Bacillus Cereus Sensu Lato Research Articles

Spores and vegetative cells of phenotypically and genetically diverse Bacillus cereus sensu lato are common bacteria in fresh water of northeastern Poland.

Gram-positive rods Bacillus cereus sensu lato (sl) are common in natural habitats and food products. It is believed that they are restricted to spores; however, their ecology in aquatic habitats is still poorly investigated. Thus, the aim of the study was to assess the rain-dependent fluctuations in the concentration of B. cereus sl vegetative cells and spores, with evaluation of their phylogenetic and population structure in relation to the toxicity and psychrotolerance. We proved that vegetative cells of B. cereus sl are widely distributed in fresh water of rivers and lakes, being as common as spores. Moreover, heavy rain has a huge impact on their concentration in undisturbed environments. The diversity of B. cereus sl reflects the multiple sources of bacteria and the differences between their distinct environments. Next, their diverse genetic structure and phenotypes better fit their ecological properties than their taxonomic affiliation.

Characterization of Enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus Isolates and Associated Enterotoxin Production Dynamics in Milk or Meat-Based Broth.

Bacillus cereus sensu lato species, as well as Staphylococcus aureus, are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA, hblA and entFM toxin genes, with lower prevalence of bceT and hlyII. When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log10(CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices.

Molecular Characterization and Risk Assessment of Bacillus cereus Sensu Lato Isolated from Ultrahigh-Temperature and Pasteurized Milk Marketed in Rio de Janeiro, Brazil

The presence of Bacillus cereus in milk is a major concern in the dairy industry. In this study 27 Bacillus cereus sensu lato isolates from pasteurized and ultrahigh-temperature (UHT) milk (24 whole UHT and 4 pasteurized samples) collected at supermarket chains in Rio de Janeiro, Brazil, were evaluated to assess the potential risk for food poisoning. Toxigenic and virulence profiles were defined by gene-specific PCR. Affiliation to phylogenetic groups was assigned by panC sequencing. Microbiological analysis revealed the presence of B. cereus s.l. in eight (33.3%) brands (six brands of UHT and two brands of pasteurized milk). Twenty-seven isolates were recovered (13 B. cereus and 14 Bacillus thuringiensis ). Predominant toxigenic patterns were type I (contains all toxin genes except ces) and type II (does not contain cytK and ces), with seven (25.9%) isolates each. Predominant virulence patterns were type 2 (does not contain hlyII or shp) and type 3 (contains all virulence genes), with five (18.5%) isolates each. All isolates belonged to phylogenetic groups III and IV. Presence of hbl, piplc, and sph was associated with group IV isolates. Our results suggest that B. thuringiensis and B. cereus sensu stricto should be considered potential foodborne pathogens. Because the majority of the milk isolates studied have the potential to cause food poisoning because of the high prevalence of toxin and virulence genes and the specific phylogenetic group affiliations, these milk products can be potentially hazardous for human consumption.

Sporeforming bacteria in beer: Occurrence, diversity, presence of hop resistance genes and fate in alcohol-free and lager beers

The aim of this study was to assess the occurrence of sporeforming bacteria in different types of beers (n = 163) and to assess the presence of hor genes in the isolates. Additionally, the study aimed to evaluate the fate of five representative sporeforming bacteria harboring horA and horC genes in alcohol-free and lager beers. Two hundred and sixty (n = 260) sporeforming bacteria belonging to eight different genera were isolated from beers, i.e., Bacillus (n = 118), Paenibacillus (n = 89) and Brevibacillus (n = 41), Lysinbacillus (n = 6), Cohnella (n = 3), Rummeliibacillus (n = 1), Alicyclobacillus (n = 1), and Anoxybacillus (n = 1), respectively. A predominance of members within the Bacillus cereus sensu lato (n = 72; 27.1%), followed by B. megaterium (n = 18; 7%), P. validus (n = 16; 6.1%), P. humicus (n = 13; 5%), P. alginolyticus (n = 13; 5%) and Br. brevis (n = 13; 5%) was observed in beer samples analyzed. Only 5% (n = 14) out of 260 sporeforming bacterial isolates recovered from beers harbored one or both horA and horC genes. Only one (0.3%) isolate, i.e., Bacillus cereus sensu lato (identified as B. thuringiensis LMQA 206) presented both horA and horC genes. None of the five bacterial sporeforming strains harboring horA or horC genes inoculated was able to grow in the beers throughout the storage period studied, and no spoilage was detected. The results of this study indicated a widespread occurrence of sporeforming bacteria in several types of beers from different brands, highlighting that measures should be taken to reduce the occurrence of sporeforming bacteria considering stability and safety concerns.

Milk-originated Bacillus cereus sensu lato strains harbouring Bacillus anthracis-like plasmids are genetically and phenotypically diverse

Bacillus cereus sensu lato is widely distributed in food products, including raw and processed milk. Plasmids often determine bacterial virulence and toxicity, but their role in the evolution of B. cereus sensu lato is only partly known. Here, we observed that nearly 8% of B. cereus sensu lato isolates were positive for pXO1-like plasmids and 12% for pXO2-like plasmids in raw and ultra-heat-treated (UHT) milk from one dairy plant. However, pXO1-like plasmids were significantly more frequent in raw milk, while pXO2-like plasmids were more frequent in processed milk. Strains from raw and UHT milk were enterotoxigenic, with up to one-fifth of the isolates being psychrotolerant. Phylogenetic assessment using multi-locus sequence typing revealed a polyphyletic structure for these bacilli, with distinct groups of cold-adapted isolates and pathogenic strains (including emetic B. cereus). Populations corresponding to both sampling sites exhibited significant linkage disequilibrium and the presence of purifying selection. The far-from-clonal population structure indicated the presence of sequence types or ecotypes adapted to specific conditions in the dairy industry. A high recombination-to-mutation ratio suggested an important role for horizontal gene transfer among B. cereus sensu lato isolates in milk.

Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verticillioides in maize plants.

Maize is an economically important crop in northern Mexico. Different fungi cause ear and root rot in maize, including Fusarium verticillioides (Sacc.) Nirenberg. Crop management of this pathogen with chemical fungicides has been difficult. By contrast, the recent use of novel biocontrol strategies, such as seed bacterization with Bacillus cereus sensu lato strain B25, has been effective in field trials. These approaches are not without their problems, since insufficient formulation technology, between other factors, can limit success of biocontrol agents. In response to these drawbacks, we have developed a powder formulation based on Bacillus B25 spores and evaluated some of its characteristics, including shelf life and efficacy against F. verticillioides, in vitro and in maize plants. A talc-based powder formulation containing 1 × 10(9) c.f.u. g(-1) was obtained and evaluated for seed adherence ability, seed germination effect, shelf life and antagonism against F. verticillioides in in vitro and in planta assays. Seed adherence of viable bacterial spores ranged from 1.0 to 1.41 × 10(7) c.f.u. g(-1). Bacteria did not display negative effects on seed germination. Spore viability for the powder formulation slowly decreased over time, and was 53 % after 360 days of storage at room temperature. This formulation was capable of controlling F. verticillioides in greenhouse assays, as well as eight other maize phytopathogenic fungi in vitro. The results suggest that a talc-based powder formulation of Bacillus B25 spores may be sufficient to produce inoculum for biocontrol of maize ear and root rots caused by F. verticillioides.

Incidence, Antibiotic Susceptibility, and Toxin Profiles of Bacillus cereus sensu lato Isolated from Korean Fermented Soybean Products.

Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho-kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty-eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho-kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty-seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β-lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<10(4) CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available.

Melanin-Like Pigment Synthesis by Soil Bacillus weihenstephanensis Isolates from Northeastern Poland.

Although melanin is known for protecting living organisms from harmful physical and chemical factors, its synthesis is rarely observed among endospore-forming Bacillus cereus sensu lato. Here, for the first time, we reported that psychrotolerant Bacillus weihenstephanensis from Northeastern Poland can produce melanin-like pigment. We assessed physicochemical properties of the pigment and the mechanism of its synthesis in relation to B. weihenstephanensis genotypic and phenotypic characteristics. Electron paramagnetic resonance (EPR) spectroscopy displayed a stable free radical signal of the pigment from environmental isolates which are consistent with the commercial melanin. Fourier transform infrared spectroscopy (FT-IR) and physicochemical tests indicated the phenolic character of the pigment. Several biochemical tests showed that melanin-like pigment synthesis by B. weihenstephanensis was associated with laccase activity. The presence of the gene encoding laccase was confirmed by the next generation whole genome sequencing of one B. weihenstephanensis strain. Biochemical (API 20E and 50CHB tests) and genetic (Multi-locus Sequence Typing, 16S rRNA sequencing, and Pulsed-Field Gel Electrophoresis) characterization of the isolates revealed their close relation to the psychrotrophic B. weihenstephanensis DSMZ 11821 reference strain. The ability to synthesize melanin-like pigment by soil B. weihenstephanensis isolates and their psychrotrophic character seemed to be a local adaptation to a specific niche. Detailed genetic and biochemical analyses of melanin-positive environmental B. weihenstephanensis strains shed some light on the evolution and ecological adaptation of these bacteria. Moreover, our study raised new biotechnological possibilities for the use of water-soluble melanin-like pigment naturally produced by B. weihenstephanensis as an alternative to commercial non-soluble pigment.

Bacillus cereus sensu lato strain B25 controls maize stalk and ear rot in Sinaloa, Mexico

Fungal pathogens causing maize stalk and ear rot are a potential threat to grain production in regions where monoculture extensions can reach over 500,000ha per year. This particular problem is observed in northern Sinaloa, Mexico with the fungal pathogen Fusarium verticillioides. Three native Bacillus spp. strains isolated from the maize rhizosphere were tested for their potential as biocontrol agents (BCAs) against fusariosis in maize, during the 2011–2012 fall-winter growing season. Based on its performance, the Bacillus cereus sensu lato strain B25 was selected for further analysis. The effectiveness of maize seed inoculation with this strain was examined in two more consecutive growing seasons. The potential for B25 to control Fusarium stalk rot (FSR) and Fusarium ear rot (FER) of maize, as well as the accumulation of fumonisins in kernels, was thus assessed with white maize hybrids grown under different field conditions in northern Sinaloa, Mexico. FSR and FER incidence and severity were substantially reduced as compared to controls in all trials conducted. Fumonisin contamination in maize grains was also reduced (up to 93.9%) by B25 application, as compared to the control. Furthermore, B25 significantly increased grain yield in several trial sites or crop seasons, above the average of the untreated controls and consistently above the average of F. verticillioides-inoculated controls. Based on these findings, we propose that seed bacterization with strain B25, combined with adequate crop management practices, may become a useful tool for avoiding Fusarium stalk and ear rot of maize. This practice will also provide safe fumonisin grain levels for maize production in northern Sinaloa.

Pathogenicity and characterization of a novel Bacillus cereus sensu lato isolate toxic to the Mediterranean fruit fly Ceratitis capitata Wied.

The lethal and sub-lethal effects of sporulated cultures of a novel Bacillus cereus sensu lato strain lacking detectable cry genes and identified through morphological and genetic analyses, have been studied on the Mediterranean fruit fly Ceratitis capitata. The lethal effects on young larvae were concentration dependent, with a median lethal concentration (LC50) of 4.48×108spores/g of diet. Sporulated cultures of this strain significantly extended development time and reduced immature survival, and the size of emerging fly adults. Besides spores, the toxicity has been associated to the insoluble extra-spore fraction characterized through a proteomic approach. The profile of the extra-spore protein fraction (ES) showed major protein bands within the 35–65kDa range. The results of mass spectrometry analysis highlighted the presence of putative virulence factors, including members of protein families previously associated to the insecticidal action of other microbial entomopathogens. These proteins include metalloproteases, peptidases and other enzymes.

Modular Genetic Architecture of the Toxigenic Plasmid pIS56-63 Harboring cry1Ab21 in Bacillus thuringiensis subsp. thuringiensis strain IS5056

Bacillus thuringiensis subsp. thuringiensis IS5056, a strain highly toxic to Trichoplusia ni larvae, produces the newly described Cry1Ab21 delta-endotoxin encoded by a gene located in the 63.8 kb pIS56-63 plasmid. In this report we present the structure and functional similarity of this plasmid to other B. thuringiensis large toxigenic plasmids with particular interest focused on its modular architecture. The 61 open reading frames (ORFs) of the plasmid made four functional modules: (i) M1-mic, the mobile insertion cassette harboring cry1Ab21; (ii) M2-tra, the putative conjugative element; (iii) M3-reg, regulation sequence; and (iv) M4-rep, the ori44 replicon. These modules display similarity to corresponding sequences in distinct B. thuringiensis plasmids, but, in general, not to plasmid of other Bacillus cereus sensu lato. The nucleotide sequence and organization of genes in pIS56-63 were highly similar (80-100%) to those in pHT73 of B. thuringiensis HT73, and in p03 of B. thuringiensis HD771, particularly within the M3-reg and M4-rep modules, and slightly less in M2-tra, the latter of which is composed of two segments exhibiting homology to sequences in pBMB28, pAH187_45, pCT83, and pIS56-85 or to pCT72, pBMB67, p04, and pIS56-68. The tetrapartite structure of the toxigenic pIS56-63 plasmid strongly suggests that its hybrid nature is a result of recombination of various genetic elements originating from different extrachromosomal and chromosomal sources in B. thuringiensis. The presence of cry1Ab21 in the mobile cassette suggests that its occurrence on pIS56-63 resulted from recombination and transposition events during the evolution of the plasmid.

Bacillus cereus Sensu Lato Genomes: Basis for Identifying Anthrax Disease Strain Sources

Why would one want to sequence the genomes of 94 additional, mostly soil and water isolates of Bacillus cereus sensu lato (1)? The answer is of course to be able to make a rapid analysis during a bioterrorism event, to develop a portable kit for the rapid identification of anthrax spores (by proteomic analysis), and to determine their precise origin (by means of DNA genomic polymorphisms). The draft genome sequences of 94 environmental Bacillus cereus sensu lato strains were deposited in GenBank (1) to facilitate the identification of the closely related subspecies B. cereus, Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis and toxicity determinants. Strains of this species often harbor pathogenicity plasmids and show different envirotypes and pathotypes.

Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk

The Bacillus cereus sensu lato group includes potentially pathogenic bacteria that are ubiquitous in the environment and, importantly, could also be present in food products. This study focuses on emetic isolates which presumably could cause acute food poisoning and emetic syndrome. Here, we evaluate the ability of psychrotolerant Bacillus weihenstephanensis MC118 (isolated from soil) and mesophilic B. cereus BOD3/9 isolated from milk to germinate and multiply at 7 and 30°C. Whereas the rates of germination at 30°C in milk and nutrient broth of MC118 and BOD3/9 were similar, MC118, but not BOD3/9, proliferated to achieve relatively high numbers (∼10(6) colony-forming units/g) within 7days of incubation at 7°C. Mesophilic BOD3/9 showed a slight decrease of cell concentration in similar studies at 7°C. Genotyping with repetitive extragenic palindromic sequence-based PCR and pulsed field gel electrophoresis revealed significant similarities between BOD3/9 and emetic reference B. cereus F4810/72 strain, while the B. weihenstephanensis MC118 isolate was more similar to the B. weihenstephanensis non-emetic reference DSMZ11821 strain. Our data suggest that emetic isolates that are also psychrotolerant, such as MC118, could constitute a hazard in the dairy industry, where milk could be a suitable medium for germination and growth.

Molecular identification and safety of Bacillus species involved in the fermentation of African oil beans (Pentaclethra macrophylla Benth) for production of Ugba

Molecular identification of Bacillus spp. involved in the fermentation of African oil bean seeds for production of Ugba, as well as ability of the Bacillus spp. isolated to produce toxins, were investigated. Forty-nine bacteria were isolated from Ugba produced in different areas of South Eastern Nigeria and identified by phenotyping and sequencing of 16S rRNA, gyrB and rpoB genes. Genotypic diversities at interspecies and intraspecies level of the isolates were screened by PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence-based PCR (rep-PCR). The ability of the bacteria to produce toxins was also investigated by detection of genes encoding production of haemolysin BL (HblA, HblC, HblD), non-haemolytic enterotoxin (NheA, NheB, NheC), cytotoxin K (CytK) and emetic toxin (EM1) using PCR with specific primers. Moreover, a Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) was used to screen ability of the isolates to produce haemolysin in broth and during fermentation of African oil bean seeds.The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. They were identified as Bacillus cereus sensu lato (42), Lysinibacillus xylanilyticus (3), Bacillus clausii (1), Bacillus licheniformis (1), Bacillus subtilis (1), and Bacillus safensis (1). B. cereus was the predominant Bacillus species and was present in all samples studied. Using ITS-PCR, interspecies diversity was observed among isolates, with six clusters representing each of the pre-cited species. Rep-PCR was more discriminatory (eight clusters) and allowed further differentiation at intraspecies level for the B. cereus and L. xylanilyticus isolates with two genotypes for each species. Genes encoding production of non-haemolytic enterotoxin (NheA, NheB, NheC) and cytotoxin K (CytK) genes were detected in all B. cereus isolates, while Hbl genes (HblA, HblC, HblD) were detected in only one isolate. The emetic-specific gene fragment was not detected in any of the isolates studied. None of the toxin genes screened was detected in isolates belonging to other Bacillus species. Using RPLA, haemolysin production was detected in one isolate of B. cereus, which showed positive amplicons for Hbl genes, both during cultivation in broth and during fermentation of oil bean seeds.

Divergence of the SigB regulon and pathogenesis of the Bacillus cereus sensu lato group

BackgroundThe Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species.ResultsDuring the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence.ConclusionsFour lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.

Genomic characterization of theBacillus cereussensu lato species: Backdrop to the evolution ofBacillus anthracis

The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

English

&nbsp; Bacterial contamination of pasteurized milk may originate from different sources: raw milk, process equipment surfaces and packaging materials. It is hypothesized that post-pasteurization contamination along the milk processing lines is responsible of reducing shelf life of Algerian pasteurized milk. This assumption was investigated through assessment of both the microflora of biofilms in milk pipeline systems at five dairy plants of Northwestern Algeria and the effectiveness of a quaternary ammonium based compound used for the disinfection of the plant equipment. Samples were collected before and after cleaning-in-place (CIP) systems from different segments of pasteurization lines with sterile cotton swabs. Quantitative assessment showed little reduction of the total bacteria count after CIP. On the average bacterial numbers were 5.6 &times; 103, 1.2 &times; 104, 5.1 &times; 104, 2.5 &times; 105&nbsp;and 9.7 &times; 107&nbsp;cfu/cm2, respectively, in the different units. Phenotypic identification of isolates revealed predominance of Gram-positive bacilli belonging to&nbsp;Bacillus&nbsp;and notably the&nbsp;Bacillus cereus&nbsp;group,&nbsp;at maximal levels of 72 and 21% respectively. The other Gram-positive microflora included&nbsp;Staphylococcus&nbsp;(30%) andMicrococcus&nbsp;(10%). In contrast, the incidence of the Gram-negative bacteria was relatively low. Two genera, identified as&nbsp;Pseudomonas&nbsp;(9%) and&nbsp;Enterobacter&nbsp;(6%), were found only in two dairies. Three dairies were Gram-negative bacteria-free. That should be the result of common contamination sources or highly environmental selective pressure. Further studies have to address these hypotheses. Treatment of experimental&nbsp;Bacillus cereus&nbsp;sensu lato&nbsp;strains biofilms with a 50, 100 and 150 ppm of quaternary ammonium disinfectant, showed a significant resistance of biofilms to this product even after long exposure time (15 min). This study emphasized the importance of aerobic spore-forming bacteria in dairy-processing equipment as they are able to built recalcitrant biofilms&nbsp;on the inside equipment surfaces&nbsp;with subsequent resistance to conventional CIP system and potential transfer to pasteurized milk. Therefore, in order to reduce the contamination levels of spore-forming bacteria and improve the quality and shelf life of the product, these dairies have, besides improvement in the hygienic status of the plant equipments, also to monitor either the pasteurization process or the contamination from raw material (that is, milk powder). &nbsp; Key words:&nbsp;Milk contamination, pasteurization, biofilm, cleaning-in-place (CIP).

Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group

BackgroundThe Bacillus cereus sensu lato group consists of six species (B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the Bc species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of B. subtilis.ResultsPhylogenetic analysis of the Bc species-group utilizing 157 single-copy genes of the family Bacillaceae suggests that several taxonomic revisions of the genus Bacillus should be considered. Within the Bc species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the Bc species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the Bc species-group.ConclusionsExpansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in B. subtilis. Divergence of the sigma-controlled transcriptional regulons among various members of the Bc species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the Bc species-group.

Characterization of Bacilli Isolated from the Confined Environments of the Antarctic Concordia Station and the International Space Station

Bacillus and related genera comprise opportunist and pathogen species that can threaten the health of a crew in confined stations required for long-term missions. In this study, 43 Bacilli from confined environments, that is, the Antarctic Concordia station and the International Space Station, were characterized in terms of virulence and plasmid exchange potentials. No specific virulence feature, such as the production of toxins or unusual antibiotic resistance, was detected. Most of the strains exhibited small or large plasmids, or both, some of which were related to the replicons of the Bacillus anthracis pXO1 and pXO2 virulence elements. One conjugative element, the capacity to mobilize and retromobilize small plasmids, was detected in a Bacillus cereus sensu lato isolate. Six out of 25 tested strains acquired foreign DNA by conjugation. Extremophilic bacteria were identified and exhibited the ability to grow at high pH and salt concentrations or at low temperatures. Finally, the clonal dispersion of an opportunist isolate was demonstrated in the Concordia station. Taken together, these results suggest that the virulence potential of the Bacillus isolates in confined environments tends to be low but genetic transfers could contribute to its capacity to spread.