Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Isolation and identification of guaiacol producing Alicyclobacillus fastidiosus strains from orchards in Poland.

The genus Alicyclobacillus comprises a group of Gram-positive, thermo-acidophilic bacteria that are capable of producing highly resistant endospores during unfavorable environmental conditions. The members of this genus inhabit natural environments, including hot springs and soils. The main reason behind the spoilage of final commercial fruit products by Alicyclobacillus is the contamination of fruits with soil at the time of harvesting. Some of the Alicyclobacillus species, including Alicyclobacillus acidoterrestris, are categorized as spoilage bacteria due to their ability to produce off-flavor compounds (e.g., guaiacol and halophenols) that adversely affect the taste and aroma of beverages. In our study, Alicyclobacillus species were isolated from Polish orchard soils and fruits and were subjected to 16S rDNA sequencing. The results of the analysis showed that the isolated strains belonged to A. acidoterrestris and Alicyclobacillus fastidiosus species. All the three isolated strains of A. fastidiosus (f1, f2, f3) exhibited similar morphological and biochemical properties as the strain described in the literature. However, these isolated strains were able to produce guaiacol at temperatures of 20°C, 25°C, and 45°C. Thus, the strains of A. fastidiosus discovered in the present study can be included in the group of spoilage species as they possessed the gene responsible for the production of guaiacol.

Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

Effect of ultraviolet (UV-C) radiation on spores and biofilms of Alicyclobacillus spp. in industrialized orange juice.

Bacteria of the genus Alicyclobacillus pose serious quality problems for the juice processing industries that have sought effective alternatives for its control. The present study evaluated the effect of UV-C radiation on the reduction of spores and biofilm formation of Alicyclobacillus spp. on stainless steel and rubber surfaces using industrialized orange juice as a culture medium. Four reference Alicyclobacillus spp. species and different UV-C dosages were investigated. After exposed for 20 min (16.8 kJ/m2) to UV-C, the spores of Alicyclobacillus acidoterrestris, Alicyclobacillus herbarius, and Alicyclobacillus cycloheptanicus decreased drastically more of 4 log CFU/mL, with counts below the detection limit of the method (<1.7 log CFU/mL), while the Alicyclobacillus acidocaldarius spores were more sensitive to UV-C, once this spore reduction was observed within 15 min (12.6 kJ/m2). Morphological changes in the Alicyclobacillus acidoterrestris spores were observed by scanning electron microscopy. A reduction of biofilm formation was observed for all UV-C treatments, and the higher reductions (approximately 2 log CFU/mL) were found for the Alicyclobacillus acidocaldarius species after 30 min (26.2 kJ/m2), on the stainless steel and rubber surfaces. The results suggest that UV-C can be used to reduce the biofilm formation and could be a promising alternative for controlling Alicyclobacillus spp. spores in industrialized orange juice.

A single enzyme PCR-RFLP assay targeting V1-V3 region of 16S rRNA gene for direct identification of Alicyclobacillus acidoterrestris from other Alicyclobacillus species.

Alicyclobacillus acidoterrestris is considered to be one of the most important target microorganisms in quality control of heat-processed acidic foods. This species can cause spoilage by forming spores with very high heat resistance; however, no gas production or visible changes occur in the contaminated product during storage. Thus, the differentiation of A. acidoterrestris from other Alicyclobacillus species is of great importance. Τhe present study aims to find a rapid method for the identification of A. acidoterrestris. To achieve this, 78 Alicyclobacillus isolates were subjected to PCR-RFLP (restriction fragment length polymorphism). Specifically, PCR products of amplified V1-V3 region of the 16S rRNA gene were digested with restriction endonuclease HhaI. According to the obtained results, all A. acidoterrestris isolates showed similar restriction fragments of the 16S rRNA gene and different from A. acidocaldarius and A. hesperidum. In conclusion, a single enzyme PCR-RFLP assay was developed and showed rapid, inexpensive and direct identification of Alicyclobacillus isolates. The application of this method will be useful to identify this contaminant in fruit juices.

Description of Alicyclobacillus montanus sp. nov., a mixotrophic bacterium isolated from acidic hot springs.

Three morphologically similar thermo-acidophilic strains, USBA-GBX-501, USBA-GBX-502 and USBA-GBX-503T, were isolated from acidic thermal springs at the National Natural Park Los Nevados (Colombia). All isolates were spore-forming, Gram-stain-positive and motile, growing aerobically at 25-55 °C (optimum ~45 °C) and at pH 1.5-4.5 (optimum pH ~3.0). Phylogenetic analysis of the 16S rRNA gene sequences of these isolates showed an almost identical sequence (99.0 % similarity) and they formed a robust cluster with the closest relative Alicyclobacillus tolerans DSM 16297T with a sequence similarity of 99.0 %. Average similarity to other species of the genus Alicyclobacillus was 93.0 % and average similarity to species of the genus Effusibacillus was 90 %. In addition, the level of DNA-DNA hybridization between strain USBA-GBX-503T and Alicyclobacillus tolerans DSM 16297T was 31.7 %. The genomic DNA G+C content of strain USBA-GBX-503T was 44.6 mol%. The only menaquinone was MK-7 (100.0 %). No ω-alicyclic fatty acids were detected in strain USBA-GBX-503T, and the major cellular fatty acids were C18 : 1ω7c, anteiso-C17 : 0 and iso-C17 : 0. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis and DNA-DNA relatedness values, along with low levels of identity at the whole genome level (ANIb and ANIm values of <67.0 and <91.0 %, respectively), it can be concluded that strain USBA-GBX-503T represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus montanus sp. nov. is proposed. The type strain is USBA-GBX-503T (=CMPUJ UGB U503T=CBMAI1927T).

Dipicolinic Acid Release and the Germination of Alicyclobacillus acidoterrestris Spores under Nutrient Germinants.

The presence of Alicyclobacillus, a thermoacidophilic and spore-forming bacterium, in acidic fruit juices poses a serious problem for the processing industry. A typical sign of spoilage in contaminated juices is a characteristic phenolic off-flavour associated with the production of guaiacol. Spores are formed in response to starvation and in a natural environment re-access the nutrients, e.g.: L-alanine and AGFK - a mixture of asparagine, glucose, fructose and potassium, triggers germination. The aim of this study was to estimate the impact of L-alanine and AGFK on the germination of the spores of two Alicyclobacillus acidoterrestris strains and to evaluate the relationship of the germination rate with dipicolinic acid (DPA) release. The spores were suspended in apple juice or in buffers at pH 4 and pH 7, followed by the addition of L-alanine and AGFK. Suspensions were or were not subjected, to a temperature of 80°C/10 min and incubated for various periods of time at 45°C. Optical density (OD660) was used to estimate the number of germinated spores. The amount of DPA released was determined using HPLC. The results indicate that the degree of germination of A. acidoterrestris spores depended on the strain and time of incubation and the nutritious compounds used. The data obtained show that the amount of DPA released correlated to the number of A. acidoterrestris spores germinated.

PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

Culture dependent and independent genomic identification of Alicyclobacillus species in contaminated commercial fruit juices.

Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming, bacteria species which occasionally cause spoilage of heat-processed fruit juices by producing guaiacol taint. In this study, Alicyclobacillus contamination of commercial fruit juices in West Africa was investigated using culture-dependent and -independent approaches. Firstly, a total of 225 fruit juice products from Ghana (n = 39) and Nigeria (n = 186) were enriched with yeast-starch-glucose (YSG) broth (pH 3.7) following heat shock at 80 °C for 10 min. Alicyclobacillus was detected in 11.6% (26) of samples. Isolates were identified to the genus taxonomic level by genus-specific PCR which targeted the squalene-hopene-cyclase (shc) gene followed by analysis of the almost-complete 16S ribosomal RNA (rRNA) gene sequences that identified 16 Alicyclobacillus acidoterrestris, 7 Alicyclobacillus acidocaldarius and 3 Alicyclobacillus genomic species 1 (Alicyclobacillus sp. 1). Whole-genome fingerprinting using PCR-RAPD primers Ba-10, F-61 and F-64 grouped the 16 A. acidoterrestris isolates into two genetic clusters. Furthermore, high performance liquid chromatographic (HPLC) analyses revealed the activity of vanillic-acid decarboxylase (vdc) in all A. acidoterrestris isolates due to guaiacol production from vanillic-acid. Lastly, species-specific PCR-DGGE targeting the 16S rRNA gene clearly discriminated between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius group. Information provided by this study is fundamental to the development of effective strategies for the improvement of quality and shelf-life of processed tropical fruit juices in W. Africa.

Genotypic and Phenotypic Heterogeneity in Alicyclobacillus acidoterrestris: A Contribution to Species Characterization.

Alicyclobacillus acidoterrestris is the main cause of most spoilage problems in fruit juices and acidic products. Since soil borne species often contaminate fruit juices and do not need strict extreme requirements for survival, it is a great concern to investigate whether and how soil species could evolve from their ecological niches in microbial community to new environments as fruit juices. In this study, 23 isolates of thermo-acidophilic, spore-forming bacteria from soil were characterized by cultural and molecular methods. In addition, 2 strains isolated from a spoilage incident in pear juice were typed. Strains phenotyping showed that they could be grouped into 3 different clusters, and some isolates showed identical or quite similar patterns. Analyzing pH and temperature ranges for growth, the majority of strains were able to grow at values described for many species of Alicyclobacillus. Qualitative utilization of lysine, arginine and indole production from tryptophan revealed, for the first time, deamination of lysine and decarboxylation of arginine. Resistance to 5% NaCl as well as the ability to hydrolyze starch and gelatin, nitrate reduction, catalase and oxidase activities confirmed literature evidences. Examining of 16S rRNA, showed that isolates were divided into three blocks represented by effectively soil species and strains that are moving from soil to other possible growing source characterized by parameters that could strongly influence bacterial survival. RAPD PCR technique evidenced a great variability in banding patterns and, although it was not possible to obtain genotypically well-distinguished groups, it was feasible to appreciate genetic similarity between some strains. In conclusion, the investigation of a microbial community entails a combination of metagenomic and classic culture-dependent approaches to expand our knowledge about Alicyclobacillus and to look for new subspecies.

Alicyclobacillus fodiniaquatilis sp. nov., isolated from acid mine water.

Two novel, Gram-stain-variable, moderately thermophilic, acidophilic, rod-shaped, endospore-forming bacteria, G45-16T and G45-17, were isolated from acid mine water of Zijin copper mine in Fujian Province, China. Phylogenetic analysis of 16S rRNA gene sequences showed that they were closely related to Alicyclobacillus acidoterrestris ATCC 49025T with sequence similarities of 96.8 %. Cells grew aerobically at 20-45 °C (optimum, 40 °C), at pH 2.5-5.5(optimum, pH 3.5) and in the presence of 0-4.0 % (w/v) NaCl. Strains contained MK-7 as the major menaquinone and the major cellular fatty acids were ω-cyclohexane C19 : 0 and ω-cyclohexane C17 : 0. The DNA G+C content was 51.3 and 49.8 mol% (Tm) for G45-16T and G45-17, respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with their relatives and DNA-DNA relatedness values, it is concluded that strains G45-16T and G45-17 represent a novel species within the genus Alicyclobacillus, for which the name Alicyclobacillus fodiniaquatilis sp. nov. is proposed; the type strain is G45-16T(=CGMCC 1.15049T=NBRC 111483T).

Genotypic and phenotypic diversity of Alicyclobacillus acidocaldarius isolates.

UNLABELLED: The fruit juice industry recognizes Alicyclobacillus as a major quality control target micro-organism. In this study, we analysed 19 bacterial isolates to identify Alicyclobacillus species by polymerase chain reaction (PCR) and sequencing analyses. Phenotypic and genomic diversity among isolates were investigated by API 50CHB system and ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR) respectively. All bacterial isolates were identified as Alicyclobacillus acidocaldarius, and almost all showed identical DNA sequences according to their 16S rRNA (rDNA) gene partial sequences. Only few carbohydrates were fermented by A. acidocaldarius isolates, and there was little variability in the biochemical profile. Genotypic fingerprinting of the A. acidocaldarius isolates showed high diversity, and clusters by ERIC-PCR were distinct to those obtained from the 16S rRNA gene phylogenetic tree. There was no correlation between phenotypic and genotypic variability in the A. acidocaldarius isolates analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection of Alicyclobacillus strains is imperative in fruit concentrates and juices due to the production of guaiacol. Identification of the genera originates rejection of the product by processing industry. However, not all the Alicyclobacillus species are deteriorative and hence the importance to differentiate among them. In this study, partial 16S ribosomal RNA sequence alignment allowed the differentiation of species. In addition, ERIC-PCR was introduced for the genotypic characterization of Alicyclobacillus, as an alternative for differentiation among isolates from the same species.

Tumebacillus algifaecis sp. nov., isolated from decomposing algal scum.

Bacterial strain THMBR28(T) was isolated from decomposing algal scum that was collected during an algal bloom in Taihu lake, China. Cells of strain THMBR28(T) were Gram-staining-positive, facultatively anaerobic and rod-shaped. Growth was observed at 20-45 °C (optimum, 30 °C), at pH 5.0-9.5 (optimum, pH 6.5-7.5), and in the presence of 0-1.0% (w/v) NaCl (optimum, 0.5%). Strain THMBR28(T) contained MK-7 as the major menaquinone and iso-C15 : 0 as the major cellular fatty acid. The polar lipid profile contained phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and six unidentified polar lipids. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. The DNA G+C content was 57.6 mol% (Tm). Phylogenetic analysis of 16S rRNA gene sequences showed that strain THMBR28(T) belonged to the genus Tumebacillus, most closely related to Tumebacillus ginsengisoli DSM 18389(T) (95.0%) and Tumebacillus permanentifrigoris Eur1 9.5(T) (93.4%). Based on phylogenetic and phenotypic characterization, it is concluded that strain THMBR28(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus algifaecis sp. nov. is proposed, with THMBR28(T) ( = CGMCC 1.10949(T) = NBRC 108765(T)) as the type strain.

Food Targeting: A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification of Alicyclobacillus spp. Spores after Aptamer-Based Enrichment.

Spore-forming Alicyclobacillus spp. are able to form metabolites that induce even in small amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations could occur by endospores, which overcame the pasteurization process. The current detection method for Alicyclobacillus spp. can take up to 1 week because of microbiological enrichment. In a previous study, DNA aptamers were selected and characterized for an aptamer-driven rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation. In the present work, a direct quantification assay for Alicyclobacillus spp. spores was developed to complete the two-step approach of enrichment and detection. After mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR-assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the European Network of Genetically Modified Organisms Laboratories (ENGL). Hence, the presented method is applicable for direct spore detection from orange juice in connection with an enrichment step.

[Differentiation and Identification of Alicyclobacillus Strains by Fourier Transform Near-Infrared Spectroscopy].

Fourier transform near-infrared spectroscopy (FT-NIR) can reflect the overall molecular composition of microbial cells to identify different types of microorganisms. To establish an accurate, effective method about the differentiation and identification of Alicyclobacillus strains between different species, the present research performed the following studies by FT-NIR: (1) The FT-NIR spectra about seven type stains was clustered for data analysis. After preprocessing, reduction of data was performed by Principal Component Analysis (PCA) and Linear Discriminant Analysis(LDA), exploring the feasibility of differentiation and identification between different species, the result suggested that the PCA model can cluster the seven species of Alicyclobacillus strains correctly and the LDA model I can predict the unknown species with 100% accuracy. It evidenced that the method could identify different species of Alicyclobacillus strains preliminary. (2)In order to improve the robustness and practicability of the model, a total of 41 Alicyclobacillus strains including type and isolated strains were prepared for LDA model II, using the same methods as mentioned before. The result indicated that the LDA model validated by fifteen sample with 86.67% accuracy. It was more perfect and more comprehensive. As a result, the FT-NIR technology combined with chemometrics method can accurately and effectively identify Alicyclobacillus strains between different microbial species.

Alicyclobacillus dauci sp. nov., a slightly thermophilic, acidophilic bacterium isolated from a spoiled mixed vegetable and fruit juice product.

A novel, moderately thermophilic, acidophilic, Gram-variable, rod-shaped, endospore-forming bacterium was isolated from a spoiled mixed vegetable and fruit juice product that had the off-flavour of guaiacol. The bacterium, strain 4F(T), grew aerobically at 20-50 °C (optimum 40 °C) and pH 3.0-6.0 (optimum pH 4.0) and produced acid from glycerol, d-galactose and d-glucose. It contained menaquinone-7 (MK-7) as the major isoprenoid quinone and the DNA G+C content was 49.6 mol%. The predominant cellular fatty acids of strain 4F(T) were ω-alicyclic (ω-cyclohexane fatty acids), which are characteristic of the genus Alicyclobacillus. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belongs to the Alicyclobacillus cluster, and is related most closely to the type strains of Alicyclobacillus acidoterrestris (97.4 % similarity) and Alicyclobacillus fastidiosus (97.3 %). Strain 4F(T) produced guaiacol from vanillic acid. It can be distinguished from related species by its acid production type and guaiacol production. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness values, it can be concluded that the strain represents a novel species of the genus Alicyclobacillus, for which the name Alicyclobacillus dauci sp. nov. is proposed; the type strain is 4F(T) ( = DSM 28700(T) = NBRC 108949(T) = NRIC 0938(T)).

Alicyclobacillus tengchongensis sp. nov., a thermo-acidophilic bacterium isolated from hot spring soil.

A thermo-acidophilic bacterium, designated strain ACK006(T), was isolated from the soil of a hot spring at Tengchong in China. Cells were Gram-staining-positive, motile, catalase-positive and oxidase-negative, spore-forming rods. The isolate grew aerobically at 30-50°C (optimum at 45°C), pH 2.0-6.0 (optimum pH 3.2) and 0-5.0% (w/v) NaCl (optimum 1% NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain ACK006(T) belongs to the genus Alicyclobacillus with the sequence similarity of 92.3, 92.4, 92.5, and 92.8% to Alicyclobacillus cycloheptanicus SCH(T), Alicyclobacillus ferrooxydans TC-34(T), Alicyclobacillus contaminans 3-A191(T) and Alicyclobacillus disulfidooxidans SD-11(T), respectively. Similarity to other species of the genus Alicyclobacillus was 90.3-92.8% and similarity to species of the genus Tumebacillus was 85.9-87.8%. The genomic DNA G+C content was 53.7 mol%. The predominant menaquinone was MK-7. Major fatty acids were ω-cycloheptane C18:0, iso-C17:0 and anteiso-C17:0. The cell-wall peptidoglycan was the A1γ type; containing meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic analysis from this study, strain ACK006(T) represents a novel species of the genus Alicyclobacillus for which the name Alicyclobacillus tengchongensis sp. nov. is proposed. The type strain is ACK006(T) (=KCTC 33022(T) =DSM 25924(T)).

Proposal of Effusibacillus lacus gen. nov., sp. nov., and reclassification of Alicyclobacillus pohliae as Effusibacillus pohliae comb. nov. and Alicyclobacillus consociatus as Effusibacillus consociatus comb. nov.

A novel thermophilic, facultatively anaerobic bacterium, strain skLN1(T), was isolated from the sediment of a freshwater lake in Japan. Cells of strain skLN1(T) were rod-shaped and Gram-stain-variable. A KOH lysis test suggested that the cell wall of the isolate has a Gram-positive structure. For aerobic growth, the optimum pH was pH 7.25-7.50 and the optimum temperature was 50-52 °C. The G+C content of the genomic DNA was 50.8 mol%. Nitrate was reduced to nitrite. Alicyclic fatty acids were not detected, and branched-chain fatty acids were major components in the cellular fatty acid profile. MK-7 was the predominant respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolated strain was related most closely to Alicyclobacillus consociatus CCUG 53762(T) (95% similarity). This analysis also showed that the monophyly of the genus Alicyclobacillus had been lost. On the basis of phylogenetic and phenotypic characterization, Effusibacillus lacus gen. nov., sp. nov. is proposed. The type strain of Effusibacillus lacus is skLN1(T) ( = NBRC 109614(T) = DSM 27172(T)). It is also proposed that Alicyclobacillus pohliae and Alicyclobacillus consociatus should be reclassified to the genus Effusibacillus as Effusibacillus pohliae comb. nov. and Effusibacillus consociatus comb. nov., respectively.

Alicyclobacillus cellulosilyticus sp. nov., a thermophilic, cellulolytic bacterium isolated from steamed Japanese cedar chips from a lumbermill.

A thermophilic bacterium, strain Sueoka(T), was isolated from steamed Japanese cedar chips from a lumber mill in Gobo, Japan. The strain was able to grow on carboxymethyl cellulose at 60 °C, was Gram-stain-negative, and grew between 40.0 and 67.5 °C (optimum at 55 °C) and between pH 3.5 and 6.5 (optimum at pH 4.8). Comparative analysis of 16S rRNA gene sequences revealed 91.9 , 90.9 , and 90.8% similarity to Alicyclobacillus macrosporangiidus(T), Alicyclobacillus pomorum(T), and Alicyclobacillus acidocaldarius(T), respectively. The major quinone was MK-7 and the predominant cellular fatty acids were ω-cyclohexane C19 : 0 and ω-cyclohexane C17 : 0. The DNA G+C content was 60.8 mol%. Based on the results of this study, strain Sueoka(T) is a novel species of the genus Alicyclobacillus, and the namehttp://dx.doi.org/10.1601/nm.5071Alicyclobacillus cellulosilyticus sp. nov. (type strain Sueoka(T)  = JCM 18487(T)  = KCTC 33007(T)) is proposed.

Alicyclobacillus contamination in the production line of kiwi products in China.

Alicyclobacillus are spoilage microbes of many juice products, but contamination of kiwi products by Alicyclobacillus is seldom reported. This study aims to investigate the whole production line of kiwi products in China to assess the potential risk of their contamination. A total of 401 samples from 18 commercial products, 1 processing plant and 16 raw material orchards were tested, and 76 samples were positive, from which 76 strains of microbes were isolated and identified as 4 species of Alicyclobacillus, including Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Alicyclobacillus herbarius and Alicyclobacillus cycloheptanicus, and another 9 strains as 3 species of Bacillus by sequencing of their 16S rDNA. Through phylogenetic tree construction and RAPD-PCR amplification, it was found that there exist genotypic diversities to some extent among these isolates. Four test strains (each from one species of the 4 Alicyclobacillus species isolated in this study) could spoil pH adjusted kiwi fruit juice and some commercial kiwi fruit products with producing guaiacol (11-34 ppb).