Exploring the biosynthetic gene clusters in Brevibacterium: a comparative genomic analysis of diversity and distribution.

Exploring Brevibacterium strains from various ecosystems may lead to the discovery of new antibiotic-producing strains. Brevibacterium sp. H-BE7, a strain isolated from marine sediments from Northern Patagonia, Chile, had its genome sequenced to study the biosynthetic potential to produce novel natural products within the Brevibacterium genus. The genome sequences of 98 Brevibacterium strains, including strain H-BE7, were selected for a genomic analysis. A phylogenomic cladogram was generated, which divided the Brevibacterium strains into four major clades. A total of 25 strains are potentially unique new species according to Average Nucleotide Identity (ANIb) values. These strains were isolated from various environments, emphasizing the importance of exploring diverse ecosystems to discover the full diversity of Brevibacterium. Pangenome analysis of Brevibacterium strains revealed that only 2.5% of gene clusters are included within the core genome, and most gene clusters occur either as singletons or as cloud genes present in less than ten strains. Brevibacterium strains from various phylogenomic clades exhibit diverse BGCs. Specific groups of BGCs show clade-specific distribution patterns, such as siderophore BGCs and carotenoid-related BGCs. A group of clade IV-A Brevibacterium strains possess a clade-specific Polyketide synthase (PKS) BGCs that connects with phenazine-related BGCs. Within the PKS BGC, five genes, including the biosynthetic PKS gene, participate in the mevalonate pathway and exhibit similarities with the phenazine A BGC. However, additional core biosynthetic phenazine genes were exclusively discovered in nine Brevibacterium strains, primarily isolated from cheese. Evaluating the antibacterial activity of strain H-BE7, it exhibited antimicrobial activity against Salmonella enterica and Listeria monocytogenes. Chemical dereplication identified bioactive compounds, such as 1-methoxyphenazine in the crude extracts of strain H-BE7, which could be responsible of the observed antibacterial activity. While strain H-BE7 lacks the core phenazine biosynthetic genes, it produces 1-methoxyphenazine, indicating the presence of an unknown biosynthetic pathway for this compound. This suggests the existence of alternative biosynthetic pathways or promiscuous enzymes within H-BE7's genome.

The impact of Brevibacterium aurantiacum virulent phages on the production of smear surface-ripened cheeses.

Phages are ubiquitous and are particularly abundant in environments where their bacterial hosts thrive, such as those in the cheese industry. Although it is well documented that phages infect lactic acid bacteria, their impact has been notably overlooked on cheese ripening strains, such as Brevibacterium aurantiacum. Here, we aimed to study the impact of B. aurantiacum phages on the production of smear-ripened cheeses. We used model cheeses in industrial settings to monitor the development of the color of the cheese rind as well as of its microbial composition in presence or absence of virulent B. aurantiacum phages. Our results showed that the presence of B. aurantiacum phages significantly slowed down the development of the orange rind color in the model cheeses. In the final days of cheese ripening, phages were also detected in the control curds. By analyzing a hypervariable region of B. aurantiacum phage genomes, we detected phages with tandem repeat patterns that were different from those used in the phage-inoculated cheeses. Our results highlight the risks of using a phage-sensitive strain in smear-ripened cheese production. This is the first study to report on the impact of B. aurantiacum phages on smear-ripened cheeses.

Brevibacterium spongiae sp. nov., isolated from marine sponge Hymeniacidon sp.

A novel bacterial strain, designated as WHS-Z9T, was isolated from marine sponge Hymeniacidon sp. collected from Weihai (37° 25' N, 121° 58' E), Shandong Province, PR China. Cells of strain WHS-Z9T were Gram-stain-positive, non-spore-forming, non-motile, short-rod-shaped and light yellow-pigmented. The strain could grow at 10-40 °C (optimum, 20 °C), pH 4.5-9.5 (optimum, pH 8.5) and 2-14 % (w/v) NaCl (optimum, 4 %). The 16S rRNA gene sequence of strain WHS-Z9T showed 98.7  % similarity to that of Brevibacterium epidermidis NBRC 14811T, 98.5  % to Brevibacterium sediminis FXJ8.269T and 98.4 % to Brevibacterium oceani BBH7T. The phylogenetic tree based on 16S rRNA gene sequences revealed that strain WHS-Z9T was clustered with Brevibacterium limosum o2T. The whole genome of WHS-Z9T was approximately 4 217 721 bp in size with a G+C content of 65.2  %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among WHS-Z9T and other Brevibacterium type strains were 83.3-85.5 % (ANI based on blast), 86.4-87.9  % (ANI based on MUMmer) and 41.9-57.5 % (dDDH). Percentage of conserved protein values between the genomes of strain WHS-Z9T and members of genera Brevibacterium were 76.8-82.9 %, while the average amino acid identity (AAI) values were 83.7-87.0  %. The dDDH, ANI, AAI and POCP values were below the standard cut-off criteria for the delineation of bacterial species. The sole respiratory quinone in strain WHS-Z9T was MK-8(H2), and the predominant fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The major polar lipids of WHS-Z9T consisted of diphosphatidylglycerol and glycolipid. The diagnostic cell-wall diamino acid of strain WHS-Z9T was meso-diaminopimelic acid. Based on the data obtained in this study, strain WHS-Z9T (=MCCC 1K07845T=KCTC 49848T) should be classified as the type strain of a novel species of the genus Brevibacterium, for which the name Brevibacterium spongiae sp. nov. is proposed.

Heterologous Expression and Function of Cholesterol Oxidase: A Review.

Cholesterol was first found in gallstones as an animal sterol; hence it is called cholesterol. Cholesterol oxidase is the chief enzyme in the process of cholesterol degradation. Its role is obtained by the coenzyme FAD, which catalyzes the isomerization and oxidation of cholesterol to produce cholesteric 4-ene-3-ketone and hydrogen peroxide at the same time. Recently, a great advance has been made in the discovery of the structure and function of cholesterol oxidase, and it has proven added value in clinical discovery, medical care, food and biopesticides development and other conditions. By recombinant DNA technology, we can insert the gene in the heterologous host. Heterologous expression (HE) is a successful methodology to produce enzymes for function studies and manufacturing applications, where Escherichia coli has been extensively used as a heterologous host because of its economical cultivation, rapid growth, and efficiency in offering exogenous genes. Heterologous expression of cholesterol oxidase has been considered for several microbial sources, such as Rhodococcus equi, Brevibacterium sp., Rhodococcus sp., Streptomyces coelicolor, Burkholderia cepacia ST-200, Chromobacterium, and Streptomyces spp. All related publications of numerous researchers and scholars were searched in ScienceDirect, Scopus, PubMed, and Google Scholar. In this article, the present situation and promotion of heterologous expression of cholesterol oxidase, the role of protease, and the perspective of its possible applications were reviewed.

Phenotypic and genomic characteristics of Brevibacterium zhoupengii sp. nov., a novel halotolerant actinomycete isolated from bat feces.

Two strictly aerobic, Gram-staining-positive, non-spore-forming, regular rod-shaped (approximately 0.7 × 1.9 mm) bacteria (HY170T and HY001) were isolated from bat feces collected from Chongzuo city, Guangxi province (22°20'54″N, 106°49'20″E, July 2011) and Chuxiong Yi Autonomous Prefecture, Yunnan province (25°09'10″N, 102°04'39″E, October 2013) of South China, respectively. Optimal growth is obtained at 25-28°C (range, 4-32°C) on BHI-5% sheep blood plate with pH 7.5 (range, 5.0-10.0) in the presence of 0.5-1.0% NaCl (w/v) (range, 0-15% NaCl [w/v]). The phylogenetic and phylogenomic trees based respectively on the 16S rRNA gene and 845 core gene sequences revealed that the two strains formed a distinct lineage within the genus Brevibacterium, most closely related to B. aurantiacum NCDO 739T (16S rRNA similarity, both 98.5%; dDDH, 46.7-46.8%; ANI, 91.9-92.1%). Strain HY170T contained MK-8(H2), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG), galactose and ribose as the predominant menaquinone, major polar lipids, and main sugars in the cell wall teichoic acids, respectively. The meso-diaminopimelic acid (meso-DAP) was the diagnostic diamino acid of the peptidoglycan found in strain HY170T. Anteiso-C15:0 and anteiso-C17:0 were the major fatty acids (> 10%) of strains HY170T and HY001, with anteiso-C17:1A predominant in strain HY170T but absent in strain HY001. Mining the genomes revealed the presence of secondary metabolite biosynthesis gene clusters encoding for non-alpha poly-amino acids (NAPAA), ectoine, siderophore, and terpene. Based on results from the phylogenetic, chemotaxonomic and phenotypic analyses, the two strains could be classified as a novel species of the genus Brevibacterium, for which the name Brevibacterium zhoupengii sp. nov. is proposed (type strain HY170T = CGMCC 1.18600T = JCM 34230T).

Reclassification of Brevibacterium frigoritolerans as Peribacillus frigoritolerans comb. nov. based on phylogenomics and multiple molecular synapomorphies.

The taxonomic assignment of Brevibacterium frigoritolerans together with the in-house environmental isolate EB93 was reassessed in this study using phylogenetic and phylogenomic approaches, and the detection of multiple molecular synapomorphies. Results from the reconstructed phylogenetic trees based on the 16S rRNA gene sequences, the concatenated protein sequences of GyrA-GyrB-RpoB-RpoC, and the whole-genome sequences revealed the consistent exclusion of B. frigoritolerans and the environmental isolate EB93 from the cluster formed by the type strains of the genus Brevibacterium. In addition, B. frigoritolerans and the environmental isolate EB93 were both observed to form a clade together with the type strains of the genus Peribacillus. The results from the analysis of the digital DNA-DNA hybridization, average nucleotide identity, average amino acid identity and the difference in the G+C content also corroborated with the phylogenetic inference, and that B. frigoritolerans and the environmental isolate EB93 were of the same species. Furthermore, the presence of the molecular synapomorphies in the protein sequences noted in the description of the genus Peribacillus were also observed in B. frigoritolerans, further strengthening its taxonomic affiliation in the genus. Based on the evidence from the multiple lines of analyses, we propose the reclassification of Brevibacterium frigoritolerans as a member of the genus Peribacillus and assume the name Peribacillus frigoritolerans comb. nov. (type strain DSM 8801 T=ATCC 25097T=CCUG 43489T=CIP 67.20T=JCM 11681T).

Biological recovery of phosphorus from waste activated sludge via alkaline fermentation and struvite biomineralization by Brevibacterium antiquum.

Struvite biomineralization is a promising method for phosphorus recovery from wastewater treatment plant streams, and the growth of responsible microorganisms in mixed cultures is one of the most critical points for applying this process in pilot and full-scale. This study aimed to investigate the growth and bio-struvite production of Brevibacterium antiquum in mixed sludge culture. Alkaline fermentation was applied at different pH conditions to enhance the phosphorus content of sludge for an efficient recovery, and pH 8 was determined as the most feasible considering the phosphorus release and sludge characteristics. Growth optimization studies showed that NaCl's presence decreases the growth rate of Brevibacterium antiquum and bio-struvite production. At the same time, pH in the range of 6.8-8.2 did not alter the growth significantly. In addition, studies showed the ability of Brevibacterium antiquum in unsterilized fermented sludge centrate to grow and recover the phosphorus as struvite. Thus, our results indicated the potential of struvite biomineralization in full-scale wastewater treatment plants.

ACC deaminase-producing Brevibacterium linens RS16 enhances heat-stress tolerance of rice (Oryza sativa L.).

The rapid rise in global temperature has adverse effects on rice productivity. The lack of eminent resources for heat stress alleviation is threatening the agricultural sector. Heat stress alleviation by endophytic plant growth-promoting bacteria (PGPB) can be a sustainable and eco-friendly approach. The present study was conducted to check the colonization of Brevibacterium linens RS16 producing ACC (1-aminocyclopropane-1-carboxylate) deaminase in the rice endosphere and to characterize its efficiency in enhancing stress tolerance. The ethylene emission pathway, reactive oxygen species (ROS) concentrations, proline accumulation, expression of glutathione S-transferase (GST), and small heat shock proteins (sHSPs) were monitored at two different levels of heat stress (40°C and 45°C). Bacterial inoculation decreased ethylene emission levels by 26.9% and 24.4% in rice plants exposed to 40°C and 45°C, respectively, compared with the non-inoculated plants. B. linens RS16 also enhanced the expression profiles of glutathione S-transferase. The collective effect of GST expression profiles and decrease in ethylene emission due to bacterial ACC deaminase activity subsequently resulted in a decrease in ROS concentrations. Additionally, HSP16 and HSP26 increased expression in heat-stressed plants inoculated with B. linens RS16 resulted in enhanced stress tolerance (i.e., lesser proline accumulation) than non-inoculated plants. Hence, this study demonstrates the bacteria-mediated tolerance against heat stress by regulating the ethylene emission pathway and upregulating antioxidant enzymes and heat shock proteins.

Cervical Squamous Intraepithelial Lesions Are Associated with Differences in the Vaginal Microbiota of Mexican Women.

Cervical cancer is an important health concern worldwide and is one of the leading causes of death in Mexican women. Previous studies have shown changes in the female genital tract microbe community related to human papillomavirus (HPV) infection and cervical cancer; yet, this link remains unexplored in many human populations. This study evaluated the vaginal bacterial community among Mexican women with precancerous squamous intraepithelial lesions (SIL). We sequenced the V3 region of the 16S rRNA gene in cervical samples from 228 Mexican women, including 121 participants with SIL, most of which were HPV positive, and 107 healthy women without HPV infection or SIL. The presence of SIL was associated with changes in composition (beta diversity) and with a higher species richness (Chao1). A comparison of HPV-positive women with and without SIL showed that microbiota changes occurred even in the absence of SIL. Multivariate association with linear models (MaAsLin) analysis yielded independent associations between HPV infection and an increase in the relative abundance of Brachybacterium conglomeratum and Brevibacterium aureum as well as a decrease in two Lactobacillus iners operational taxonomic units (OTUs). We also identified a positive independent association between HPV-16, the most common HPV subtype linked to SIL, and Brachybacterium conglomeratum. Our work indicates that HPV infection leading to SIL is primarily associated with shifts in vaginal microbiota composition, some of which may be specific to this human population. IMPORTANCE Human papillomavirus (HPV) plays a critical role in cervical carcinogenesis but is not sufficient for cervical cancer development, indicating the involvement of other factors. The vaginal microbiota is an important factor in controlling infections caused by HPV, and, depending on its composition, it can modulate the microenvironment in vaginal mucosa against viral infections. Ethnic and sociodemographic factors influence differences in vaginal microbiome composition, which underlies the dysbiotic patterns linked to HPV infection and cervical cancer across different populations of women. Here, we provide evidence for associations between vaginal microbiota patterns and HPV infection linked to ethnic and sociodemographic factors. To our knowledge, this is the first report of the species Brevibacterium aureum and Brachybacterium conglomeratum linked to HPV infection or squamous intraepithelial lesions (SIL).

Brevibacterium casei endophthalmitis after intravitreal dexamethasone implant.

CLINICAL CASE: 49-year-old man with diabetic macular edema refractory to antiangiogenics, it is decided to perform therapy with intravitreal dexamethasone implant (Ozurdex; Allergan, California, United States of America). Seven days after treatment, he showed acute endophthalmitis suggestive signs. Despite the intravitreal injection of antibiotics, the patient got worse. Vitreous sampling was repeated for Gram and cultures, and vitrectomy was performed via pars plana. The culture suggested the development of Brevibacterium species. Through an additional test, the presence of Brevibacterium casei was confirmed. Despite the treatment adjusted by antibiogram, retinal ischemia and macular atrophy was evident. DISCUSSION: Brevibacterium casei is a Gram-positive bacterium, barely pathogenic, that mainly affects immunodepressed patients. Only two cases of endophthalmitis are described, one endogenous and the other one secondary to vegetal trauma. This is the first case of endophthalmitis, secondary to an ophthalmological procedure.

Brevibacterium limosum sp. nov., Brevibacterium pigmenatum sp. nov., and Brevibacterium atlanticum sp. nov., three novel dye decolorizing actinobacteria isolated from ocean sediments.

During a study of the marine actinobacterial biodiversity, a large number of Brevibacterium strains were isolated. Of these, five that have relatively low 16S rRNA gene similarity (98.5-99.3%) with validly published Brevibacterium species, were chosen to determine taxonomic positions. On the basis of 16S rRNA gene sequence analysis and BOX-PCR fingerprinting, strains o2T, YB235T, and WO024T were selected as representative strains. Genomic analyses, including average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), clearly differentiated the three strains from each other and from their closest relatives, with values ranging from 82.8% to 91.5% for ANI and from 26.7% to 46.5% for dDDH that below the threshold for species delineation. Strains YB235T, WO024T, and o2T all exhibited strong and efficient decolorization activity in congo red (CR) dyes, moderate decolorization activity in toluidine blue (TB) dyes and poor decolorization in reactive blue (RB) dyes. Genes coding for peroxidases and laccases were identified and accounted for these strains' ability to effectively oxidize a variety of dyes with different chemical structures. Mining of the whole genome for secondary metabolite biosynthesis gene clusters revealed the presence of gene clusters encoding for bacteriocin, ectoine, NRPS, siderophore, T3PKS, terpene, and thiopeptide. Based on the phylogenetic, genotypic and phenotypic data, strains o2T, YB235T and WO024T clearly represent three novel taxa within the genus Brevibacterium, for which the names Brevibacterium limosum sp. nov. (type strain o2T = JCM 33844T = MCCC 1A09961T), Brevibacterium pigmenatum sp. nov. (type strain YB235T = JCM 33843T = MCCC 1A09842T) and Brevibacterium atlanticum sp. nov. (type strain WO024T = JCM 33846T = MCCC 1A16743T) are proposed.

The mechanisms of struvite biomineralization in municipal wastewater.

The mechanisms of struvite production by biomineralization were investigated for five microorganisms (Bacillus pumilus, Brevibacterium antiquum, Myxococcus xanthus, Halobacterium salinarum and Idiomarina loihiensis) in municipal wastewater. The microbial exponential phase of growth occurred within the first 48 h of incubation, with growth rates varying from 0.02-0.08 1/h. These five microorganisms removed 23-27 mg/L (66-79%) of ortho-phosphate from wastewater, which was recovered as biological struvite (i.e., bio-struvite) identified by morphological, X-ray diffraction and elemental analysis. Bio-struvite crystals occurred in a low extracellular supersaturation index (0.6-0.8 units). Bio-struvite formation in B. pumilus M. xanthus, H. salinarum cultures was linked to biologically induced mineralization. Whereas B. antiquum and I. loihiensis produced bio-struvite through biologically controlled mineralization mechanism because the crystals presented homogeneity in morphology and size, and intracellular vesicle-like cell structures were observed enclosing electron-dense granules/materials. Nutrient recovery through biomineralization has potential application in wastewater streams promoting circularity within the wastewater industry.

Antibiotic Therapy of an Infant With a Brevibacterium casei Ventriculoperitoneal Shunt Infection.

We describe a newborn infant with hydrocephalus and a ventriculoperitoneal shunt infection caused by Brevibacterium casei. Essential for correct diagnosis was rapid species identification by matrix-assisted laser desorption/ionization time-of-flight, after initial report of coryneform bacteria. The patient responded well to vancomycin and rifampicin for 15 days. The shunt was not removed. Repeated cerebrospinal fluid cultures up to 4 months after therapy remained negative.

Understanding the mechanisms of biological struvite biomineralisation.

The mechanisms of struvite production through biomineralisation were investigated for five microorganisms (Bacillus pumilus, Brevibacterium antiquum, Myxococcus xanthus, Halobacterium salinarum and Idiomarina loihiensis). After 72-96 h of incubation, the microbial strains tested increased the solution pH from 7.5 to 7.7 to 8.4-8.7, and removed ortho-phosphate (63-71%) and magnesium (94-99%) by biomineralisation. The minerals formed were identified as struvite (i.e. bio-struvite). Within the initial 24 h of incubation, microbial growth rates of 0.16-0.28 1/h were measured, and bio-struvite production was observed when the solution supersaturation index with respect to struvite achieved 0.6-0.8 units. The crystals produced by B. pumilus, H. salinarum and M. xanthus were thin trapezoidal-platy shaped and presented a gap size about 200 µm for intervals between cumulative volume undersize distribution at 50% and 90%. While B. antiquum and I. loihiensis produced crystals of coffin-lid/long-bar shape and a narrow size gap around 100 µm for intervals between cumulative volume percentage of 50% and 90%, indicating homogeneous crystal size distribution. Intracellular supersaturation of struvite phase was achieved within B. antiquum and I. loihiensis cells, corresponding to observation of intracellular vesicle-like structures occupied with electron-dense granules/materials. This study suggests that B. antiquum and I. loihiensis produced bio-struvite through biologically controlled mineralisation. This mechanism is the preferred for recovering nutrients from streams such as wastewater because it allows a link between manipulation of microbial growth conditions and bio-struvite production, even in highly complex streams like wastewater.

Unraveling the world of halophilic and halotolerant bacteria in cheese by combining cultural, genomic and metagenomic approaches.

Halophilic/halotolerant bacteria are generally assumed to live in natural environments, although they may also be found in foods such as cheese and seafood. These salt-loving bacteria have been occasionally characterized in cheese, and studies on their ecological and technological functions are still scarce. We therefore selected 13 traditional cheeses to systematically characterize these microorganisms in their rinds via cultural, genomic and metagenomic methods. Using different salt-based media, we identified 35 strains with unique 16S rRNA and rpoB gene sequences, whose whole genome was sequenced. Twenty are Gram-positive species including notably Brevibacterium aurantiacum (6) and Staphylococcus equorum (3), which are also frequently added as starters. ANI and pan-genomic analyses confirm the high genetic diversity of B. aurantiacum and reveal the presence of two subspecies in S. equorum, as well as the genetic proximity of several cheese strains to bovine isolates. Additionally, we isolated 15 Gram-negative strains, potentially defining ten new species of halophilic/halotolerant cheese bacteria, in particular for the genera Halomonas and Psychrobacter. The use of all the genomes sequenced in this study as a reference to complement those existing in the databases allowed us to study the representativeness of 66 species of halophilic/halotolerant bacteria in 74 cheese rind metagenomes. While Gram-positive strains may flourish in the different types of technologies, Gram-negative species are particularly abundant in cheeses with high moisture, such as washed-rind cheeses. Finally, analyses of co-occurrences reveal assemblies, including the frequent coexistence of several species of the same genus, forming moderately complex ecosystems with functional redundancies that probably ensure stable cheese development.

Effect of Metabolites Produced by Bacillus atrophaeus and Brevibacterium frigoritolerans Strains on Postharvest Biocontrol of Alternaria alternata in Tomato (Solanum lycopersicum L.).

In the present study, the antifungal activity of metabolites produced by Bacillus atrophaeus B5 and a new Brevibacterium strain against Alternaria alternata was evaluated. Assays in vitro and in vivo on tomato fruit during postharvest were made. Based on the 16S rDNA gene sequence analysis, the new strain (strain B7) was identified as Brevibacterium frigoritolerans. Metabolites produced by both bacterial strains reduced the spore germination of A. alternata in vitro and decreased the severity of the alternaria rot disease on tomato fruit during postharvest. This is the first report that demonstrates the potential of B. frigoritolerans B7 as a biocontrol agent against this fungal phytopathogen. The use of metabolites produced by B. atrophaeus B5 and B. frigoritolerans B7 represents a new approach to reduce the use of chemical pesticides and control fungal decay during the postharvest stage.

Nickel bioaccumulation by a marine bacterium Brevibacterium sp. (X6) isolated from Shenzhen Bay, China.

Nickel bioaccumulation capacity of a marine Brevibacterium sp., designated as X6, was evaluated to explore its potential application in the bioremediation of Ni2+ pollutants in marine environments. The minimum Ni2+ inhibitory concentration and maximum Ni2+ bioaccumulation of X6 were 1000 mg/L and 100.95 mg/g, respectively, higher than most reported strains. Among the co-existing metal ions in seawater, K+ caused a slight adverse impact on Ni2+ uptake, followed by Na+ and Ca2+, whereas Mg2+ drastically inhibited Ni2+ bioaccumulation. Other heavy metals such as Co2+, Zn2+ and Cd2+ moderately affected Ni2+ binding, but the adverse effect of Cu2+ was severe. The investigation of the mechanism of Ni2+ bioaccumulation revealed that 66.34% of the accumulated Ni2+ was bound to the cell surface. Carboxylic, hydroxyl, amino and thiol groups participated in Ni2+ binding, while carboxylic group contributed the most, while thiol group may be more involved in Ni2+ binding at low Ni2+ concentrations.

Temporal differences in microbial composition of Époisses cheese rinds during ripening and storage.

Époisses is a protected designation of origin smear-ripened cheese from the Burgundy region in France. It has an orange color and a strong flavor, both of which are generated by surface microorganisms. The objective of the present study was to investigate the microbial dynamics at the surface of Époisses cheese during ripening and postmanufacturing storage at low temperatures. Rind samples were analyzed by enumeration on agar plates and by 16S rRNA gene and internal transcribed spacer amplicon sequencing. During most of the ripening process, the counts of yeasts, which corresponded to the species Debaryomyces hansenii and Geotrichum candidum, were higher than those of the aerobic acid-sensitive bacteria. Debaryomyces hansenii reached a level of about 3 × 108 cfu/cm2, and its viability strongly decreased in the late stage of ripening and during storage at 4°C. Two of the inoculated bacterial species, Brevibacterium aurantiacum and Staphylococcus xylosus, did not establish themselves at the cheese surface. At the end of ripening, among the 18 most abundant bacterial species detected by amplicon sequencing, 14 were gram-negative, mainly from genera Psychrobacter, Vibrio, Halomonas, and Mesonia. It was hypothesized that the high moisture level of the Époisses rinds, due the humid atmosphere of the ripening rooms and to the frequent washings of the curds, favored growth of these gram-negative species. These species may be of interest for the development of efficient ripening cultures. In addition, because the orange color of Époisses cheeses could not be attributed to the growth of Brevibacterium, it would be interesting to investigate the type and origin of the pigments that confer color to this cheese.