Bacillus sporothermodurans, a New Species Producing Highly Heat-Resistant Endospores

Abstract

Bacteria that differentiate into highly heat-resistant endospores (HHRS strains) may survive ultrahightemperature treatment of milk and germinate in the final product. They do not noticeably spoil the milk and are nonpathogenic. The complete (>96%) 16s rRNA genes from three HHRS strains were identical, and phylogenetic analysis placed them alongside Bacillus firmus in the B. meguterium group of the genus Bacillus. Moreover, the approximately 550 nucleotides between regions U2 and U5 were invariant for seven HHRS strains. However, three cloned 16s rRNA genes from one HHRS strain, M215, showed marked size and sequence variations within the V1 and V2 regions. DNA reassociation assays confirmed the distinction between a reference HHRS strain and closely related members of the B. meguterium group, notably, B.firmus (30%), B. benzoevorans (28%), and B. circuhns (20%). Ribotyping and pyrolysis mass spectrometry both indicated that the HHRS strains belong to a homogeneous, species-ranked taxon, an exception being strain TP1248, which is slightly atypical. The HHRS strains are unusual in that they grow poorly, if at all, on nutrient agar; good growth is obtained on brain heart infusion agar. On subculture, most HHRS strains form long, filamentous rods which stain unevenly in the Gram reaction. They are strictly aerobic and do not produce acid from sugars. We propose the name Bacillus sporothermoduruns for these bacteria, which are phenotypically and phylogenetically distinct from other Bacillus species. The type strain is M215 (= DSMZ 10599).High-temperature processing of milk takes several forms, including autoclaving to produce sterilized milk and ultrahightemperature (UHT) treatment to provide a product which can be stored without refrigeration for prolonged periods, generally, several months. The typical heating regimen for autoclaving is 115 to 120°C for 15 to 20 min or 109 to 115°C for 20 to 40 rnin, and for UHT treatment the range is 135 to 142°C for a few seconds, which should result in the destruction of any vegetative cells and endospores present in the raw material. Occasionally, spoilage can occur in sterilized and UHT-treated milk, usually as a result of contamination during filling operations. Members of the genus Bacillus, notably, Bacillus badius, B. cereus, B. licheniformis, B. polymyxa, B. subtilis, and B. stearothermophilus, have been identified in such situations (10, 23, 36).In certain circumstances, exceptionally heat-resistant endospores survive the UHT or autoclaving treatment and pass into the final product. These endospores may subsequently germinate and grow in the stored milk (8, 15). These mesophilic bacteria, which differentiate into highly heat-resistant spores (HHRS), were detected first in UHT-treated milk from southern Europe in 1985 and in UHT-treated milk from Germany in 1990. Today, the problem is more widespread and has been noted in several other European countries, including the Benelux countries, France, and Spain, as well as in some nonEuropean dairies (15). HHRS bacteria multipl...

Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov.

“…On the basis of phylogenetic evidence and supported by the availability of taxonomically salient characteristics, the genus Bacillus has been dissected into novel genera (Shida et al, 1996 ;Heyndrickx et al, 1998 ;Touzel et al, 2000 ;Fortina et al, 2001), new species have been described (Combet-Blanc et al, 1995 ;Pettersson et al, 1996 ;Heyndrickx et al, 1999) and invalid species have been validated (Sunna et al, 1997 ;Manachini et al, 2000). Only recently, Ahmad et al (2000) described a new species of Saccharococcus, Saccharococcus caldoxylosilyticus.…”

Ureibacillus gen. nov., a new genus to accommodate Bacillus thermosphaericus (Andersson et al. 1995), emendation of Ureibacillus thermosphaericus and description of Ureibacillus terrenus sp. nov.

“…Indeed, in recent years there has been a proliferation of new species described (Manachini et al, 1985 ;Demharter & Hensel, 1989 ;Combet-Blanc et al, 1995 ;Meier-Stauffer et al, 1996 ;Pettersson et al, 1996) and, through more detailed phylogenetic and chemosystematic studies, the proposal of new genera (Wisotzkey et al, 1992 ;Shida et al, 1996 ;Heyndrickx et al, 1998, Wainø et al, 1999. In this framework Bacillus thermosphaericus represents a thermophilic Bacillus species which is not closely related to members of any of the newly described genera that accommodate former Bacillus species.…”

Bacillus thermodenitrificans sp. nov., nom. rev.

“…Following extensive phylogenetic analyses, the genus Bacillus has undergone major taxonomic revisions, leading to the establishment of novel genera (Wisotzkey et al, 1992 ;Shida et al, 1996 ;Heyndrickx et al, 1998) and the recognition of new affiliations at the level of genera (Ash et al, 1991 ;Rheims et al, 1999) and species (Sunna et al, 1997), while at the same time the list of species, including thermophilic representatives, has been extended (Manachini et al, 1985 ;Demharter & Hensel, 1989 ;Andersson et al, 1995 ;Combet-Blanc et al, 1995 ;Meier-Stauffer et al, 1996 ;Pettersson et al, 1996). Despite the progress in rapidly recognizing close degrees of strain relatedness through analysis of 16S rDNA similarities, species delineation requires additional evidence.…”