Reclassification of Micrococcus aloeverae and Micrococcus yunnanensis as later heterotypic synonyms of Micrococcus luteus.

Micrococcus aloeverae,Micrococcus endophyticus, Micrococcus luteus and Micrococcus yunnanensis are phenotypically and genotypically closely related, and together comprise the M. luteus group. In this study, the taxonomic relationships among Micrococcus aloeverae, M. luteus and M. yunnanensis were re-evaluated by using polyphasic approaches. The similarity values of the concatenated housekeeping gene (gyrB, recA and rpoB) sequences shared by the type strains of M. aloeverae, M. luteus and M. yunnanensis ranged from 98.3 to 99.4 %. The average nucleotide identity, average amino acid identity and digital DNA‒DNA hybridization values among these three taxa were greater (97.1‒98.1 %, 96.8‒98.1 % and 75.0‒83.5 %, respectively) than the thresholds for bacterial species delineation, indicating that they belong to the same species, whereas those for M. endophyticus were clearly lower than the thresholds. In addition, phenotypic and chemotaxonomic characterization results also support the synonymy of these three taxa. Therefore, we propose that M. aloeverae and M. yunnanensis should be reclassified as later heterotypic synonyms of M. luteus.

Structural characterization and antioxidant potential of a novel anionic exopolysaccharide produced by marine Microbacterium aurantiacum FSW-25.

An exopolysaccharide (EPS) producing strain FSW-25 was isolated from the Rasthakaadu beach Kanyakumari, Tamil Nadu India. Based on polyphasic taxonomy, the strain FSW-25 was assigned to the genus Microbacterium and found to be the closest relative of the species aurantiacum. Large quantity of EPS (7.81 g/l) was secreted by the strain upon fermentation using Reasoner's 2A medium enriched with 2.5% glucose and was designated as Mi-25. FT-IR spectrum revealed presence of hydroxyl, carbonyl, carboxyl, methyl and sulfate functional groups in purified EPS. The EPS Mi-25 has a molecular weight of 7.0 × 106 Da and mainly comprises of glucuronic acid followed by glucose, mannose and fucose. Rheological study revealed that Mi-25 possesses significant viscosity with pseudoplastic nature. Interestingly, it was observed that the EPS Mi-25 has higher antioxidant activity as compared to xanthan. The characteristics of EPS Mi-25 suggested that, it can be used as a potential antioxidant with viscosifier properties in diverse industrial sectors.

The dorsal skin structure contributes to the surface bacteria populations of Sunda Porcupine (Hystrix javanica).

Skin becomes the largest organ in the body and protects its own inner layer. The structure and chemical composition of the skin contribute to skin condition and affect the habitat of certain bacteria. The Sunda Porcupine is one of endemic animals of Indonesia which possesses quill as the main derivate of its skin and as a defence tool against predators. The present study used nine adults (five females and four males) of Sunda Porcupine and aimed to observe the correlation of skin structure with bacterial population at the surface level. The skin was wavy due to the protrusion of quill follicle orifices on the skin surface and formed clusters. The skin of Sunda Porcupine was also wrinkled and had a lot of flakiness. Histologically, the skin was composed of epidermis, dermis, hypodermis and subcutaneous muscle. The quill follicles and their properties were the dominant structure component of the skin. No sweat gland was observed in the skin of the Sunda Porcupine, and sebaceous gland was found only around quill and hair follicles. The bacterias identified in the skin were Staphylococcus aureus, S. epidermidis, Micrococcus sp. and Salmonella sp. When compared, the bacterial population was higher in the lumbosacral region than in the thoracodorsal region, but the difference was not significant. The density of quill clusters was negatively correlated to the bacterial population. It was suggested the structure of the skin has contribution to bacterial population in dorsal trunk of the Sunda Porcupine.

Bioprocess enhancement of feather degradation using alkaliphilic microbial mixture.

1. The main aim of this work is to develop a robust method to generate a microbial mixture which can successfully degrade poultry feathers to overcome environmental problems. 2. Four different alkaliphilic microbes were isolated and shown to degrade poultry feathers. 3. Two of the isolates were phylogenetically identified as Lysinibacillus and the others were identified as Nocardiopsis and Micrococcus. 4. The best microbial co-culture for white and black feather degradation was optimised for pH, temperature and relative population of the isolates to achieve almost 96% of degradation compared with a maximum of 31% when applying each isolate individually. 5. The maximum activity of keratinase was estimated to be 1.5 U/ml after 3 d for white feathers and 0.6 U/ml after 4 d for black feathers in a basal medium containing feather as the main carbon source. Additionally, non-denaturing polyacrylamide gel electrophoresis showed 4 and 3 protease activity bands for white and black feather, respectively. 6. This study provides a robust method to develop potential new mixtures of microorganisms that are able to degrade both white and black feathers by applying a Central Composite Design.

Evaluation of autochthonous micrococcus strains as starter cultures for the production of Kedong sufu.

AIMS: The technological properties of 22 micrococcus strains from traditional fermented Kedong sufu were evaluated in order to develop autochthonous starter cultures. METHODS AND RESULTS: The proteolytic, autolytic and lipolytic activity, salt tolerance, production and degradation of the biogenic amines of six Micrococcus luteus, nine Kocuria kristinae and seven Kocuria rosea were evaluated. The results indicated that these micrococcus strains exhibited a certain technological diversity, and the results also indicated the best properties to be used in mixed starter cultures. Based on the above findings, two sets of autochthonous starters were formulated. Considering the physicochemical properties and sensory characteristics of sufu, the maturation period of sufu was shortened by 30 days. The profiles of free amino acids and peptides partly revealed the mechanism of sensory quality and shorter ripening time of sufu manufactured using autochthonous mixed starters. Compared to back-slopping fermentation, sufu manufactured with selected autochthonous starter cultures exhibited lower levels of total biogenic amines. CONCLUSIONS: The selected strains could be used as starter to avoid the accumulation of high concentrations of biogenic amines while also maintaining typical sensory characteristics and preserving the autochthonous strains of the traditional Kedong sufu. The maturation times of Kedong sufu were shortened by 30 days with application of the autochthonous starter. SIGNIFICANCE AND IMPACT OF THE STUDY: Autochthonous mixed starters can reduce the generation of biogenic amines, speed up the sufu maturation process and preserve typical sensory quality. Furthermore, the rotation of two sets of mixed starter cultures can effectively resist phage attack during the production of sufu.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

Classification and identification of pigmented cocci bacteria relevant to the soil environment via Raman spectroscopy.

A soil habitat consists of a significant number of bacteria that cannot be cultivated by conventional means, thereby posing obvious difficulties in their classification and identification. This difficulty necessitates the need for advanced techniques wherein a well-compiled biomolecular database consisting of the already cultivable bacteria can be used as a reference in an attempt to link the noncultivable bacteria to their closest phylogenetic groups. Raman spectroscopy has been successfully applied to taxonomic studies of many systems like bacteria, fungi, and plants relying on spectral differences contributed by the variation in their overall biomolecular composition. However, these spectral differences can be obscured due to Raman signatures from photosensitive microbial pigments like carotenoids that show enormous variation in signal intensity hindering taxonomic investigations. In this study, we have applied laser-induced photobleaching to expel the carotenoid signatures from pigmented cocci bacteria. Using this method, we have investigated 12 species of pigmented bacteria abundant in soil habitats belonging to three genera mainly Micrococcus, Deinococcus, and Kocuria based on their Raman spectra with the assistance of a chemometric tool known as the radial kernel support vector machine (SVM). Our results demonstrate the potential of Raman spectroscopy as a minimally invasive taxonomic tool to identify pigmented cocci soil bacteria at a single-cell level.

[Quantitative and qualitative composition of axilla microbiota in practically healthy individuals].

AIM: Characteristics of quantitative and qualitative composition of cultured microorganisms isolated from axilla skin of practically healthy individuals. MATERIALS AND METHODS: 77 practically healthy individuals aged 18 to 40 years were examined. Species identification of microorganisms was carried out byculture-morphologic, tinctorial and biochemical properties using time-of-flight mass spectrometer and rpoB gene amplification with subsequent direct sequencing. RESULTS: Quantitative evaluation of microbial composition of axilla skin microbiota in most of the practically healthy individuals varied in the 4-5 lg CFU/ml interval, whereas seeding of skin by this microbiota at the level of 8 lg CFU/ml was not detected. 158 strains of 24 microorganism species were identified in this biotope. Most of these strains (68.9%) belonged to Corynebacterium genus, 21.6% of strains--to Staphylococcus genus, 7.6% of strains--to Micrococcus genus and 1.9% of strains--Candida albicans. 16 species of corynebacteria were isolated with predomination of C. tuberculostearicum (40.3%), C. amycolatum (18.4%) and C. ureicelerivorans (14.8%) strains. The microbial landscape in most of the examined individuals (77.9%) was presented by microorganism association. CONCLUSION: Quantitative and qualitative species composition of cultured microorganisms isolated from axilla skin biotope of practically healthy individuals was characterized for the first time.

Description of Micrococcus aloeverae sp. nov., an endophytic actinobacterium isolated from Aloe vera.

A yellow Gram-stain-positive, non-motile, non-endospore -forming, spherical endophytic actinobacterium, designated strain AE-6(T), was isolated from the inner fleshy leaf tissues of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. Strain AE-6(T) grew at high salt concentrations [10% (w/v) NaCl], temperatures of 15-41 °C and a pH range of 5-12. It showed highest (99.7%) 16S rRNA gene sequence similarity with Micrococcus yunnanensis YIM 65004(T) followed by Micrococcus luteus NCTC 2665(T) (99.6%) and Micrococcus endophyticus YIM 56238(T) (99.0%). Ribosomal protein profiling by MALDI-TOF/MS also showed it was most closely related to M. yunnanensis YIM 65004(T) and M. luteus NCTC 2665(T). Like other members of the genus Micrococcus, strain AE-6(T) had a high content of branched chain fatty acids (iso-C15:0 and anteiso-C15:0). MK-8(H2) and MK-8 were the predominant isoprenoid quinones. Cell wall analysis showed an 'A2 L-Lys-peptide subunit' type of peptidoglycan and ribose to be the major cell wall sugar. The DNA G+C content was 70 mol%. Results of DNA-DNA hybridization of AE-6(T) with its closest relatives from the genus Micrococcus produced a value of less than 70%. Based on the results of this study, strain AE-6(T) could be clearly differentiated from other members of the genus Micrococcus. We propose that it represents a novel species of the genus Micrococcus and suggest the name Micrococcus aloeverae sp. nov., with strain AE-6(T) ( = MCC 2184(T) = DSM 27472(T)) as the type strain of the species.

Degradation of benzo[a]pyrene by bacterial isolates from human skin.

Polycyclic aromatic hydrocarbons (PAHs) are some of the most widespread xenobiotic pollutants, with the potentially carcinogenic high-molecular-weight representatives being of particular interest. However, while in eukaryotes, the cytochrome P450 (CYP)-mediated activation of benzo[a]pyrene (B[a]P) has become a model for metabolism-mediated carcinogenesis, the oxidative degradation of B[a]P by microorganisms is less well studied. This should be reason for concern as the human organ most exposed to environmental PAHs is the skin, which at the same time is habitat to a most diverse population of microbial commensals. Yet, nothing is known about the skin's microbiome potential to metabolise B[a]P. This study now reports on the isolation of 21 B[a]P-degrading microorganisms from human skin, 10 of which were characterised further. All isolates were able to degrade B[a]P as sole source of carbon and energy, and degradation was found to be complete in at least four isolates. Substrate metabolism involved two transcripts that encode a putative DszA/NtaA-like monooxygenase and a NifH-like reductase, respectively. Analysis of the 16S-rRNA genes showed that the B[a]P-degrading isolates comprise Gram(+) as well as Gram(-) skin commensals, with Micrococci being predominant. Moreover, microbial B[a]P-degradation was detected on all volunteers probed, indicating it to be a universal feature of the skin's microbiome.

[Micrococcus sp.--the pathogen of leaf necrosis of horse-chestnuts (Aesculus L.) in Kiev].

A group of phytopathogenic bacteria was isolated from patterns of drying horse-chestnuts (Aesculus L.), which grow in Kyiv. The properties of slowly growing, highly aggressive microorganisms have been described in the paper. They grow up on the 8-10th day after sowing. The investigated microorganisms form very small (0.5-1 mm in diameter) colonies on the potato agar. Bacteria are protuberant, shining, smooth with flat edges, they are pale yellow, yellow, or pink. The bacteria are Gram-positive, spherical, are disposed in smears singly, in pairs, as accumulations, or netting. They are aerobes, do not form spores, are not mobile. They are inert in respect of different sources of carbon. They reduce nitrates, do not dilute gelatin, do not hydrolyze starch, do not release hydrogen sulphide and indole. The bacteria are catalase-positive, oxidase-negative. They do not cause potato and carrot rot. They lose quickly their viability under the laboratory conditions. The saturated acids C 14:0; C 15:0; C16:0; C18:0 have been revealed in the composition of cellular fatty acids. Microorganisms are identified as Micrococcus sp. Under artificial inoculation this highly aggressive pathogen causes drying of the horse-chestnut buds and necrosis, which occupies 1/3-1/2 of the leaf plate. A wide zone of chlorosis, surrounding necrosis, may occupy the whole leaf surface. The infected leaves use to twist up from the top (apex) or along a midrib and to dry.

Micrococcus cohnii sp. nov., isolated from the air in a medical practice.

Three Gram-reaction-positive bacteria, isolated from the air in a medical practice (strains WS4601(T), WS4602) or a pharmaceutical clean room (strain WS4599), were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences of the three novel strains showed that they formed a distinct lineage within the genus Micrococcus, sharing 16S rRNA gene sequence similarities of 96.1-98.0 % with other species of this genus. Chemotaxonomic features also supported the classification of the three novel strains within the genus Micrococcus. The major cellular fatty acids of strain WS4601(T) were anteiso-C(15 : 0) and iso-C(15 : 0), the cell-wall peptidoglycan was of type A3α (L-Lys-L-Ala), and the predominant respiratory quinones were MK-7(H(2)) and MK-8(H(2)). The polar lipid profile contained diphosphatidylglycerol and phosphatidylglycerol, but no phosphatidylinositol. The G+C content of the genomic DNA was 70.4 mol%. Numerous physiological properties were found that clearly distinguished strains WS4599, WS4601(T) and WS4602 from established members of the genus Micrococcus. Based on the phenotypic and phylogenetic data, strains WS4599, WS4601(T) and WS4602 are considered to represent three different strains of a novel species of the genus Micrococcus, for which the name Micrococcus cohnii sp. nov. is proposed. The type strain is WS4601(T) (=DSM 23974(T)=LMG 26183(T)).

Putrescine biosensor based on putrescine oxidase from Kocuria rosea.

The novel putrescine oxidase based amperometric biosensor selectively measures putrescine, which can be considered as an indicator of microbial spoilage. Putrescine oxidase (PUOX, EC 1.4.3.10) was isolated from Kocuria rosea (Micrococcus rubens) by an improved and simplified purification process. Cells were grown on brain heart infusion medium supplemented with putrescine. Cell-free extract was prepared in Tris buffer (pH 8.0) by Bead-beater. A newly elaborated step based on three-phase partitioning (TPP) was applied in the purification protocol of PUOX. The purified enzyme was immobilized on the surface of a spectroscopic graphite electrode in redox hydrogel with horseradish peroxidase, Os mediator and poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as crosslinking agent. This modified working electrode was used in wall-jet type amperometric cell together with the Ag/AgCl (0.1M KCl) reference electrode and a platinum wire as auxiliary electrode in flow injection analysis system (FIA). Hydrogel composition, pH and potential dependence were studied. Optimal working conditions were 0.45 mLmin(-1) flow rate of phosphate buffer (66 mM, pH 8.0) and +50 mV polarizing potential vs. Ag/AgCl. The linear measuring range of the method was 0.01-0.25 mM putrescine, while the detection limit was 5 µM. Beer samples were investigated by the putrescine biosensor and the results were compared by those of HPLC reference method.

A simple and cost-effective cover-glass test for the differentiation between staphylococci and micrococci in clinical laboratory.

A cover-glass placed on a heavily inoculated culture plate clearly differentiates facultatively anaerobic staphylococci growing underneath the cover-glass after overnight incubation from nongrowing aerobic micrococci. Even if there are some exceptions, all medically significant staphylococci can grow in the test. Thus, the test provides a cost-effective and highly specific tool for separation of both genera which fundamentally differ in their pathogenicity.

Micrococcus lactis sp. nov., isolated from dairy industry waste.

A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).

Biodegradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1.

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.

Extraction, purification and characterization of a protease from Micrococcus sp. VKMM 037.

The haloalkaliphilic bacterium Micrococcus sp. VKMM 037, isolated from an effluent of the caustic soda industry, was found to produce a protease. Maximal proteolytic activity was observed in cell culture grown at 40 degrees C using 2% (w/v) glycerol, 2% (w/v) beef extract and 2% (w/v) peptone as nutrients in medium also containing 0.85 M NaCl with a pH of 10.0. An efficient purification procedure combining ammonium sulphate precipitation and Q-Sepharose ion-exchange chromatography was developed. The purified 41 kDa protease was stable in a temperature range between 20 degrees C and 60 degrees C. The protease remained active over a wide range of pH values (4.0-12.0) and NaCl concentrations (0-3.42 M) with an optimum at pH 10.0 and 0.85 M NaCl, respectively. Furthermore, the enzyme remained stable or was only marginally inhibited in the presence of various organic solvents, surfactants and reducing agents. The purified protease of Micrococcus sp. VKMM 037 efficiently removed blood stains within 40 minutes of treatment. Given the biochemical characteristics determined, this novel protease could be exploited as an additive in the detergent industry and also for the synthesis of biomolecules and the degradation of protein.

Isolation and characterization of novel plant growth promoting Micrococcus sp NII-0909 and its interaction with cowpea.

A phosphate-solubilizing bacterial strain NII-0909 isolated from the Western ghat forest soil in India was identified as Micrococcus sp on the basis of phenotypic characteristics, carbon source utilization pattern, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The strain exhibited the plant growth-promoting attributes of phosphate solubilization, auxin production, 1-aminocyclopropane-1-carboxylate deaminase activity, and siderophore production. It was able to solubilize (122.4µg of Ca(3)PO(4) ml(-1)), and produce IAA (109µgml(-1)) at 30°C. P-solubilizing activity of the strain NII-0909 was associated with the release of organic acids and a drop in the pH of the NBRIP medium. HPLC analysis detected two organic acids in the course of P-solubilization. A significant increase in the growth of cow pea was recorded for inoculations under controlled conditions. Scanning electron microscopic study revealed the root colonization of strain on cow pea seedlings. These results demonstrate that isolates NII-0909 has the promising PGPR attributes to be develop as a biofertilizer to enhance soil fertility and promote the plant growth.