C. There are species alkaliphilic, alkali tolerant or neutrophilic, but most species can grow at neutral pH. Chemo-organotrophic, aerobes, facultative aerobes or facultative anaerobes, the metabolism is fermentative. The genus contains saccharolytics and proteolitic species.

 Anoxybacillus contaminans: A novel species isolated from gelatin in a French production plant. Species description from Pikuta (2009): The cells are motile rods, curved or frankly curled with round ends, Gram variable, occurring singly, in pairs or in short chains. Endospores are oval, subterminal or terminal and the sporangia are slightly swelled. Colonies on solid media are circular with regular margins and raised centers and edges, opaque, glossy and cream-colored. Catalase is positive, oxidase is negative, nitrate is reduced to nitrite. Moderately thermophilic, the maximum temperature for growth varies between 40°C and 60°C and the optimum is 50°C. Alkali tolerant, the optimum pH for growth is 7.0, with a minimum at 4.0 to 5.0 and a maximum at 9.0 to 10.0. NaCl is not required for growth which occurs between 0 and 5%. Chemoheterotrophic, facultative anaerobic, small amounts of acid without gas is produced from glucose and other carbohydrates.

1.2.5 Bacillus Cohn 1872

Nomenclature update from Euzéby (2012):  Bacillus is one of the original genera of spore-forming bacteria which often occur in foods, but several species were reclassified into the new genera  Aeribacillus,  Alicyclobacillus,  Aneurinibacillus,  Brevibacillus,  Geobacillus, Lysinibacillus,  Paenibacillus and  Virgibacillus. Among the species that remain in the genus  Bacillus the most important are B. cereus and the facultative thermophilic B. coagulans, B. smithii and  B. sporothermodurans. Other bacilli found in food and food production environment are, according data surveyed by Scheldeman et al. (2005),  B. licheniformis and  B. subtilis, found in raw milk, milking equipment and other samples from dairy cattle farms. Isolation of these species was achieved both at 20 as at 37 and at 55°C. In commercially sterile ingredients (sugar, starch, cereals, seasonings, spices and herbs, milk powder, cocoa, gelatin, tomato and other vegetables) the most frequent species are  B. coagulans, B. licheniformis, B. subtilis, B. circulans and B. pumilus (isolated at 55°C) (Richmond & Fields, 1966). Data gathered by Kalogridou-Vassiliadou (1992) mention the involvement of B. licheniformis and  B. subtilis in “flat sour” deterioration of evaporated milk. Data collected by Scheldeman et al. (2006) report the involvement of B. sphaericus and B. licheniformis in the contamination of milk sterilized by the conventional or UHT sterilization process.

Genus description by Logan and De Vos (2009b): Cells are motile or nonmotile, rod-shaped, occurring singly and in pairs, some in chains, and occasionally in long filaments. Gram stain is positive or Gram positive only in early stages of growth, or Gram negative. Endospores are formed, only one per cell. The sporangial morphology (ellipsoidal, oval, spherical, kidney-shaped, banana-shaped), the position of the endospore (central, paracentral, subterminal, terminal) and the swelling of the sporangia (present or absent) are characteristic of the species. Most species common in foods grow well on routine media such as Nutrient Agar. Colony characteristics on solid media vary between and within species. Large colonies with irregular edges, sometimes spreading, sometimes colored, sometimes flat, mucoid, dry and adherent often occur.  B. licheniformis pro-duce colonies which are variable in appearance and the cultures often appear to be mixed. Aerobes or facultative anaerobes, most species are catalase positive and oxidase positive or negative. Most species use glucose and/or other fermentable carbohydrates as sole source of carbon and energy. Some species do not utilize carbohydrates. Mesophiles are most common among the members of the genus, with optimum temperature between 25°C and 40°C (typically around 30°C), mini-mum between 5°C and 20°C, and maximum between 35°C and 55°C. However, several species are moderately thermophilic (optimum 40–55°C, maximum 55–65°C and minimum 25–40°C), some are true thermophiles (optimum 55–70°C, maximum 65–75°C and mini-mum 37°C) and some are psychrotrophics or true psychrophiles. The optimum pH of most of these species is in the neutral range, but some species are Aciduric and some are alkaliphilic. The heat resistance of the spores varies with the species and, in some cases, between the strains of the same species.

 Bacillus coagulans: B. coagulans has been known for decades as an economically very important food spoil-age agent in slightly acidic canned products. According to Evancho and Walls (2001), this species causes “flat sour” deterioration, which is characterized by acidification without gas production, resulting in spoiled product but not swollen packages. The canned foods most commonly affected are tomato products (canned whole tomatoes, tomato juice, tomato puree, tomato soup, tomato-vegetables juice mixes) but has also been found in dairy products (cream, evaporated milk, cheese), fruits, and vegetables B. coagulans is also a producer of commercially valuable products such as lactic acid, thermostable enzymes, and the antimicrobial peptide coagulin, and is probiotic for chickens and piglets (Logan and De Vos (2009b). The specie description was corrected by De Clerck et al. (2004) because many strains isolated and identified as B. coagulans, were reclassified as  Bacillus smithii,  Bacillus licheniformis or  Geobacillus stearothermophilus. Species description from Logan and De Vos (2009b): Cells are motile rods, Gram-positive. Spore forming, but some strains do not sporulate readily. Endospores are ellipsoidal (in some cases appear spherical), subterminal (occasionally paracentral or terminal) in slightly swollen sporangia. Colonies on Trypticase Soy Agar (TSA 40°C/48 h) are white (cream-colored with age), convex with entire margins and smooth sur-face. Facultative thermophile, growth occurs between 30°C and 57–61°C, but not at 65°C, with an optimum between 40°C and 57°C. Slightly aciduric, growth occurs between pH 4.0 and 10.5–11.0 with an optimum at 7.0. It does not grow in presence of 5% NaCl. Catalase-positive, facultative anaerobe, acid but not gas is produced from carbohydrates. The D_121.1ºC value reported by Stumbo (1973) varies between 0.01 and 0.07 min.

 Bacillus smithii: B. smithii is a new species, proposed to reclassify strains formerly classified as  B. coagulans. Species description by Logan and De Vos (2009b): Cells are motile Gram-positive rods, spore forming, producing ellipsoidal to cylindrical endospores, terminal or sub-terminal in non-swollen or slightly swollen sporangia. Colonies are not pigmented, translucent, thin, smooth, circular, entire, and about 2 mm in diameter. B. smithii is a facultative thermophile, growing between 25 and 60°C, though the majority of the strains also grow at 65°C. Growth occurs at pH 5.7, but not at 4.5 or lower. Catalase and oxidase reactions are positive. No growth occurs in presence of 3% NaCl or 0.001% lysozyme. Facultative anaerobic, this species uses carbohydrates to produce acid, but no gas (“flat sour”). It has been isolated from evaporated milk, canned foods, cheese and sugar beet juice.

 Bacillus sporothermodurans:  B. sporothermodurans is a new species proposed by Pettersson  et al. (1996) based on genetically homogeneous isolates from UHT-milk. According to a review published by Scheldeman et al. (2006), this new species was discovered in contaminated lots of UHT and sterilized milk in Italy, Austria and Germany in 1985, 1990 and 1995. The problem subsequently affected other products including whole, skimmed, evaporated or reconstituted UHT milk, UHT cream and chocolate milk, UHT-treated coconut cream and also milk powders. The microorganism usually reaches 105 vegetative cells and 103 spores/ml of milk after incubation at 30°C for 15 days. The pH of the milk is not affected and the stability or sensory qualities usually are not altered. Spores of strains isolated from UHT milk survive ultra-high temperature treatment (UHT) and the studies available suggest that this highly heat-resistant spores were adapted and selected by sub lethal stress in the industrial process. The best method for isolation from raw milk or other farm sources is: Autoclave the sample for 5 min or heat at 100°C for 30–40 min, plate on Brain Heart Infusion (BHI) supplemented with vitamin B₁₂ (1mg/l) and incubate at 37°C/48 h. Species description as emended by Heyndrickx et al. (2011): Cells are motile Gram-positive thin rods, occurring in chains. Colonies on Plate Count Agar (PCA) are pinpoint because vita-min B₁₂ is required for satisfactory growth. After two days on Brain Heart Infusion (BHI) Agar supplemented with MnSO₄ (5mg/l) and vitamin B₁₂ (1mg/l), colonies are 1–2 mm in diameter, flat, circular, entire, beige or cream and smooth or glossy. Endospores are spherical to ellipsoidal, paracentral and subterminal (sometimes terminal) in slightly swollen or unswollen sporangia. Sporulation is infrequent but can be enhanced by using BHI supplemented with soil extract, vitamin B₁₂ and MnSO₄. Aerobic, oxidase and catalase reactions are positive. Mesophilic, growth may occur between 20°C and 55°C, with an optimum of about 37°C. Growth occurs between pH 5.0 and 9.0 and NaCl is tolerated up to 5% (w/v). Acid but not gas is produced from carbohydrates.

1.2.6  Brevibacillus Shida et al. 1996

Nomenclature update from Euzéby (2012): This genus was proposed to reclassify the strains previously classified as Bacillus brevis (which was subdivided into nine new species) and later other new species were discovered. A data survey by Scheldeman et al. (2006) showed the presence of  B. brevis,  B. agri,  B. borstelensis and other brevibacilli in raw milk, and on equipment and in the environment of dairy farms. They also showed the involvement of B. brevis and/or B. borstelensis in the contamination of milk sterilized by the UHT or conventional process. A data survey by Logan and De Vos (2009c) reported the isolation of  B. laterosporus from water, sweet curdling milk spoilage, bread dough and spontaneously fermenting soybeans,  B. centrosporus from spinach,  B. parabrevis from cheese,  B. agri from sterilized milk, a gelatin processing plant and a public water supply (the later involved in an outbreak of water-borne illness) and B. brevis and B. laterosporus from food packaging's of paper and board.

Genus description by Logan and De Vos (2009c): Cells are Gram positive, Gram variable, or Gram negative rods forming ellipsoidal spores in swollen sporangia. Most species grow on Nutrient Agar (NA) and Trypticase Soy Agar (TSA) producing flat, smooth, yellowish-gray colonies. One species produces red pigment. Most species are strictly aerobic and one species is facultative anaerobic. Most species are catalase positive and the oxidase reaction varies between species. Voges Proskauer (VP) reaction is negative, nitrate reduction and casein, gelatin and starch hydrolysis varies between species. Growth is inhibited by 5% NaCl. Optimum growth occurs at pH 7.0 and the growth at pH 5.5 varies among the species. Carbohydrates may be assimilated, but acid is produced weakly or not produced by most species. The growth temperatures vary considerably; most species grow at 20°C, but not at 50°C or 55°C, with the optimum at 28–30°C. The species growing at 50–55°C also have a higher optimum temperature (>40°C). Most species do not grow at pH 5.5 or below.

1.2.7  Clostridium Prazmowski 1880

Nomenclature update from Euzéby (2012): Clostridium is one of the original genera of spore-forming bacteria which often occur in foods, but some species usually found in foods were reclassified into the new genera Desulfotomaculum,  Moorella and  Thermoanaerobacterium. Among the species that remain in the genus Clostridium the pathogenic species transmitted by foods are C. botulinum  and C. perfringens. In addition to these, Scott et al. (2001) reports three important groups causing food spoilage: the proteolytic species C. botulinum, C. sporogenes, C. bifermentans, C. putrefasciens and C. histolyticum, the saccharolytic species  C. butyricum, C. pasteurianum,  C. tyrobutyricum,  C. beijerinckii and C. acetobutylicum, and the psychrophilic or psychrotrophic species  C. estertheticum,  C. algidicarnis and C. gasigenes, treated in this chapter.

Genus description from Rainey et al. (2009): Cells are obligate anaerobic rods, motile or nonmotile. The Gram stain is usually Gram positive (at least in the very early stages of growth) but in some species Gram positive cells have not been seen. The majority of species form oval or spherical endospores that usually swell the sporangia. Usually chemoorganotrophic, some species are chemoautotrophic or chemolitotrophic as well. Usually organic acids and alcohols are produced from carbohydrates or peptones. The species may be proteolytic, saccharolytic, neither or both. Usually catalase negative, although trace amounts of catalase may be detected in some strains.

 Clostridium botulinum: This species is a very serious public health hazard, producing highly potent toxins that cause botulism. According to FDA/CFSAN (2009) the toxins act on the nervous system and are lethal by ingestion of a few nanograms. Thermolable, they are destroyed by heating to 65–80ºC/30 min or 100ºC/5 min. The disease is caused by the ingestion of foods contaminated with the toxin, being characterized by selective neurological manifestations, dramatic evolution and high mortality rate. The disease can start with marked lassitude, weakness and vertigo. Next, double vision and progressive difficulty in speaking and swallowing are observed. Difficulty in breathing, weak-ness of other muscles, abdominal distention, and constipation may also be common symptoms. This overall phase or form of the disease causes respiratory and cardio-vascular difficulties, ultimately leading to death by cardiorespiratory collapse. Spores of  C. botulinum are widely distributed in nature, and may occur in almost all foods, including foods of plant origin and foods of animal origin. A great many foods have already been implicated in the transmission of this bacterium, including sausages and stuffed meat products, candies, leafy vegetables, and canned legumes (heart of palms, asparagus, mushrooms, artichokes, sweet peppers, egg plant, garlic, pickles, etc.), fish, seafood, and others. A survey conducted by the Food Safety Inspection Service of the United States Department of Agriculture (FSIS/USDA, 1997) reports cases occurred in several countries, involving peppers preserved in oil, cooked meat, salmon, tuna fish, soups, mushrooms and others. Although it does not grow in acid foods, sporadic involvement of acidified preserves in cases of botulism have been reported. In the FSIS/USDA (1997) survey, the main cause of these events has been the multiplication of other microorganisms in the product, which increase the pH up to the growth range of C. botulinum.

In function of the antigenic properties of the toxins produced, the strains of C. botulinum have been classified into seven types (A, B, C, D, E, F, G). Data from the International Commission on Microbiological Specifications for Foods (ICMSF, 1996) indicate that strains of type A affect humans and chickens, and is more common in parts of North America and in countries that formerly pertained to the former Soviet Union. The most common vehicles of transmission are homemade vegetable preserves, fruits, meats and fish. Strains of type B affects humans, cattle and horses and are more common in North America, Europe and countries of the former Soviet Union (non-proteolytic strains). The most common vehicles of transmission are prepared meats, particularly pork meat. Strains of  types C and D affect water fowl, cattle and horses, but not humans. Strains of type E affects humans and fish and the most common vehicles of transmission are fish and seafood. It occurs mainly in regions where consumption of these products is high, including Japan, Denmark, Sweden, Alaska, Labrador and countries of the former Soviet Union. Strains of type F affects humans and are more common in Denmark, North America, South America and Scotland. The most frequent vehicles of transmission are meat products. Strains of type G was isolated from the soil in Argentina and there are no outbreaks confirmed of botulism caused by this type in humans. It was never encountered in foods and, in 1988, was reclassified as Clostridium argentinense. Based on metabolic characteristics, the strains were divided into three groups (I, II and III) and the strains of group G were placed in a separate group (Group IV). Rainey  et al. (2009) summarized the characteristics of these groups:

Characteristics of  C. botulinum Group I from Rainey  et al. (2009): Group I includes the strains of type A and proteolytic strains of types B and F. Cells are straight to slightly curved rods, motile, producing spores oval, subterminal in swollen sporangia. The optimum temperature for growth is 30–40°C. Some strains grow well at 25°C and a few at 45°C. Growth is inhibited by 6.5% NaCl, 20% bile, and at pH 8.5. Toxin production is delayed in atmosphere of 100% CO₂ and pressurized CO₂ may be lethal depending on the amount of pressure and length of exposure. Gelatin, milk and meat are digested (proteolytic). Ammonia and H₂S are produced. Fermentation products include large amounts of acid and gas H₂. Data from ICMSF (1996): The spores of this group have the highest level of resistance among the spores produced by C. botulinum species. The D121.1ºC value reported in different substrates varies between 0.05 and 0.32 min. The minimum temperature for growth is 10–12ºC. They do not grow in the presence of 10% NaCl. Under optimal conditions of temperature and water activity, the minimum pH value for growth is 4.6. Multiplication is characterized by putrid odor and production of gas.

Characteristics of  C. botulinum Group II from Rainey  et al. (2009): Group II includes the strains of type E and saccharolytic strains of types B and F. Cells are straight rods, motile, producing spores oval, central to subterminal, usually in swollen sporangia. The optimum temperature for growth ranges from 25°C to 37°C. Poor or no growth occurs at 45°C. Growth is stimulated by a fermentable carbohydrate and is inhibited by 6.5% NaCl, 20% bile, and at pH 8.5. Fermentation products include acid and gas H₂. Data from ICMSF (1996): The spores are less heat resistant than those of Group I; the D82.2ºC related in different substrates varies between 0.25 and 73.61 min. The mini-mum temperature for growth is 3.3ºC, although there are reports of growth under refrigeration. They do not grow in the presence of 5% NaCl. Under optimal conditions of temperature and water activity, the minimum pH for growth is 5.2. Multiplication is characterized by the production of gas, but without the development of putrid odor.

Characteristics of  C. botulinum Group III from Rainey et al. (2009): Group III includes the strains of type C and D. Cells are straight rods, motile, producing spores oval, subterminal, in swollen sporangia. The optimum temperature for growth is 30–37°C. Most strains grow well at 45°C and grow poorly or do not grow at 25°C. Growth is stimulated by a fermentable carbohydrate and is inhibited by 6.5% NaCl, 20% bile, and at pH 8.5. Gelatin is digested; milk is acidified, curdled, and digested by 20 of 29 strains tested; meat is digested by 20 of 28 strains tested. Production of ammonia and H₂S varies among strains. Fermentation products include acid and large amounts of gas H₂.

Data from ICMSF (1996): The minimum temperature for growth is 15ºC and they do not grow in the presence of 3% NaCl. Characteristics of  C. botulinum Group IV from Rainey et al. (2009): Group IV includes the strains of type G. Cells are straight rods, motile, producing spores oval, subterminal, in swollen sporangia. The optimum temperature for growth is 30–37°C and good growth is observed at 25°C and 45°C. Growth is inhibited by 6.5% NaCl and 20% bile. Gelatin and casein are digested rapidly; milk and meat are digested within three weeks. Ammonia and H₂S are produced. Fermentation products include acid and large amounts of gas H₂. Data from ICMSF (1996): The minimum temperature for growth is 12ºC and they do not grow in the presence of 3% NaCl.

 Proteolytic clostridia (data from Scott et al., 2001): This group also called putrefactive clostridia includes the mesophilic spore forming anaerobes that digest proteins with putrid odor. Species associated with foods include  C. botulinum types A and B,  C. sporogenes, C. bifermentans,  C. putrefasciens and  C. histolyticum. These species are the main cause of spoilage of low-acid canned foods under-processed. They do not grow at a pH lower than 4.6 (with the exception of  C. putrefaciens) and may deteriorate any type of food with pH 4.8 or higher. Mesophilic, the temperature range for growth is between 10 and 50°C, with an optimum between 30 and 40ºC. C. putrefaciens is psychrotrophic, and grows between 0 and 30°C, with an optimum between 15 and 22ºC. They are Gram-positive rods, except for C. putrefasciens, which forms long curved filaments. Motile or non-motile, catalase-negative, they produce spores oval (C. putrefaciens oval or spherical), most frequently sub-terminal (eventually central or terminal) in swollen or not swollen sporangia. C. bifermentans and C. sporogenes produce acetic acid and large quantities of gas hydrogen during growth.  C. histolyticum produces moderate amounts of hydrogen and does not produce acids from carbohydrates.  C. putrefasciens does not produce acids nor hydrogen from carbohydrates. The classical condition encountered in spoiled products is swollen pack-ages and putrid odor, but changes without gas may also occur.

 Saccharolytic clostridia (data from Scott  et al., 2001): This group also called non-proteolytic clostridia includes the mesophilic spore forming anaerobes that do not digest proteins and, therefore, do not produce putrid odor. They ferment carbohydrates and the fermentation end products include butyric and acetic acids, carbon dioxide and hydrogen. The species most common in foods are  C. butyricum,  C. pasteurianum, C. tyrobutyricum,  C. beijerinckii and  C. acetobutylicum, capable of growing at pH 4.2–4.4 and deteriorate slightly acid canned foods (slightly acid tomato-based products and slightly acid fruits). Mesophiles, their optimum growth temperature varies from 30 to 40°C. They are Gram-positive rods that form oval, central or subterminal spores usually with a swollen sporangium. The spores are not very heat-resistant, compared to those of the putrefactive species and are more commonly involved in post-processing contamination (leakage) than in deterioration by under-processing.

Their growth is characterized by butyric odor and pro-duction of gas. Psychrophilic and psychrotrophic clostridia that cause the spoilage of refrigerated vacuum-packed meats. The first reports associating psychrophilic clostridia with the deterioration of refrigerated vacuum-packed meats were published by Dainty et al. (1989) and Kalchayan and et al. (1989). The isolated cells were later characterized as new species and denominated  Clostridium estertheticum (Collins et al., 1992) and Clostridium laramie (Kalchayanand  et al. 1993). Later studies have demonstrated a straight relationship between these strains, which were reclassified into one single species, divided into two subspecies –  Clostridium estertheticum subsp. estertheticum and  Clostridium estertheticum subsp.  laramiense (Spring et al. 2003). Both are true psychrophiles, subsp. estertheticum with an optimum growth temperature from 6ºC to 8ºC, maximum temperature 13ºC and a minimum of 1°C.  C. estertheticum subsp.  laramiense grows from minus 3ºC to 21°C, with an optimum at 15ºC. They are motile, Gram-positive and form spores that resist heat treatments of 80°C/10 min, but not of 90°C/10 min. Saccharolytic in nature, the main fermentation products of  C. estertheticum  subsp. estertheticum are butyric and acetic acid (4:1 ratio), in addition to hydrogen gas (30%) and CO2 (70%). Furthermore, they also produce butanol, butyl butanoate, butyl acetate and a complex mixture of esters and sulphurous compounds, principally H₂S and methanethiol. In fermentation by  C. estertheticum subsp. laramiense predominate butyric acid, 1-butanol and the gases hydrogen and CO₂, in addition to the production of lactic acid, acetic acid, formic acid and ethanol. The spoilage of vacuum-packed meats, known as “blown-pack”, is not related to conditions of temperature abuse and occurs in products stored under adequate conditions of refrigeration. It causes foul odor and pronounced blowing of the packages. In the case of deterioration by  C. estertheticum subsp. estertheticum, the odor detected immediately upon opening of the packaging is described as sulphurous, changing to the smell of fruit and solvent odor after 5 min of exposure to ambient temperature. Over the next 10 min it further changes to a strong cheese smell and butanoic odor.

In 1994 a new species of  Clostridium was reported isolated from spoiled samples of cooked, vacuum-packed refrigerated pork meat, denominated  Clostridium algidicarnis (Lawson  et al. 1994). This species is psychrotrophic with optimum temperature in the 25 to 30°C range, with a maximum of 37°C and a minimum (tested) of 4°C. Saccharolytic, it ferments carbohydrates with the production of acids (predominantly butyric and acetic) and gas. A third new species of Clostridium causing deterioration of vacuum-packed, refrigerated meat kept under conditions of temperature abuse was reported in 1999 and denominated  Clostridium frigidicarnis (Broda et al. 1999). This species is psychrotrophic, with optimum temperature in the 30–38.5°C range, maximum of 40.5°C and minimum of 3.8°C. It is saccharolytic, fermenting carbohydrates with the production of acetic, butyric, lactic and other acids, along with ethanol, and the gases hydrogen and CO₂. Deterioration in vacuum-packed meats is of the “blown-pack” type. The fourth new species of Clostridium that causes spoilage of vacuum-packed refrigerated meats was reported in 2000, and was denominated Clostridium gasigenes (Broda et al. 2000). This species is psychrotrophic, with optimum temperature in the 20–22°C range, maximum of 26°C and minimum of 1.5°C. It is saccharolytic, fermenting carbohydrates with the production of ethanol, acetic, butyric, and lactic acid, butyric esters and the gases hydrogen and CO₂. It causes spoilage of vacuum-packed meats of the “blown-pack” type, although with less pronounced blowing than that caused by  C. estertheticum. Deterioration is not related to conditions of temperature abuse and occurs in products stored under adequate conditions of refrigeration.

1.2.8  Cohnella Kämpfer et al. 2006

Nomenclature update from Euzéby (2012): The genus Cohnella was created with the description of two novel species:  C. thermotolerans, isolated from a starch-producing company in Sweden and C. hongkongensis, isolated as  “Paenibacillus hongkongensis” (this name has never been validly published) from a boy with neutropenic fever and pseudobacteremia. Later new species was assigned to the genus which has its description emended by García-Fraile et al. (2008) and Khianngam et al. (2010). Cohnella fontinalis was isolated from fresh water from a fountain (Shiratori et al., 2010).

Genus description from Kampfer  et al. (2006), García-Fraile et al. (2008) and Khianngam et al. (2010): Cells are motile or non-motile spore forming rods and stain Gram-positive or Gram negative. Aerobic or facultatively anaerobic. Most species are thermotolerant and good growth occurs after 24 h incubation on TSA and Nutrient agars at 25–30°C and also at 55°C. Some species grow at 10 or 60°C. Some species grow in the presence of 3% NaCl.

1.2.9   Desulfotomaculum Campbell and Postgate 1965

Nomenclature update from Euzéby (2012): The genus Desulfotomaculum was created to accommodate anaerobic spore forming bacteria capable to reduce sulphate producing hydrogen sulfide gas (H₂S).  Desulfotomaculum nigrificans, previously denominated Clostridium nigrificans is the species usually found in foods.

Genus description from Kuever and Rainey (2009): Cells are straight or curved rods occurring singly or in pairs, motile (motility can be lost during cultivation), with a Gram-positive cell wall but a variable Gram-staining reaction. Spores are oval or round, terminal to central and swell the sporangia. Catalase-negative, strict anaerobes with a respiratory type of metabolism. Chemoorganotrophic or chemoautotrophs, simple organic compounds are used as electron donor and carbon sources and are either completely oxidized to CO₂, or incompletely to acetate. Some species can grow on H₂ autotrophically with CO₂ as the sole carbon source. Sulfate, and usually sulfite and thiosulfate, serve as terminal electron acceptors and are reduced to H₂S. Sulfur and nitrate are not used as electron acceptors. Fermentative growth has been observed for some species. The optimum temperature range for growth is 30–37°C for mesophilic species and 50–65°C for thermophilic species. The pH range for growth is 5.5–8.9 and the optimum is 6.5–7.5.

 Desulfotomaculum nigrificans: According to Don-nelly & Hannah (2001), sugar and starch are the main sources of this microorganism in foods. In canned foods they cause sulfidric spoilage with darkening of the internal content, without swelling the packages. The darkening is generated by the reaction of H₂S with the iron of the cans. This is not a common occurrence, and takes place when the product remains for a prolonged period of time at temperatures higher than 43°C, allowing the surviving spores to germinate. It can be caused by slow cooling and/or storage at temperatures above 43°C (vending machines). Species description from Kuever and Rainey (2009): The spores are oval, subterminal, in swollen sporangia. Thermophile, the temperature range for growth is 45 to 70°C, with an optimum at 55°C. The spores are highly heat-resistant, with a D121.1ºC = 2 at 3 min (Stumbo, 1973).

1.2.10 Geobacillus Nazina et al. 2001

Nomenclature update from Euzéby (2012): This genus was proposed to reclassify the thermophilic species of Bacillus. G. stearothermophilus, previously denominated Bacillus stearothermophilus is the species usually found in foods but  G. kaustophilus, previously named  Bacillus kaustophilus, was first isolated from pasteurized milk and have been found in spoiled, canned food. G. tepidamans was isolated from sugar beet juice (Logan et al., 2009).

Genus description from Logan  et al. (2009): Cells are rod-shaped occurring singly or in short chains, with a Gram-positive cell wall, but a Gram staining reactions positive or negative. They can be motile or non-motile and form terminal or subterminal ellipsoidal or cylindrical spores in slightly swollen or non-swollen sporangium. Chemo-organotrophic, aerobic or facultative anaerobic, most species are catalase positive and produce acid but not gas from carbohydrates. Growth occurs between 35°C and 75°C, with an optimum at 55–65°C. Neutrophilic, the growth pH varies between 6.0 and 8.5 with an optimum between 6.2 and 7.5. Oxidase reaction varies; vitamins or growth factors are not required for growth.

Geobacillus stearothermophilus: The taxonomy of  G. stearothermophilus was reviewed by Logan  et al. (2009), who concluded that there is not a practically useful description for this species at present.  G stearothermophilus is recognized as a typical “flat sour” spoil-age agent in the canned food and dairy industries. Their spores are extremely heat resistant (D121,1ºC = 4–5 min according to Stumbo, 1973) and may survive in commercially sterile low-acid foods, although multiplication only occurs if the product is kept at temperatures higher than 37°C.  G. stearothemophilus may represent up to third of thermophiles isolates from foods and approaching two-thirds of the thermophiles in milk. However, the collection of strains gathered along the years before the creation of the genus Geobacillus (named  Bacillus stearothermophilus) was markedly heterogeneous and the species description given by the 8th Edition of  Bergey’s Manual of Determinative Bacteriology (Gibson and Gordon, 1974) recognized as  Bacillus stearothermophilus only the strictly thermophiles strains able to grow at 65°C. This restriction has the effect of excluding strains with maximum temperature between 55°C and 65°C, although they cannot be distinguished from the strictly thermophiles by any other property. The 1st Edition of Bergey’s Manual of Systematic Bacteriology (Claus and Berkeley, 1986) did not change the taxonomy of Bacillus stearothermophilus and when the species was transferred to the genus  Geobacillus, the description given by Nazina  et al. (2001) was almost the same given by Claus and Berkeley (1986). According to the description given by Claus and Berkeley (1986), the temperature range for growth is 37 to 70°C, with an optimum at 60–65°C and 90% or more of the strains are incapable to grow at pH 5.7. White et al. (1993), on the other hand, found that 88% of the strains were able to grow at pH 5.5. Some strains are strictly aerobes while others are facultative anaerobes. Nazina et al. (2001) reported 11 to 89% of the strains as facultative anaerobes, while White et al. (1993) found 99% of the strains facultative anaerobes. Claus and Berkeley (1986) reported catalase, oxidase and citrate tests either negative or positive, but White et al. (1993) found that 99% of the strains tested negative for these three characteristics.

1.2.11   Jeotgalibacillus Yoon et al. 2001 emend. Chen et al. 2010

Nomenclature update from Euzéby (2012): The genus Jeotgalibacillus was created by Yoon  et al. (2001) with the description of Jeotgalibacillus alimentarius sp. nov. as the sole recognized species of the genus. Later new species was assigned to the genus which has its description emended by Chen et al (2010).

Genus description from Yoon  et al. (2001) and Chen et al. (2010): Jeotgalibacilli are aerobic or facultative anaerobic. Cells are rod-shaped forming round or ellipsoidal endospores, central, subterminal or terminal, in swollen or unswollen sporangia. Catalase reaction is positive and oxidase may be positive or negative. They grow in the presence of 18–20% NaCl with an optimum NaCl concentration of 2–10%. Growth occurs at 5–10 to 40–50°C with an optimum at 25–30°C to 30–35°C. The pH for growth is 6–6.5 to 10–10.5, with an optimum at pH 7 to 8. Jeotgalibacillus alimentarius, a new species isolated from jeotgal (traditional Korean fermented seafood) was described by Yoon et al. (2001): Facultative anaerobic, cells are motile rods Gram-variable. Spores are round, subterminal or terminal, in swollen sporangia. Colonies on marine agar are smooth, glistening, irregular, and orange-yellow. They grow in presence of 19% NaCl and weakly in the presence of 20% NaCl. Growth occurs at 10 and 45°C, but not at 4 or 50°C. Optimum growth temperature is 30–35°C. Optimum pH for growth is pH 7–8 and no growth is observed at pH 6. Catalase and oxidase-positive. Acid is produced from glucose, galactose, fructose, sucrose, maltose and other sugars.

1.2.12   Lentibacillus Yoon et al. 2002 emend. Jeon et al. 2005

Nomenclature update from Euzéby (2012): The genus Lentibacillus was created with the description of Lentibacillus salicampi sp. nov. as the sole recognized species of the genus, isolated from fish sauce in Thailand. Later new species was assigned to the genus, some isolated from foods:  Lentibacillus halophilus was isolated from fish sauce, Lentibacillus kapialis from fermented shrimp paste and Lentibacillus jeotgali from jeotgal, traditional Korean fermented seafood.

Genus description from Heyrman and De Vos (2009a): Rod-shaped cells, forming terminal endospores that swell the sporangia. Gram variable, motile or non-motile. Colonies are white to cream-colored, smooth and circular to slightly irregular. Catalase positive, oxidase variable and urease negative. Moderately to extremely halophilic, generally show slow growth on media with low NaCl content and good growth on media with higher NaCl content. The temperature range for growth is 10–50°C.

1.2.13  Lysinibacillus Ahmed et al. 2007

Nomenclature update from Euzéby (2012): This genus was proposed to reclassify Bacillus species (B. fusiformis and  B. sphaericus) which have lysine and aspartate in the peptidoglycan of the cell wall and characteristically can grow in the presence of 60mM boron or more. Data collected by Scheldeman et al. (2006) report the involvement of  L. sphaericus in the contamination of milk sterilized by the conventional or UHT sterilization process.

Genus description from Ahmed et al. (2007): Cells are Gram positive motile rods producing ellipsoidal or spherical endospores in swollen sporangia. Oxidase and catalase tests are positive. The growth temperature range is 10 to 45°C. The growth pH range is 5.5 to 9.5. The species Lysinibacillus sphaericus was described in the 2nd edition of Bergey’s Manual of Systematic Bacteriology in the genus  Bacillus with its formerly name Bacillus sphaericus (Logan and De Vos, 2009b): Aerobic, cells are Gram positive, motile rods, forming spherical spores, terminal, in swollen sporangia. Colonies are opaque, unpigmented, smooth, often glossy and usually entire. Minimum growth temperature is 10–15°C and maximum is 30–45°. Grows at pH 7.0 to 9.5; some strains grow at pH 6.0. Catalase and oxidase positive. Grow in the presence of 5% NaCl but not in 7% NaCl. No acid or gas is produced from glucose or other common carbohydrates.

1.2.14 Moorella Collins et al. 1994

Nomenclature update from Euzéby (2012): The genus Moorela was proposed to reclassify the homoacetogenic species of  Clostridium (Clostridium thermoaceticum and  Clostridium thermoautotrophicum), which differ from other clostridia in their high DNA base compositions (approximately 53 to 55 mol% G+C) and in the presence of LL-diaminopimelic acid in their cell wall peptidoglycans.  Moorella species have been isolated mainly from hot springs, but also have been found in horse manure, sewage sludge, freshwater sediments and canned food samples. Carlier and Bedora-Faure (2006) isolated six strains from various spoiled cans including fish soups and cooked meats. Prevost et al. (2010) evaluated 34 canned products which had failed the stability test performed at 55°C and found  M. thermoacetica/thermoautotrophica in 14 samples including fish dump-ling, pre-cooked meal with meat, pre-cooked meal with chicken, cooked vegetables, green peas and carrots, green peas and mixed vegetables.

Genus description from Wiegel (2009): Cells are straight rods occurring singly, in pairs or in chains. Under stress conditions they show a tendency to polymorphism. Generally Gram positive, but older cultures may stain gram negative. Spores are round to slightly oval, terminal or subterminal, in swollen sporangia. Obligate anaerobic, thermophilic, chemolithoautotrophic and/or hetero-trophic, acetate is produced as sole or main fermentation product from sugars, C1 carbon sources and other substrates (homoacetogenic). The optimum temperature for growth is 56 to 60°C, the maximum is 65 to 68°C and the minimum is 40–47°C. Byrer et al. (2000) evaluated the spore resistant of two strains of  Moorella thermoacetica which were isolated from 0.1% (wt/vol) yeast-extract-containing media that had been autoclaved at 121°C for 45 min. The spores of the two strains required heat activation at 100°C of more than 2 min and up to 90 min for maximal percentage of germination. The D121ºC value varied between 23 and 111 min depending on sporulation conditions. The spores obtained at 60°C from the two strains grown chemoorganoheterotrophically had D_121ºC of 44 min and 38 min; spores obtained at 60°C from cells grown chemolithoautotrophically had D_121ºC of 83 min and 111 min. These spores are amongst the most heat-resistant noted to date.

1.2.15   Oceanobacillus Lu et al. 2002 emend. Lee et al. 2006

Nomenclature update from Euzéby (2012): The genus Oceanobacillus was created with the description of Oceanobacillus iheyensis sp. nov. as the sole recognized species of the genus, an extremely halotolerans and alkaliphilic bacteria isolated from deep-sea sediment. Later new species was assigned to the genus, some isolated from foods and food production environment: O. kapialis from fermented shrimp paste,  O. kimchii from kimchi (a Korean food produced from cabbage, radishes and cucumbers) and O. soja isolated from soy sauce production equipment.

Genus description from Heyrman and De Vos (2009b): Cells are motile Gram positive rods forming ellipsoidal subterminal or terminal endospores in swollen sporangia. Colonies on solid media are circular and white to beige. Aerobic or facultative anaerobic, catalase positive, oxidase variable, alkaliphilic, mesophilic. Halo-tolerant, the optimum NaCl concentration for growth is 3–10% (w/v) and growth occurs at up to 20%. The pH range for growth is 6.5–10.0 and the temperature range is 5–42°C.

1.2.16   Paenibacillus Ash et al. 1994 emend. Shida et al. 1997

Nomenclature update from Euzéby (2012): This genus was created to reclassify mesophilic species of  Bacillus which are typically capable of hydrolyzing complex carbohydrates (starch, pectin, carboxymethyl cellulose, chitin and others). The proposal to create the genus was made by Ash et al. (1993), but the name was only validated in1994. The species that have been reported as food spoilage microorganisms or food contaminants are  P. polymyxa, P. macerans and  P. lactis. Stevenson & Segner (2001) reported occasional loss of container vacuum or bulging of the container because of growth and gas production by P. macerans and  P. polymyxa in low acid canned foods. According to the authors spores of  P. macerans or  P. polymyxa strains commonly have D100ºC values from 0.1 to 0.5 minutes. Data collected by Scheldeman et al. (2006) showed the presence of  P. lactis in raw milk and dairy farm equipment, as well as involvement in cases of contamination of milk sterilized by the conventional or UHT process.

Genus description from Priest (2009): Cells are motile rods with a Gram positive cell wall but a variable or negative Gram stain reaction. Oval endospores are formed in swollen sporangia. Aerobic or facultative anaerobic, most species are catalase positive. Colonies are generally small, smooth and translucent, light brown, white, or some-times light pink or yellow in color.  P. alvei forms motile microcolonies which spread over agar media. Other species also form motile colonies. Optimum growth generally occurs at 28–40°C and pH 7.0 and is inhibited by 10% NaCl. The end products of carbohydrate utilization vary;  Paenibacillus macerans (previously named  Bacillus macerans) is anaerobic facultative and convert glucose initially to ethanol, acetic acid, and small amounts of formate. As the culture ages, the formate and acetate are catabolized to H₂, CO₂ and acetone, but the production of acid and gas are the characteristics noted in diagnostic tests.  Paenibacillus polymyxa (previously named Bacillus polymyxa) is also facultative anaerobic and produces 2,3-butanediol, ethanol, CO₂ and H₂ from carbohydrates.  P. lactis is strictly aerobic and produces acid but not gas from carbohydrates.

1.2.17   Sporolactobacillus Kitahara and Suzuki 1963

Nomenclature update from Yanagida and Suzuki (2009): The genus  Sporolactobacillus was first established as a subgenus of the genus  Lactobacillus, to accommodate a novel lactic acid bacteria (Sporolactobacillus inulinus) capable of spore formation. Later subgenus was elevated to the genus level and received new species and transferred species.

According to Stevenson and Segner (2001)  Sporolactobacillus does not have a great importance in food spoilage, since they form spores comparatively low resistant to heat and are apparently distributed in low numbers in food. Banks (1989) reported a D90ºC value of 4 to 7 min for  S. inulinus and summarized a few occurrences in products such as pickles, concentrated fruit juice, dairy products and fermented musts. Fujita et al. (2010) isolated the new species  S. putidus from spoiled orange juice.

Genus description from Yanagida and Suzuki (2009): Cells are Gram positive spore-forming straight rods occurring singly, in pairs and rarely in short chains, mostly motile. The spores are rarely observed and resist to heating at 80°C/10 min. Facultative anaerobic or microaerophilic, homofermentative, lactic acid is produced from glucose and a limited number of other carbohydrates. Carbohydrates are essential for growth; good growth occurs on media containing glucose, but poor or no growth occurs in Nutrient Broth (NB). Mesophilic, catalase and oxidase negative.

1.2.18   Thermoanaerobacter Wiegel and Ljungdahl 1982 emend. Lee et al. 2007

Nomenclature update from Euzéby (2012): The genus Thermoanaerobacter was created with the description of  Thermoanaerobacter ethanolicus sp. nov. as the sole recognized species of the genus. Later new species was assigned to the genus which has its description emended by Lee et al. (2007). Dotzauer et al. (2002) isolated  Thermoanaerobacter spp. from various spoiled canned food samples (meat/vegetables, food with meat and rice, tomato puree, noodles/vegetables, spinach, potatoes). The type strain of T. mathranii subsp.  alimentarius was isolated at 55°C from spoiled meat and   T. thermohydrosulfuricus have been isolated from many sources, including extraction juices of beet sugar factories (Onyenwoke and Wiegel, 2009a).

Genus description from Onyenwoke and Wiegel (2009a): Cells are rod with a Gram positive cell wall, but the Gram-stain reaction is variable. Most species are motile exhibiting a sluggish motility. Endospore formation has been observed except for  T. acetoethylicus, T. ethanolicus, T. kivui, T. matharanii subsp. alimentarius and T. sulfurophilus. However, a spore-specific gene has been demontrated for several species in which no spores have been observed. These species are regarded as “asporogenic”, to distinguishe them from non-sporogenic species (that lack sporulation genes). All species are obligatory anaerobic thermophiles. The optimum temperature is 55 to 75°C, with growth ranges of 35–78°C. The pH for growth ranges from 4.0 to 9.9 with an optimum of 5.8 to 8.5.

1.2.19   Thermoanaerobacterium Lee et al. 1993

Nomenclature update from Euzéby (2012): This genus was created to reclassify thermophilic and saccharolytic species of Clostridium, which produces thermostable saccharolytic enzymes of interest for industrial applications.  According to Onyenwoke and Wiegel (2009b) the known habitat of these bacteria are geothermal environ-ments but they have also been found in association with fruit juice waste products and tartrate infusion of grape residues. One species is commonly found in foods, T. thermosaccharolyticum and Dotzauer  et al. (2002), investigating the loss of vacuum in several canned foods, also found  T. saccharolyticum,  T. thermosulfurigenes and Thermoanaerobacterium spp. in low-acid products and in tomato puree.

Genus description from Onyenwoke and Wiegel (2009b): Cells are motile rods with a Gram positive cell wall, but many strains stain Gram negative. Endospores are present in some species and others have sporulation genes but do not sporulate (asporulating species). Obligate anaerobes, catalase negative, extreme thermophiles with optimum growth temperature between 55 and 70°C. The temperature range for growth is 35 to 75°C and the pH range is 3.2 to 8.5. The lowest pH optimum is 5.2 (for T. aotearoense) and the lowest pH minimum is 3.2 (for  T. aciditolerans). Chemoorganotrophs, the most common end products of glucose fermentation are acetic acid, ethanol, lactic acid, H₂ and CO₂.

 Thermoanaerobacterium thermosaccharolyticum: This species (formerly Clostridium thermosaccharolyticum) has been called “the swelling can food spoiler” (Onyenwoke and Wiegel (2009b) because of the spoil-age of thermally processed foods with gas formation. According to Ashton and Bernard (2001) the spores of this species exhibit a great heat resistance (D_121.1ºC = 3 to 4 min according to Stumbo, 1973) and their survival in canned foods is not unexpected. Spoilage occurs when the finished product is improperly cooled or is held for extended periods at elevated temperatures favorable to strictly thermophilic bacteria. Cans contamination by leakage may also occur if the microorganism grows and accumulates in the cooling area of hydrostatic cookers. Ingredients such as sugar, dehydrated milk, starch, flour, cereals, and alimentary pastes have been found to be the predominant sources of T. thermosaccharolyticum, which occurs widely in soil and therefore is found on raw materials that have contact with the soil. Species description from Onyenwoke and Wiegel (2009b): The spores are round or oval, terminal in swollen sporangia, sometimes with elongation of the mother cell. The pH optimum for sporulation is 5.0–5.5 and the cells do not sporulate in medium containing glucose. No growth occurs in the absence of a fermentable carbohydrate. The optimum temperature for growth is 55–62°C, with some growth at 37°C and poor if any growth at 30°C. Growth also occurs at 69°C but not at 70°C. The pH range for growth is 6.5–8.5 with an optimum at 7.8.

1.2.20   Virgibacillus Heyndrickx et al. 1998 emend. Wainø et al. 1999 emend. Heyrman et al. 2003

Nomenclature update from Euzéby (2012): This genus was proposed to reclassify strains previously classified as Bacillus pantothenticus, which depend on pantothenic acid, thiamine and biotin for growth. A survey conducted by Scheldeman  et al. (2005) detected the presence of species of this genus in raw milk, and on equipment and in the environment of dairy farms.  V. pantothenticus was originally isolated from soil but has also been found in canned chicken; V. proomii has been isolated from soil, infant bile and a water supply (Heyrman  et al., 2009). Tanasupawat et al. (2010) isolated V. dokdonensis, V. halodenitrificans, V. marismortui, V. siamensis and Virgibacillus sp. from a fermented fish (plara) in Thailand. Kim et al. (2011) isolated V. alimentarius from traditional salt-fermented seafood in Korea.

Genus description from Heyrman  et al. (2009): Cells are motile Gram positive rods occurring singly, in pairs, in chains or, especially in older cultures, in filaments. The endospores are spherical to ellipsoidal, terminal (sometimes subterminal or paracentral) and swell the sporangia. Some species are strictly aerobic and others are weakly facultative anaerobic. Catalase positive, salt-tolerant, the growth is stimulated by 4–10% NaCl. Several species will tolerate 20–25% NaCl concentrations and some species do not grow or grow poorly in the absence of salt. Growth may occur between 10°C and 50°C, with an optimum of 28°C or 37°C.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .

Ahmed, I., Yokota, A., Yamazoe, A. & Fujiwara, T. (2007) Proposal of  Lysinibacillus boronitolerans gen. nov., sp. nov., and transfer of  Bacillus fusiformis to  Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to  Lysinibacillus sphaericus comb. nov.  Inter-national Journal of Systematic and Evolutionary Microbiology, 57, 1117–1125.

Ash, C., Priest, F.G. & Collins, M.D. (1993) Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Antonie van Leeuwenhoek, 64, 253–260.

Ashton, D. & Bernard, D.T. (2001) Thermophilic anaerobic sporeformers. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 26, pp. 249–252.

Banat, I. M., Marchant, R. & Rahman, T. J. (2004)  Geobacillus debilis sp. nov., a novel obligately thermophilic bacterium isolated from a cool soil environment, and reassignment of Bacillus pallidus to Geobacillus pallidus comb. nov. International Journal of Systematic and Evolutionary Microbiology, 54:2197–2201.

Banks, J.G. (1989)  The spoilage potencial of Sporolactobacillus –Technical Bulletin N° 66. England, Campden Food and Drink Research Association.

Broda, D.M., Lawson, P.A., Bell, R.G. & Musgrave, D.R. (1999) Clostridium frigidicarnis sp. nov., a psychrotolerant bacterium associated with “blown pack” spoilage of vacuum-packed meats. International Journal of Systematic Bacteriology, 49, 1539–1550.

Broda, D.M., Saul, D.J., Lawson, P.A., Bell, R.G. & Musgrave, D.R. (2000) Clostridium gasigenes  sp. nov., a psychrophile caus-ing spoilage of vacuum-packed meats.  International Journal of Systematic and Evolutionary Microbiology, 50, 107–118.

Byrer, D.E., Rainey, F.A & Wiegel, J. (2000) Novel strains of Moorella thermoacetica form unusually heat-resistant spores. Archives of Microbiology, 174, 334–339.

Carlier, J.P. & Bedora-Faure, M. (2006) Phenotypic and genotypic characterization of some Moorella sp. strains isolated from canned foods. Systematic and Applied Microbiology, 29, 581–588.

Chen, Y.G., Peng, D.J., Chen, Q.H., Zhang, Y.Q., Tang, S.K., Zhang, D.C., Peng, Q.Z. & Li, W.J. (2010)  Jeotgalibacillus soli sp. nov., isolated from non-saline forest soil, and emended description of the genus  Jeotgalibacillus.  Antonie Van Leeuwen-hoek, 98, 415–421.

Claus, D. & Berkeley, R.C.W. (1986) Genus Bacillus Cohn 1872. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E. & Holt, J.G. (eds). Bergey’s Manual of Systematic Bacteriology. 1st edition, Volume 2. Baltimore, Williams & Wilkins. pp. 1105–1139.

Collins, M.D., Rodrigues, U.M., Dainty, R.H., Edwards, R.A. & Roberts, T.A. (1992). Taxonomic studies on a psychrophilic Clostridium from vacuum-packed beef: description of Clostridium estertheticum sp. nov. FEMS Microbiology Letters, 96, 235–240.

Costa, M.S., Rainey, F.A. & Albuquerque, L. (2009) Genus Alicyclobacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 229–243.

Dainty, R.H., Edwards, R.A. & Hibbard, C.M. (1989) Spoilage of vacuum-packed beef by a Clostridium sp. Journal of the Science of Food and Agriculture, 49, 473–486.

De Clerck, E., Rodríguez-Díaz, M., Forsyth, G., Lebbe, L., Logan, N. & De Vos, P. (2004) Polyphasic characterization of  Bacillus coagulans strains, illustrating heterogeneity within this species, and emended description of the species.  Systematic and Applied Microbiology, 27, 50–60.

Donnelly, L.S. & Hannah, T. (2001) Sulfide spoilage sporeformers. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 27, pp. 253–255.

Dotzauer, C., Ehrmann, M.A. & Vogel, R.F. (2002) Occurrence and detection of  Thermoanaerobacterium and  Thermoanaerobcter in canned food. Food Technology and Biotechnology, 40(1), 21–26.

Driks, A. (2004) The  Bacillus spore coat.  Phytopathology, 94, pp. 1249–1251.

Euzéby J.P. (2012)  List of Prokaryotic Names with Standing in Nomenclature. [Online] Available from: http://www.bacterio.cict. fr/ [Accessed 20th January 2012].

Evancho, G. M. & Walls, I. (2001) Aciduric Flat Sour Sporeformers. InDownes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 24, pp. 239–244.

FDA/CFSAN (ed.) (2009)  Foodborne Pathogenic Microorganisms and Natural Toxins Handbook “Bad Bug Book”. [Online] Co-lege Park, Food and Drug Administration, Center for Food Safety & Applied Nutrition. Available from: http://www.fda.gov/food/foodsafety/foodborneillness/foodborneillnessfoodbornepa-thogensnaturaltoxins/badbugbook/default.htm [Accessed 10th October 2011].

FSIS/USDA (1997) Generic HACCP Model for Thermally Proc-essed Commercially Sterile Meat and Poultry Products. Food Safety Inspection Service, United States Department of Agri-culture. Available from:  http://haccpalliance.org/alliance/haccp-models/thermal.pdf [Accessed 10th May 2012].

Frank, J.F. & Yousef, A.E. (2004) Tests for groups of microrganisms. In: Wehr, H.M. & Frank, J.F (eds).  Standard Methods for the Examination of Dairy Products. 17th edition. Washington, Ameri-can Public Health Association. Chapter 8, pp. 227–248.

Fujita, R., Mochida, K., Kato, Y. & Goto, K. (2010) Sporolactobacillus putidus sp. nov., an endospore-forming lactic acid bacterium isolated from spoiled orange juice.  International Journal of Systematic and Evolutionary Microbiology, 60, 1499–1503.

García-Fraile, P., Velázquez, E., Mateos, P.F., Martínez-Molina, E. & Rivas, R. (2008) Cohnella phaseoli sp. nov., isolated from root nodules of Phaseolus coccineus in Spain, and emended description of the genus Cohnella. International Journal of Systematic and Evolutionary Microbiology, 58, 1855–1859.

Gibson, T. & Gordon, R.E. (1974) Bacillus Cohn 1872. In: Bucha-nan, R.E. & Gibbons, N.E. (eds). Bergey’s Manual of Determinative Bacteriology. 8th edition. Baltimore, Eilliams & Wilkins. pp. 529–550.

Goto, K., Mochida, K., Asahara, M., Suzuki, M., Kasai, H. & Yokota, A. (2003)  Alicyclobacillus pomorum   sp. nov., a novel thermo-acidophilic, endospore-forming bacterium that does nor possess  ω-alicyclic fatty acids, and emended description of the genus Alicyclobacillus. International Journal of Systematic and Evolutionary Microbiology, 53, 1537–1544.

Gouws, P.A., Gie, L., Pretorius, A. & Dhansay, N. (2005) Isolation and identification of Alicyclobacillus acidocaldarius  by 16S rDNA from mango juice and concentrate. International Journal of Food Science & Technology, 40(7), 789–792.

Heyndrickx, M., Coorevits, A., Scheldeman, P., Lebbe, L., Schu-mann, P., Rodríguez-Diaz, M., Forsyth, G., Dinsdale, A., Heyr-man, J., Logan, N.A & De Vos, P. (2011) Emended descriptions of  Bacillus sporothermodurans and  Bacillus oleronius with the inclusion of dairy farm isolates of both species. International Journal of Systematic and Evolutionary Microbiology, Published online ahead of print March 11, 2011, doi: 10.1099/ijs.0.026740–0.

Heyrman, J. & De Vos, P. (2009a) Genus Lentibacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 175–178.

Heyrman, J. & De Vos, P. (2009b) Genus Oceanibacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 181–184.

Heyrman, J., De Vos, P. & Logan, N. (2009) Genus Virgibacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds).  Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 193–228.

Hilbert, D.W. & Piggot, P.J. (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiology and Molecular Biology Reviews, 68(2), pp. 234–262.

Hunt, M.E. & Rice, E.W. (2005) Microbiological examination. In: Eaton, A.D., Clesceri, L.S., Rice, E.W. & Greenberg, A.E. (eds). Standard Methods for the Examination of Water & Wastewater. 21st edition. Washington, American Public Health Association (APHA), American Water Works Association (AWWA) & Water Environment Federation (WEF). Part 9000, pp. 9.1–9.169.

ICMSF (International Commission on Microbiological Specifica-tions for Foods) (ed) (1996) Microrganisms in Foods 5 – Microbiological Specifications of Food Pathogens. London, Blackie Academic & Professional.

International Federation of Fruit Juice Producers (2007) IFU 12:2007. Method on the detection of taint producing Alicyclobacillus in fruit juices. Paris, France.

Kalchayanand, N. Ray, B., Field, R.A. & Johnson, M.C. (1989) Spoilage of vacuum-packaged refrigerated beef by  Clostridium. Journal of Food Protection, 54, 424–426.

Kalchayanand, N., Ray, B., Field, R.A. & Johnson, M.C. (1993) Characteristics of psychrotrophic  Clostridium laramie causing spoilage of vacuum-packaged refrigerated fresh and roasted beef. Journal of Food Protection, 56, 13–17.

Kalogridou-Vassiliadou, D. (1992) Biochemical activities of Bacillus species isolated from flat sour evaporated milk. Journal of Dairy Science, 75, 2681–2686.

Kämpfer, P., Rosselló-Mora, R., Falsen, E., Busse, H.J. & Tindall, B.J. (2006) Cohnella thermotolerans gen. nov., sp. nov., and clas-sification of ‘Paenibacillus hongkongensis’ as Cohnella hongkongen-sis sp. nov.  International Journal of Systematic and Evolutionary Microbiology, 56, 781–786.

Karavaiko, G.I., Bogdanova, T.I., Tourova, T.P., Kondrateva, T.F., Tsaplina, I.A., Egorova, M.A., Krasilnikova, E.N. & Zakhar-chuk, L.M. (2005) Reclassification of ‘Sulfobacillus thermosulfidooxidans   subsp.  thermotolerans’ strain K1 as  Alicyclobacillus tolerans   sp. nov. and  Sulfobacillus disulfidooxidans   Dufresne et al. 1996 as Alicyclobacillus disulfidooxidans  comb. nov., and emended description of the genus  Alicyclobacillus.  Interna-tional Journal of Systematic and Evolutionary Microbiology, 55, 941–947.

Khianngam, S., Tanasupawat, S., Akaracharanya, A., Kim, K.K., Lee, K.C. & Lee, J.S. (2010)  Cohnella thailandensis sp. nov., a xylanolytic bacterium from Thai soil. International Journal of Systematic and Evolutionary Microbiology, 60, 2284–2287.

Kim, J., Jung, M.J., Roh, S.W., Nam, Y.D., Shin, K.S. & Bae, J.W. (2011) Virgibacillus alimentarius sp. nov., isolated from a traditional Korean food. International Journal of Systematic and Evolutionary Microbiology, 61, 2851–2855.

Kuever, J. & Rainey, F.A. (2009) Genus  Desulfotomaculum. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Man-ual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 989–996.

Lawson, P., Dainty, R.H., Kristiansen, N., Berg, J. & Collins, M.D. (1994) Characterization of a psychrotrophic  Clostridium causing spoilage in vacuum-packed cooked pork: description of Clostridium algidicarnis sp. nov.  Letters in Applied Microbiology, 19, 153–157.

Lee, Y.J., Dashti, M., Prange, A., Rainey, F.A., Rohde, M., Whit-man, W.B. & Wiegel, J. (2007) Thermoanaerobacter sulfurigignens sp. nov., an anaerobic thermophilic bacterium that reduces 1M thiosulfate to elemental sulfur and tolerates 90mM sulfite. International Journal of Systematic and Evolutionary Microbiology, 57, 1429–1434.

Logan, N.A. & De Vos, P. (2009a) Genus  Aneurinibacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 298–305.

Logan, N.A. & De Vos, P. (2009b) Genus Bacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds).  Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 21–128.

Logan, N.A. & De Vos, P. (2009c) Genus Brevibacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds).  Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 305–316.

Logan, N.A. & De Vos, P. & Dinsdale, A. (2009) Genus Geobacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds).  Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 144–160.

Miñana-Galbis, D., Pinzón, D.L., Lorén, J.G., Manresa, A. & OliartRos, R.M. (2010) Reclassification of  Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov.  International Journal of Systematic and Evolutionary Microbiology, 60, 1600–1604.

Nazina, T.N., Tourova, T.P., Poltaraus, A.B., Novikova, E.V., Grigoryan, A.A., Ivanova, A.E., Lysenko, A.M., Petrunyaka, V.V., Osipov, G.A., Belyaev, S.S. & Ivanov, M.V. (2001). Taxonomic study of aerobic thermophilic bacilli: descriptions of  Geobacillus subterraneus gen. nov., sp. nov. and  Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus,  Bacillus thermocatenulatus,  Bacillus thermoleovorans, Bacillus kaustophilus,  Bacillus thermoglucosidasius and  Bacillus thermodenitrificans to  Geobacillus as the new combinations G.  stearothermophilus,  G.  thermocatenulatus,  G.  thermoleovorans, G.  kaustophilus,  G.  thermoglucosidasius  and  G.  thermodenitrificans. International Journal of Systematic and Evolutionary Micro-biology, 51, 433–446.

Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J. & Setlow, P. (2000) Resistance of  Bacillus endospores to extreme terrestrial and extraterrestrial environments.  Microbiology and Molecular Biology Reviews, 64(3), pp. 548–572.

Olson, K.E. & Sorrells, K.M. (2001) Thermophilic flat sour spore-formers. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 25, pp. 245–248.

0nyenwoke, R.U. & Wiegel, J. (2009a) Genus Thermoanaerobacter. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds).  Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 1225–1239.

Onyenwoke, R.U. & Wiegel, J. (2009b) Genus Thermoanaerobac-terium. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 1279–1287.

Pettersson, B., Lembke, F., Hammer, P., Stackbrandt, E. & Priest, F.G. (1996) Bacillus sporothermodurans, a new species producing highly heat resistant endospores. International Journal of Systematic Bacteriology, 46, 759–764.

Pikuta, E.V. (2009) Genus  Anoxybacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteri-ology. 2nd edition, Volume 3. New York, Springer. pp. 134–141.

Prevost, S., Andre, S. & Remize, F. (2010) PCR Detection of Thermophilic Spore-Forming Bacteria Involved in Canned Food Spoilage. Current Microbiology, 61, 525–533.

Priest, F.G. (2009) Genus Paenibacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 269–295.

Rainey, F.A., Hollen. B.J. & Small, A. (2009) Genus  Clostridium. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds).  Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 738–828.

Richmond, B. & Fields, M.L. (1966) Distribution of thermophilic aerobic sporeforming bacteria in food ingredients. Applied Microbiology, 14(4), 623–626.

Scheldeman, P., Pil, A., Herman, L., De Vos, P. & Heyndrickx, M. (2005) Incidence and diversity of potentially highly heat-resist-ant spores isolated at dairy farmers.  Applied and Environmental Microbiology, 71(3), 1480–1494.

Scheldeman, P., Herman, L., Foster, S., Heyndrickx, M. (2006) Bacillus sporothermodurans and other highly heat-resistant spore formers in milk. Journal of Applied Microbiology, 101, 542–555.

Scholz, T., Demharter, W., Hensel, R. & Kandler, O. (1987) Bacillus pallidus sp. nov., a new thermophilic species from sewage. Systematic and Applied Microbiology, 9, 91–96.

Scott, V.N., Anderson, J.E. & Wang, G. (2001) Mesophilic anaerobic sporeformers. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 23, pp. 229–237.

Shiratori, H., Tagami, Y., Beppu, T. & Ueda, K. (2010)  Cohnella fontinalis sp. nov., a xylanolytic bacterium isolated from fresh water. International Journal of Systematic and Evolutionary Micro-biology, 60, 1344–1348.

Spring, S., Merkhoffer, B., Weiss, N., Kroppenstedt, R.M., Hippe, H. & Stackebrandt, E. (2003) Characterization of novel psy-chrophilic clostridia from an Antarctic microbial mat: descrip-tion of  Clostridium frigoris  sp. nov.,  Clostridium lacusfryxellense  sp. nov.,  Clostridium bowmanii  sp. nov. and  Clostridium psychrophilum  sp. nov. and reclassification of  Clostridium larami-ense  as Clostridium estertheticum  subsp. laramiense  subsp. nov. International Journal of Systematic and Evolutionary Microbiology, 53, 1019–1029.

Stevenson, K.E. & Segner, W.P. (2001) Mesophilic aerobic sporeformers. In: Downes, F.P. & Ito, K. (eds). Compendium of Methods for the Microbiological Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 22, pp. 223–227.

Stumbo, C.R. (1973) Thermobacteriology in Food Processing. 2nd edi-tion. New York, Academic Press. Tanasupawat, S., Chamroensaksri, N., Kudo, T. & Itoh, T. (2010) Identification of moderately halophilic bacteria from Thai fer-mented fish (pla-ra) and proposal of Virgibacillus siamensis sp. nov. Journal of General and Applied Microbiology, 56(5), 369–379.

White, D., Sharp, R.J. & Priest, F.G. (1993) A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area. Antonie van Leuwenhoec, 64, pp. 357–386.

Wiegel, J. (2009) Genus  Moorella. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 1247–1256.

Wisotzkey, J.D., Jurtshuk Jr., P., Fox, G.E., Deinhard, G. & Poralla, K. (1992). Comparative sequence analyses on the 16S rRNA (rDNA) of  Bacillus acidocaldarius,  Bacillus acidoterrestris, and Bacillus cycloheptanicus  and proposal for creation of a new genus, Alicyclobacillus  gen. nov. International Journal of Systematic Bac-teriology, 42, 263–269.

Yanagida, F. & Suzuki, K. (2009) Genus  Sporolactobacillus. In: DeVos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A. Schleifer, K. & Whitman, W.B. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd edition, Volume 3. New York, Springer. pp. 386–391.

Yoon, J.H., Weiss, N., Lee, K.C., Lee, I.S., Kang, K.H. & Park, Y.H. (2001)  Jeotgalibacillus alimentarius gen. nov., sp. nov., a novel bacterium isolated from jeotgal with L-lysine in the cell wall, and reclassification of Bacillus marinus Rüger 1983 as Marinibacillus marinus gen. nov., comb. nov. International Journal of Systematic and Evolutionary Microbiology, 51, 2087–2093.