Brevibacterium is a genus of Gram-positive, nonmotile, non-spore-forming bacteria in the family Brevibacteriaceae, phylum Actinobacteria, characterized by irregular rod-shaped cells that undergo a distinct rod-to-coccus growth cycle.[1] These strictly aerobic, halotolerant organisms possess a high DNA G+C content (58–71 mol%) and are notable for their ecological roles in food fermentation, particularly cheese ripening, as well as their presence as commensals on human skin and in diverse environmental niches.[2]Morphologically, Brevibacterium species appear as slender, irregular rods measuring 0.6–1.2 μm in width and 1.5–6 μm in length, and they are non-acid-fast and catalase-positive.[1] Physiologically, they thrive in a temperature range of 4–42°C with an optimum of 20–35°C (up to 37°C for human-associated isolates), at pH levels from 5.5 to 9.5, and demonstrate tolerance to high salt concentrations.[1] Key metabolic traits include the production of methanethiol from L-methionine, which contributes to distinctive sulfurous aromas, and the ability to degrade proteins and lipids.[3] Genomically, strains exhibit size heterogeneity from 2.3 to 4.5 Mbp, with cheese-adapted isolates averaging around 4 Mbp and featuring genes for osmotolerance (e.g., ectoine biosynthesis), iron acquisition via siderophores, and bacteriocin production.[2]The genus encompasses 41 recognized species, identified through 16S rRNA gene sequencing and DNA-DNA hybridization, with notable members including the type speciesB. linens, B. aurantiacum, and B. casei.[1][4] Ecologically, Brevibacterium inhabits dairy products, soil, marine environments, insects, mural paintings, and the skin of humans and animals, where it influences microbial communities through antimicrobial compounds like linecin A and linocin M18 that inhibit pathogens such as Listeria monocytogenes and Clostridium botulinum.[3] In industry, these bacteria are crucial for surface-ripened cheeses like Limburger and Munster, where they drive flavor development via volatile sulfur compounds, fatty acids, and carotenoid pigments (e.g., isorenieratene) for rind coloration, while also enhancing texture through enzymatic activity.[3] Some strains are utilized in biotechnology for producing flavor enhancers, such as glutamic acid for monosodium glutamate.[1]Although primarily non-pathogenic, certain Brevibacterium species, particularly B. casei, can cause opportunistic infections in immunocompromised patients, including catheter-related bacteremia, endocarditis, meningitis, and osteomyelitis, often requiring vancomycin treatment due to resistance to β-lactams and fluoroquinolones.[3] Additionally, skin-colonizing strains contribute to foot odor by metabolizing sweat components into volatile odorous compounds.[3] Comparative genomic studies reveal two major phylogenetic lineages, with evidence of horizontal gene transfer enhancing adaptations to cheese habitats, underscoring the genus's metabolic versatility and environmental resilience.[2]
Taxonomy
Etymology and history
The genus name Brevibacterium derives from the Latin adjective brevis, meaning short, and the Neolatin neuter noun bacterium (from Greek bakterion, small rod), alluding to the characteristically short, rod-shaped morphology of its members.[4]The genus was formally established in 1953 by Robert S. Breed in the seventh edition of Bergey's Manual of Determinative Bacteriology, where it was proposed as a new genus to accommodate certain Gram-positive, non-spore-forming rods previously scattered among other taxa.[4] The type species, Brevibacterium linens, originated from earlier work by Wolff, who isolated and described it as Bacterium linens in 1910 from the surface microflora of ripening Limburger cheese.[5] This description built on prior observations of similar organisms in dairy environments by researchers like Karl Weigmann, who had identified an "organism IX" in cheese studies around 1900.[5]Initial isolations of Brevibacterium species occurred primarily from dairy sources, such as milk, butter, and surface-ripened cheeses, during the early 20th century, with B. linens recognized for its association with smear-ripened varieties like Limburger and Brick cheese.[1] The name Bacterium linens was retained until Breed's reclassification, and the genus was officially validated under the International Code of Nomenclature in the Approved Lists of Bacterial Names published in 1980, which confirmed B. linens as the type species.Following its creation, the genus served as a repository for diverse coryneform bacteria, leading to taxonomic revisions in the 1960s through 1980s based on phenotypic traits such as cell morphology, pigmentation, and biochemical reactions; these efforts, including emendations by Collins et al. in 1980, restricted the genus to a more phylogenetically coherent group, reclassifying several misplaced species to other genera like Corynebacterium and Microbacterium.[1]
Classification and phylogeny
Brevibacterium is classified within the domain Bacteria, phylum Actinomycetota, class Actinobacteria, order Brevibacteriales, family Brevibacteriaceae, and genus Brevibacterium.[4] This hierarchical placement reflects its position among Gram-positive, high G+C content bacteria, with the family Brevibacteriaceae originally proposed by Breed in 1953 and subsequently emended based on molecular data, including the establishment of the order Brevibacteriales in 2020 (Salam et al.).[6] As of 2025, the genus encompasses 41 validly named species.[4]Phylogenetic studies utilizing 16S rRNA gene sequences position the genus Brevibacterium firmly within the classActinobacteria, where it forms a distinct clade closely related to genera such as Corynebacterium and Dietzia.[7] These analyses highlight shared evolutionary traits, including irregular rod-shaped morphology and adaptation to diverse environments, supported by sequence similarities exceeding 95% in conserved regions. The DNA of Brevibacterium species typically exhibits a high G+C content of 60–70 mol%, a hallmark of the phylumActinomycetota that aids in distinguishing it from lower G+C Gram-positive lineages.[8]Taxonomic reclassifications in the 1990s, driven by 16S rRNA-based phylogenetics, shifted Brevibacterium from the broader orderActinomycetales to Micrococcales, better aligning it with molecularly coherent groups like the suborder Micrococcineae.[6] This adjustment, proposed by Stackebrandt et al. in 1997, refined the understanding of actinobacterial diversity by emphasizing genomic and ribosomal evidence over phenotypic traits alone. A further update in 2020 reclassified it to the orderBrevibacteriales.[6] The genus itself was formally established by Breed in 1953 to accommodate short, non-spore-forming rods previously scattered across coryneform classifications.[4]
Description
Morphology
Brevibacterium species are Gram-positive, non-spore-forming bacteria characterized by irregular rod-shaped cells, often described as coryneform or club-shaped. These cells typically measure 0.6–1.2 μm in diameter and 1.5–6.0 μm in length, exhibiting variability in form during growth, including a rod-to-coccus transition in older cultures.[1][9] They frequently arrange in palisades, pairs, or angled V- and Y-shaped configurations, sometimes referred to as "Chinese letters," a feature common to coryneform bacteria.[1][8]The cell wall of Brevibacterium is composed of a thick peptidoglycan layer diagnostic of Gram-positive bacteria, featuring meso-diaminopimelic acid as the diamino acid in a directly cross-linked A1γ-type structure. This composition contributes to the retention of crystal violet during Gram staining, resulting in a positive reaction, while the absence of mycolic acids renders the cells non-acid-fast.[8][10]On solid agarmedia under aerobic conditions, Brevibacterium produces small, circular to irregular colonies measuring 0.5–2.0 mm in diameter after 24–48 hours of incubation. These colonies are convex, opaque, and range from smooth and shiny to rough in texture, often displaying yellow to orange pigmentation due to the accumulation of carotenoid compounds such as isorenieratene and dehydrogenated derivatives.[11][12][13] The coloration intensity can vary by strain and environmental factors, shifting from cream-yellow in young cultures to deeper orange-red tones with prolonged growth or light exposure.[1][14]
Physiology and biochemistry
Brevibacterium species are strictly aerobic, chemoorganotrophic bacteria that exhibit oxidative metabolism. They grow optimally at temperatures between 20°C and 30°C for environmental and food-associated strains, with some human isolates tolerating up to 37°C, and a broader growth range of 8–42°C. Optimal pH is 6.5–8.5, with tolerance from 5.5–9.5, and they are halotolerant, growing in media with up to 20% NaCl, though many strains prefer around 5%.[1][15][16]These bacteria utilize a variety of carbon sources, including glucose and fructose for energy, as well as amino acids such as L-methionine and glutamic acid, which support growth and contribute to metabolic processes like amino acidbiosynthesis. They produce small amounts of acid from carbohydrate fermentation but do not generate gas. Their metabolism emphasizes amino acid catabolism and proteolysis, enabling survival under nutrient-limited conditions through low endogenous metabolic rates and slow growth.[1][17][18]Biochemically, Brevibacterium species are catalase-positive and oxidase-negative, aiding in their identification. Urease activity is variable across species, as is nitrate reduction, which ranges from negative to weakly positive. The DNA base composition shows a high mol% G+C content, typically 59–72%, characteristic of the Actinobacteria phylum. Key enzymes include alkaline phosphatase and esterases (such as esterase C4 and esterase lipase C8), which facilitate hydrolysis of phosphates and lipids. Certain species produce sulfur-containing volatiles, notably methanethiol from L-methionine, contributing to their ecological roles.[1][16][15]
Habitat and ecology
Natural distribution
Brevibacterium species are widely distributed in terrestrial environments, particularly in soils across diverse ecosystems. They are commonly isolated from arable, forest, saline, and desert soils globally, including regions such as the Sahara and Gibson deserts, where they contribute to microbial communities adapted to arid conditions.[19] In these habitats, Brevibacterium plays roles in nutrient cycling, including nitrogen fixation and phosphate solubilization, which support soil fertility and ecosystem processes.[20]Aquatic and marine habitats also harbor Brevibacterium, with isolations reported from seawater, deep-sea sediments, and freshwater systems. For instance, species such as Brevibacterium oceani and Brevibacterium sediminis have been recovered from deep-sea sediments in the Indian Ocean and Carlsberg Ridge, respectively, demonstrating adaptation to high-pressure and saline conditions.[21][22] Some species display halophilic or halotolerant traits, enabling persistence in high-salinity environments like marine sediments and coastal areas.[23]Brevibacterium is frequently associated with plants, occurring on surfaces and in rhizospheres of various species, including Aloe vera and heavy metal-contaminated sites. In these niches, strains such as Brevibacterium casei promote plant growth through siderophore production, which enhances iron acquisition, alongside other mechanisms like indole acetic acid synthesis.[24]Overall, Brevibacterium exhibits global ubiquity in natural environments but typically at low abundance within microbial communities. Culture-independent methods, such as metagenomics, have detected the genus in soils, aquatic systems, and rhizospheres across multiple continents, underscoring its widespread yet non-dominant presence.[25]
Associations with environments
Brevibacterium species are prevalent in dairy processing environments, particularly on the rinds of surface-ripened cheeses where they form part of complex microbial consortia alongside bacteria like Arthrobacter and fungi such as Penicillium.[26] These bacteria contribute to the maturation process by colonizing cheese surfaces during ripening, with species like Brevibacterium linens and B. aurantiacum emerging dominantly in washed-rind varieties.[27] In fermented foods beyond dairy, Brevibacterium strains participate in microbial communities that influence product development, often originating from facility-specific environments.[28]As commensal members of the human skinmicrobiota, Brevibacterium species, including B. epidermidis and B. casei, are commonly found in moist areas such as the feet, where they contribute to odor production through metabolic activities.[29] They are also detected in mucosal sites and occasionally in the gastrointestinal tract, though less abundantly than on skin.[30] In clinical settings, Brevibacterium has been isolated from human blood, wounds, and peritoneal dialysis fluids, reflecting opportunistic colonization from skin flora in immunocompromised or catheterized individuals.[31][32]Brevibacterium species associate with animal-derived materials in agricultural contexts, including hides where they aid in degrading keratin-rich hair waste during leather processing.[33] Strains like B. luteolum exhibit proteolytic activity that facilitates hair breakdown, supporting eco-friendly waste management in tanning operations.[34] Additionally, Brevibacterium appears in animal gut microbiomes, such as in poultry, where it may increase in abundance under certain litter conditions, potentially influencing microbial diversity.[35]In polluted environments, certain Brevibacterium species demonstrate bioremediation potential by degrading hydrocarbons like pyrene in oil-contaminated sites.[36] For instance, B. sediminis isolates from petroleum-polluted areas efficiently metabolize polycyclic aromatic hydrocarbons.[36] Other strains, such as B. casei, tolerate heavy metals including lead, cadmium, and chromium in contaminated rhizospheres and wastewaters, aiding in metal sequestration or reduction.[37][38] While soil serves as a primary natural reservoir for Brevibacterium, human-modified sites amplify their ecological roles in remediation.[2]
Applications
Role in food production
Brevibacterium linens plays a pivotal role in the ripening of surface-ripened cheeses, particularly those with washed rinds, where it colonizes the cheese surface and contributes to the degradation of proteins and fats. As a key component of the surface microflora, B. linens performs proteolysis, breaking down caseins into free amino acids that serve as precursors for flavor development, and lipolysis, hydrolyzing triglycerides to release free fatty acids.[39] These metabolic activities enhance the texture and overall maturation of cheeses such as Limburger and Munster.[40]The bacterium is renowned for producing volatile sulfur compounds that impart the characteristic pungent, sulfurous aromas to these cheeses. Specifically, B. linens converts methionine into methanethiol and dimethyl disulfide, which are essential for the distinctive foot-like odor associated with smear-ripened varieties.[41] Additionally, its production of carotenoid pigments results in the orange-red coloration of the rind, enhancing the visual appeal and authenticity of traditional cheeses.[42] The halotolerance of B. linens enables its survival and proliferation on salted cheese surfaces during the washing process.[43]In commercial cheesemaking, B. linens strains are routinely inoculated as starter cultures to ensure consistent flavor profiles and rind development in washed-rind cheeses. Selected strains are applied directly to the curd or via brine washes to standardize the ripening process and accelerate maturation without bitterness.[44] These organisms are considered safe for use in food production by regulatory authorities such as the USDA.[45]Historically, B. linens has been integral to European cheesemaking traditions since the 19th century, naturally occurring on the rinds of soft, smear-ripened cheeses like those from Belgium and France, long before its formal isolation in 1910.[46] Its role in flavor and color formation has been harnessed in artisanal practices, contributing to the sensory diversity of regional dairy products.[47]
Industrial uses
Strains formerly classified as Brevibacterium flavum and B. lactofermentum (now Corynebacterium glutamicum) were among the first bacteria employed for industrial amino acid production in the 1960s and 1970s, with strains engineered through classical mutagenesis to overproduce L-lysine and L-threonine via fermentation processes using carbon sources like glucose or molasses.[48][49] These early efforts achieved yields of up to 65 g/L of L-lysine under optimized conditions, though production later shifted toward Corynebacterium glutamicum due to its superior efficiency and genetic tractability.[50] Metabolic engineering techniques, such as disrupting feedback inhibition in aspartate kinase and overexpressing key pathway enzymes like dihydrodipicolinate synthase, enhanced overproduction in these strains.[51]Certain Brevibacterium isolates exhibit bioremediation potential through enzymatic degradation of environmental pollutants, including sulfur-containing compounds like dibenzothiophene and petroleum hydrocarbons.[52] For instance, Brevibacterium sp. DO utilizes dibenzothiophene as a sole carbon, sulfur, and energy source, cleaving the sulfur via desulfurization enzymes to produce 2-hydroxybiphenyl without ring destruction.[53] Similarly, Brevibacterium sp. BS18 achieves up to 70% degradation of crude oil components in contaminated soils over 30 days, highlighting its role in microbial consortia for oil spill cleanup.[54]Beyond amino acids, Brevibacterium strains contribute to metabolite production for pharmaceutical and antioxidant applications, notably through carotenoid biosynthesis. B. linens naturally produces pigments like isorenieratene and β-carotene, with yields varying by strain (up to 1.5 mg/g dry cell weight) and optimized via media adjustments like yeast extract supplementation.[55] Genetic reconstruction of the crt operon in B. linens DSMZ 20426 has extended pathways to yield astaxanthin and zeaxanthin, compounds with high antioxidant activity used in nutraceuticals.[56] Research also explores enzyme production, such as proteases from B. linens for industrial hydrolysis, though yields remain modest compared to amino acid processes.[57]Recent genomic studies have identified biosynthetic gene clusters in Brevibacterium strains from diverse ecosystems, suggesting potential for novel antibiotic production. Additionally, strains such as B. casei EB3 demonstrate applications in agricultural biotechnology by enhancing rhizobacterial communities to promote plant growth.[25][58]Strain development relies on genetic tools like shuttle vectors (e.g., pCB192) for introducing plasmids into Brevibacterium, enabling stable expression of heterologous genes for pathway engineering.[59] Techniques include electroporation for transformation efficiencies up to 10^4 transformants/μg DNA and CRISPR-based editing for precise knockouts, as demonstrated in recent studies on B. linens.[60] These bacteria's tolerance to osmotic stress and high temperatures supports their viability in robust industrial fermentations.[61]
Clinical significance
Infections in humans
Brevibacterium species are opportunistic pathogens that primarily cause infections in immunocompromised individuals, with documented cases including bacteremia, endocarditis, and peritonitis, particularly in patients undergoing peritoneal dialysis. These infections arise from translocation of the bacteria, which are part of the normal human skinflora, often facilitated by invasive procedures such as central venous catheters or wounds. The first reported human infection by Brevibacterium dates back to a case of meningitis in 1969, though recognition as clinically significant pathogens increased in the 1990s with the identification of species like B. casei from clinical isolates previously classified under CDC coryneform groups.[29][62]As of February 2025, 42 cases of Brevibacterium infections in humans had been documented across the literature in a narrative review, underscoring their rarity, with a median patient age of 48 years and a slight male predominance (57.5%). Bacteremia accounts for the majority (57.1%) of cases, frequently associated with catheter-related bloodstream infections; endocarditis represents 7.1% of reports, including the first documented instance in 2002 caused by B. otitidis in a patient with prosthetic heart valves; and peritonitis comprises 16.7%, often linked to peritoneal dialysis, with an early case of B. otitidis reported in 2000. An additional case of B. ravenspurgense bacteremia was reported later in 2025 in a patient with adrenoleukodystrophy.[29][63][64][65] Common risk factors include the presence of central venous catheters (41.5% of cases), underlying malignancies (25%), and end-stage renal disease managed with peritoneal dialysis (17.5%), highlighting the role of medical interventions in predisposing vulnerable patients. Despite their low virulence, Brevibacterium species can persist in biofilms, contributing to chronic or relapsing infections in these settings.[29]The most frequently isolated species is B. casei (47.6% of cases), followed by B. epidermidis (7.1%), B. otitidis (7.1%), and B. sanguinis (2.4%), with isolates commonly recovered from blood, peritoneal fluid, and valvular tissue. A case of B. ravenspurgense was also reported in 2025. Diagnostic challenges stem from the morphological similarity of Brevibacterium to Corynebacterium species, leading to frequent initial misidentifications in routine culture; accurate confirmation typically requires advanced molecular methods such as 16S rRNA gene sequencing (used in 31% of cases) or matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS, employed in 33.3%). These techniques have been essential in distinguishing Brevibacterium from more common coryneform contaminants, enabling proper recognition of its pathogenic potential in clinical contexts.[29][65][66]
Antimicrobial susceptibility
Brevibacterium species demonstrate consistent susceptibility to vancomycin, with resistance reported in only 8.1% of isolates across reviewed cases, and no instances of high-level vancomycin resistance documented to date.[29] They also exhibit high sensitivity to linezolid, with all tested clinical isolates susceptible in multiple studies, and to tetracyclines, where resistance rates remain low at 5.3%.[29][67] In contrast, resistance to beta-lactam antibiotics is more variable, affecting 50% of isolates for penicillin and 34.8% for cephalosporins, often linked to beta-lactamase production in strains such as B. casei and B. ravenspurgense.[29][68]Resistance mechanisms primarily involve intrinsic low-level tolerance to certain agents and enzymatic inactivation via beta-lactamases, which hydrolyze penicillin and cephalosporins in susceptible strains; acquired resistance through mobile elements like plasmids appears rare in clinical contexts.[29][69] Minimum inhibitory concentrations (MICs) from susceptibility testing underscore this profile, with MIC<sub>90</sub> values typically below 4 μg/mL for vancomycin (≤0.5 μg/mL), linezolid (1-2 μg/mL), tetracyclines (≤4 μg/mL), and carbapenems like meropenem (≤0.5 μg/mL), while beta-lactams show elevated MICs in resistant cases (e.g., penicillin MIC 0.5-8 μg/mL, intermediate to resistant).[70][71][72]Treatment guidelines recommend empirical therapy with glycopeptides such as vancomycin for severe Brevibacterium infections, particularly in immunocompromised patients, pending susceptibility results to guide de-escalation.[29] Clinical outcomes are favorable, with cure rates exceeding 90% when appropriate antibiotics are administered promptly; reported mortality is low at 10.3% overall (7.7% attributable to infection) in case series up to 2025, emphasizing the importance of speciesidentification and targeted therapy.[29]
Beta-lactamase mediated; use carbapenems if needed.[29][70]
Species
Type species
Brevibacterium linens serves as the type species for the genus Brevibacterium, providing the foundational reference for taxonomic classification within this group of Gram-positive bacteria. Originally described as Bacterium linens by Wolff in 1910 from butter and cheese surfaces, it was reclassified into the genus Brevibacterium by Breed in 1953. The species description was emended by Collins et al. in 1980 to incorporate chemotaxonomic data, including cell wall composition and fatty acid profiles, and further refined in subsequent studies during the 2000s to account for genomic insights.[75] The type strain, DSM 20425 (also known as ATCC 9172), was isolated from a dairy environment and maintains the defining characteristics of the species.[76]Morphologically, B. linens consists of irregular rods that undergo a characteristic rod-coccus growth cycle, appearing as short, club-shaped cells in young cultures and transitioning to coccoid forms in older ones; it is non-motile, non-sporeforming, and strictly aerobic. Colonies are typically yellow- to orange-pigmented due to carotenoid production, and the species is notable for generating linens-specific volatiles, such as methanethiol and dimethyl sulfide, which contribute to its distinct aroma profile. This bacterium is primarily isolated from the rinds of smear-ripened cheeses, where it thrives in the surface microbial community.[77][43][1]Physiologically, B. linens exhibits strong proteolytic activity, secreting extracellular enzymes that hydrolyze caseins and other proteins into peptides and free amino acids, followed by deamination to produce ammonia as a key metabolic end product. This ammonia generation raises the pH of the surrounding environment and is central to its biochemical role. Optimal growth occurs at 20–30°C, with many strains peaking around 25–28°C, and requires moderate salinity of 5–10% NaCl, though it demonstrates halotolerance up to 15% NaCl, enabling survival in saline dairy habitats.[78][79][1]As the archetype for the Brevibacteriumgenus, B. linens exemplifies the group's coryneform morphology, salt tolerance, and enzymatic capabilities, serving as a benchmark in taxonomic delineations. It is extensively employed in comparative genomics studies to elucidate genetic adaptations across brevibacteria, including gene clusters for pigmentation and flavor compound biosynthesis. In cheese production, B. linens contributes to rind coloration and volatile aroma development during ripening.[2][80]
Diversity and notable species
As of November 2025, the genusBrevibacterium comprises 41 validly published species, according to the List of Prokaryotic names with Standing in Nomenclature (LPSN).[4] This diversity reflects ongoing discoveries from diverse habitats, with recent additions including B. album (validly published in 2008), B. ammoniilyticum (2013), and B. anseongense (2019).[4]Among these, several species stand out for their ecological or clinical significance. Brevibacterium casei, frequently isolated from human clinical specimens, is notable for causing opportunistic infections such as bacteremia and endocarditis, particularly in immunocompromised individuals; it forms white-gray colonies on culture media.[3][29]Brevibacterium epidermidis, a common skin commensal, contributes to the bacterial flora on human skin surfaces with higher pH values, such as intertriginous areas, and is characterized by its aerobic growth and association with foot odor production through volatile compounds.[81][82]Brevibacterium iodinum, often found in aquatic and soil environments including water and raw milk, produces distinctive purple phenazine pigments like iodinin and is implicated in rare opportunistic infections.[83][40]Brevibacterium mcbrellneri, isolated from clinical sites such as infected genital hair in cases of white piedra, exhibits potential clinical relevance in superficial infections, though its pathogenicity remains understudied; it is distinguished by its Gram-positive rod morphology and isolation from fungal co-infections.[84][85]Brevibacterium aurantiacum is notable for its role in cheese rind coloration due to carotenoid pigments.[1]The taxonomy of Brevibacterium has seen reclassifications and synonymies due to phylogenetic reevaluations. For instance, Brevibacterium halotolerans was reclassified as Bacillus halotolerans based on phylogenomic and DNA-DNA relatedness analyses.[86] Similarly, Brevibacterium frigoritolerans was reclassified as Peribacillus frigoritolerans comb. nov. based on phylogenomics and multiple molecular synapomorphies.[87] Invalid proposals include "Brevibacterium pigmentatum" (2021), now a synonym of an unvalidly published name.[4]Species identification within Brevibacterium relies on polyphasic taxonomy, integrating genotypic methods like 16S rRNA gene sequencing (with similarity thresholds often below 98.7% indicating novel species) and DNA-DNA hybridization (values >70% for conspecificity), alongside phenotypic traits and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid differentiation.[88][89] This approach ensures robust classification amid the genus's heterogeneity.[90]