Alicyclobacillus On Bat Agar

7.1 Introduction 160

7.2 Taxonomic History 160

7.3 Physiological and Phenotypic Characteristics 164

7.3.1 Distinguishing Features 164

7.3.2 Thermoacidophilic Growth 164

7.3.3 Alicyclic Fatty Acids in Membrane 165

7.4 Thermal Resistance Characteristics 165

7.4.2 Factors Affecting Thermal Resistance 166

7.4.3 Other Control Measures 171

7.5 Industrial Importance 172

7.5.1 Emergence as Spoilage Organisms 172

7.5.2 Types of Spoilage 172

7.5.3 Sanitation 173

7.6 Detection and Identification 174

7.6.1 Controversy 174

7.6.3 Heat Shock Conditions 177

7.6.4 Enumeration 177

7.6.5 Detection by Enrichment 178

7.6.6 Identification and Confirmation 178

7.7 Significance of Detection/Isolation from Foods 179

7.8 Future Direction 179

Acknowledgment 180

References 180


The versatility and diversity of the microbial world often lead to unique and valuable discoveries that expand our knowledge and yield advancements for humankind. Antibiotics, fermented foods, health and beauty aids, and other commonly utilized items in our everyday lives were discovered or produced based upon unusual physiological and phenotypical characteristics of microorganisms. In the past five decades the study of microbial extremophiles in geothermal sites with high temperatures and high acidity has advanced our understanding of spore-forming bacteria and led to the establishment of the new genera alicyclobacillus and sulfobacillus.

Until the mid-1980s, the presence of bacterial spore-formers in low pH foods was thought to be insignificant. The reigning dogma of the time declared that Gram-positive, sporogenous bacteria could not outgrow to any great extent at pH levels below 4.5. Therefore, the first report of spoilage in shelf-stable, low pH fruit juices by Gram-positive, spore-forming bacteria [1] was met with some skepticism. However, by the mid-1990s spoilage of acidic juice products by members of the recently named genus alicyclobacillus [2] was well established and the impact of this situation began to clarify. Fruit juice and juice-containing beverages, bottled tea, isotonic drinks, and other low pH, shelf-stable products were at risk of spoilage by a widespread thermotolerant-to-thermophilic organism that could survive pasteurization and hot-fill treatments, and was, surprisingly, acidophilic in nature. At present, roughly 20 years after the initial reports of spoilage in fruit juice, concerns by food processors about these thermoacidophilic, spore-forming bacteria remain strong, economic losses continue, and effective commercial protocols to address the situation are limited.


Ecological studies of extreme environments, such as geothermal hot springs, during the latter half of the 20th century amplified scientific awareness of unique, spore-forming, acidophilic bacteria with the ability to survive and reproduce at high temperatures (40 to 100°C). This awareness, coupled with characterization studies of various isolates, led to the discovery of one such group, now recognized as the genus alicyclobacillus. The alicyclobacilli have optimal growth conditions in warm to hot, acidic, low-nutrient environments and have been isolated from a variety of sources. These bacteria are rod-shaped, approximately 2 to 4 ^ in length and < 1 ^ in width. Cells produce swollen, terminal to subterminal sporangia with refractile endospores (Figure 7.1) that are significantly heat resistant and capable of surviving typical pasteurization and thermal concentration conditions of juice/beverage manufacturing. On agar, colonies are usually a white to cream color, slightly raised, with smooth to irregular margins (Figure 7.2). Older, larger colonies take on a translucent character and may have slightly raised edges.

FIGURE 7.1 (Color insert follows page 594) Gram stain of Alicyclobacillus acidoterres-tris. Note swollen sporangia at arrows. (Magnification x1000.)
Alicyclobacillus Acidocaldarius Agar
FIGURE 7.2 (Color insert follows page 594) Colonies of Alicyclobacillus acidoterrestris ATCC 49025 on Ali agar after 24 hours at 45°C. Key characteristics: white/cream color, smooth to irregular edges. Older, larger colonies may develop translucent quality with slightly raised margins.

In 1967 Uchino and Doi [3] reported the isolation of thermoacidophilic bacteria from geothermal hot springs in Japan. Similar organisms were subsequently isolated from other geographically distinct geothermal sources [4,5]. Brock and Darland [4] isolated thermophilic bacteria in about 300 hot springs of various pH levels in the western U.S., New Zealand, Japan, and Iceland. Microbial populations were found in "virtually every spring in the neutral and alkaline pH range'' despite extreme temperatures up to 100°C. Bacterial isolates in acidic hot springs were temperature dependent and were not apparent at 90°C in springs with pH < 4.0 or at 70°C in springs with pH < 2.0. Darland and Brock [5] named the species Bacillus acido-caldarius to represent the unusual thermoacidophilic nature of these bacteria. Hippchen et al. [6] isolated thermoacidophilic bacilli from common soil samples, and Cerny et al. [1] reported the first account of a thermoaci-dophile, later identified as B. acidoterrestris, isolated from spoiled fruit juice. De Lucca et al. [7] reported the first isolation of B. acidocaldarius from another agricultural source, sugar refineries. Other research has established the presence of these organisms in high-fructose corn syrup (HFCS) commonly used as a sweetener in beverages (R. Worobo, personal communication).

De Rosa et al. [8,9] reported the presence of several forms of ffl-cyclohexane fatty acids in the membranes of B. acidocaldarius. Poralla et al. [10] suggested a "cholesterol-like" function for hopanoids in the membranes of B. acidocaldarius. Poralla and Konig [11] isolated strains with ffl-cycloheptane fatty acids that were later designated B. cycloheptanicus by Deinhard et al. [12]. Deinhard et al. [13] also reported on the new species, B. acidoterrestris, the thermoacidophile reportedly most involved in fruit juice spoilage.

Wisotzkey et al. [2] suggested reclassification of B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus, as species of alicyclobacillus based upon their thermoacidophilic phenotype and alicyclic fatty acids in the cellular membranes. In a review of classification schemes for endospore-forming bacteria, Berkeley and Ali [14] reported high homology (98.8%) between DNA from A. acidocaldarius and A. acidoterrestris and suggested that these might belong to one species rather than two. At the present time, the species remain separate due largely to their differences in optimum growth temperature and range.

Species of alicyclobacillus (Table 7.1) have been identified in a variety of samples from six continents, including Antarctica [15]. Differentiation of isolated strains from known species has been based largely upon genotypic and phenotypic characterization with phylogenetic and chemotaxonomic analyses. Albuquerque et al. [16] isolated thermoacidophilic strains from volcanic soil in the Azores Islands and subsequently designated one as A. hesperidum. Matsubara et al. [17] isolated a new species, A. acidiphilus, from acidic beverages based upon phylogenetic analysis of the 16S rRNA gene sequence and phenotypic differences related to spore morphology, growth temperatures, and acid production from carbon sources.

Goto et al. [18] isolated the species, A. herbarius, from hibiscus-flavored herbal tea. This strain contained ffl-cycloheptane fatty acid as the major membrane lipid component and could be distinguished from other species by phylogenetic analysis of the 16S rDNA sequence. In 2003 Goto et al. [19] isolated a new species from fruit juice and named it A. pomorum. This novel species does not contain alicyclic fatty acids but clusters among the alicyclobacilli based upon phylogenetic analysis of the 16S rDNA sequence with a level of similarity between 92.5 and 95.5%. Tsuruoka et al. [20] isolated a collagenase positive strain that was closely related to species of the alicyclobacilli based on 16S rDNA sequence analysis but had less than 33%

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