Functional antagonism and insights into the biosynthetic potential of human gut-derived microbes.

The specialised small molecules encoded by commensal microbes mediate distinct functional interactions. However, there is a landscape of antagonistic interactions mediated by specialised strains and their small molecules. Herein, the antagonistic landscape within a collection of 330 human gut-derived commensal microbial strains was elucidated to evaluate antimicrobial interactions as a defensive contributor, and gain new insights into structure-related functions. The potential antagonistic gut-derived strains displayed strain-specific selective inhibition. This is in contrast to common antimicrobial drugs, which typically wipe out a broad range of species and are usually found in environmental microbes. Genome sequencing of representative gut strains revealed the presence of significant biosynthetic gene clusters (BGCs) encoding compound families that contribute to antagonistic activities, and are important in host defence and maintaining gut homeostasis. Subsets of these BGCs were abundant in metagenomic sequencing data from healthy individuals. Furthermore, the cell culture secretome of these strains revealed potential biomarkers linked to hallmark pathways. These microorganisms have biosynthetic novelty and are a source of biologically significant natural products. Such natural products are essential in the development of new antimicrobial agents to reduce the usage of broad-spectrum antibiotics and combat antimicrobial resistance.

Anaerobic growth and drug susceptibility of versatile fungal pathogen Scedosporium apiospermum.

Although severe cases of invasive mycoses of different hypoxic and anoxic body parts have been reported, growth and drug susceptibility of fungal pathogens under anaerobic conditions remains understudied. The current study evaluated anaerobic growth potential and drug susceptibility of environmental Scedosporium apiospermum isolates under aerobic and anaerobic conditions. All tested strains showed equivalent growth and higher sensitivity to tested antifungal drugs under anaerobic conditions with lower minimum inhibitory concentration (MIC) as compared to aerobic conditions. Antifungal azoles were effective against isolates under both aerobic and anaerobic conditions. Most strains were resistant to antifungal echinocandins and polyenes under aerobic conditions but exhibited sensitivity under anaerobic conditions. This study provides evidence that resistance of S. apiospermum to antifungal drugs varies with oxygen concentration and availability and suggests re-evaluating clinical breakpoints for antifungal compounds to treat invasive fungal infections more effectively.

Draft Genome Sequence of Ignatzschineria sp. Strain RMDPL8A, a Bacterium Isolated from Landfill Leachate.

The genome of Ignatzschineria sp. strain RMDPL8A was sequenced and analyzed. This draft genome sequence was 2,175,527 bp long, with a GC content of 45.12% and 1,890 protein coding genes.

Human small-intestinal gluten-degrading bacteria and its potential implication in celiac disease.

Celiac disease (CeD) is an immune-mediated chronic disorder triggered by the ingestion of wheat gluten in genetically predisposed individuals. Gluten is a major food ingredient, infamously containing proline and glutamine-rich domains that are highly resistant to digestion by mammalian proteolytic enzymes. Thus, adhering to a gluten-free diet (GFD) is the only known treatment for CeD, albeit with many complications. Therefore, any therapy that eliminates the gluten immunogenic part before it reaches the small intestine is highly desirable. Probiotic therapy containing gluten-degrading bacteria (GDB) and their protease enzymes are possibly new approaches to treating CeD. Our study aimed to identify novel GDB from the duodenal biopsy of the first-degree relative (FDR) subjects (relatives of diseased individuals who are healthy but susceptible to celiac disease) with the potential to reduce gluten immunogenicity. Using the gluten agar plate technique, bacterial strains Brevibacterium casei NAB46 and Staphylococcus arlettae R2AA77 displaying glutenase activity were screened, identified, and characterized. Whole-genome sequencing found gluten-degrading prolyl endopeptidase (PEP) in the B. casei NAB46 genome and glutamyl endopeptidase (GEP) in the S. arlettae R2AA77 genome. Partially purified PEP has a specific activity of 1.15 U/mg, while GEP has a specific activity of 0.84 U/mg, which are, respectively, 6- and 9-fold times higher after concentrating the enzymes. Our results showed that these enzymes could hydrolyse immunotoxic gliadin peptides recognized in western blot using an anti-gliadin antibody. Additionally, a docking model was proposed for representative gliadin peptide PQPQLPYPQPQLP in the active site of the enzymes, where the residues of the N-terminal peptide extensively interact with the catalytic domain of the enzymes. These bacteria and their associated glutenase enzymes efficiently neutralize gliadin immunogenic epitopes, opening possibilities for their application as a dietary supplement in treating CeD patients.

Resolution of inter/intraspecies variation in Weissella group requires multigene analysis and functional characterization.

Weissella confusa and Weissella cibaria strains isolated from the human- gut are considered as potential probiotics, but remain under-explored owing to their ambiguous taxonomic assignment. The present study assesses the taxonomic resolution of 11 strains belonging to W. confusa and W. cibaria species and highlights the inter- and intraspecies variations using an array of phenetic and molecular methods. Remarkable genomic variability among the strains was observed by phylogenetic analysis using concatenated housekeeping genes (pheS, gyrB, and dnaA) along with 16S rRNA gene sequence, suggesting intraspecies variations; which is also supported by the phenetic data. Analysis showed that 16S rRNA gene sequence alone could not resolve the variation, and among the tested marker genes, signals from pheS gene provide better taxonomic resolution. The biochemical and antibiotic susceptibility tests also showed considerable variations among the isolates. Additionally, 'quick' identification using mass spectroscopy-based matrix-assisted laser desorption/ionization-time of flight mass spectra was accurate up to genus only, and not species level, for the Weissella group. The study highlights need for inclusion of functional, phenetic, and multigene phylogenetic analysis in addition to 16S rRNA gene-based identification for the Weissella group, to provide better resolution in taxonomic assignments, which is often a prerequisite for the selection of potential strains with biotechnological applications.

Culture-based and culture-independent approach for the study of the methanogens and obligate anaerobes from different landfill sites.

The landfill is a cheap way of solid waste management in developing countries. The majority of landfills are non-sanitary and work as open garbage dumping sites and pose threats to public and environmental health. Therefore, an in-depth understanding of the chemistry and microbiology of landfills is imperative to develop the right policies for landfill management. In the current study, we investigated the chemistry and microbiology of three Indian landfill sites using culture-based and culture-independent molecular approaches. Our data indicate that the nature of landfills varies from site to site in terms of chemistry, pollutants, and pathogens. We also enriched and cultivated three methanogens using an optimized medium and constructed two high-quality draft genomes from enriched microbiomes using metagenome-assembled genome approaches. The phylogenomic study of one draft genome showed the highest 93% sequence similarity with members of Methanomassiliicoccaceae and was always enriched with Acholoplasma and Anaerohalosphaera lusitana. Despite all the efforts, we did not isolate it in pure culture and hypothesized that for the cultivation of some not-yet-cultured methanogen, the presence of other organisms plays an important role, and their syntrophic interaction must be discerned for its successful cultivation in the future. Co-cultivation of amino acid-degrading organisms indicates that their co-culture can assist in boosting the growth of methanogens. In addition, our data indicated that landfill leachate contains a heavy load of pollutants and treatment is a must before discharge in nature or use in irrigation or biofertilizer.

A culture-based and culture-independent approach to the study of landfill leachate bacterial and archaeal communities.

The landfill is a convenient and affordable method of municipal solid waste (MSW) management. Landfill leachate contains a heavy load of pollutants and pathogens. Discharge of untreated leachate is the leading cause of surface and groundwater contamination and a threat to public and environmental health. To develop an efficient leachate treatment technology, an in-depth understanding of landfill chemistry and microbiology is essential. In the present manuscript, we conducted a comparative study of three different landfill leachate samples using cultivation-based and culture-independent molecular studies. We cultivated 85 species of aerobic, anaerobic bacteria and archaea from leachate represented by a total of 200 strains using extensive culturomics approaches. Twelve out of 200 cultivated strains of bacteria showed very low 16S rRNA gene sequence similarity (84-98.6%) with their closest relatives and could be the potential novel taxa, the first time cultivated from leachate. Members of the six genera only have 2-5 representatives from past studies from other habitats but first time cultivated from leachate. In addition to bacteria, we also cultivated and characterized different groups of methanogenic archaea. Our chemistry data indicate that leachate is a highly stressed ecosystem with an assemblage of many toxic wastes like sulfur, zinc, mercury, chromium, etc. 16S rRNA gene-based amplicon analysis showed the dominance of (30-55%) methanogens and haloarachaea. Our data suggest that archaea are the significant regulators of leachate ecology, and more in-depth studies with multiple leachate samples are required to understand their role in leachate nutrient cycling and the development of effective leachate treatment technology.

Oligotrophy vs. copiotrophy in an alkaline and saline habitat of Lonar Lake.

We reported our comparative observations on oligotrophs vs. copiotrophs from a hyper-alkaline and hypersaline habitat, Lonar Lake, situated in the Buldhana district of Maharashtra, India. Cell numbers of oligotrophic and copiotrophic microbes from the sediment were enumerated by the three-tube most probable number (MPN) method using an array of nutrient-rich and oligotrophic (≈10-20 mg carbon L-1) media offering simulated natural conditions of pH and salinity. A total of 50 strains from 15 different genera and 30 different species were isolated from the highest positive dilutions of MPN to identify the taxa of oligotrophs and copiotrophic microorganisms dominating in Lonar Lake. We did not get any true oligotrophs due to their adaptation to higher carbon levels during the isolation procedure. On the contrary, several true copiotrophs, which could not adapt and survive on a low-carbon medium, were isolated. It is also observed that changes in medium composition and nutrient level altered the selection of organisms from the same sample. Our data indicate that copiotrophic microorganisms dominate the eutrophic Lonar Lake, which is also supported by the past metagenomics studies from the same site. We also reported that quick depletion of carbon from oligotrophic medium worked as a limiting factor, inducing cell death after 2-3 generations and preventing the development of visible colonies on plates and sufficient optical density in liquid medium. Therefore, a long-term supply of low levels of carbon, followed by isolation on enriched media, can serve as a good strategy in isolation of novel taxa of microorganism, with industrial or environmental importance.

A Microcosm Model for the Study of Microbial Community Shift and Carbon Emission from Landfills.

The landfill is an inexpensive way of municipal solid waste (MSW) management and contributes extensively to the total carbon budget and global climate change. Three landfills from two geographically distinct metro- cities of India were taken as model systems to create microcosms and study their physiochemistry, microbiology, and carbon emission. The microcosm experiments revealed that facultative anaerobic bacterial community showing the dominance in the beginning but with the progression of anoxia and anaerobic conditions, methanogenesis prevailed, resulting in a clear shift towards the abundance of methanogens especially the members of Methanosarcina, Methanocorpusculum, and Methanoculleus (70-90% of the total microbial population). Geochemical data showed a wide range of heterogeneity in landfills' composition located even in the same city. In past, greenhouse gas emission from landfills is mainly estimated using different models which lack accuracy. As limited information is available as of now, this study can elicit researcher interest for in-depth characterization of microbial diversity and carbon emission from landfills. The microcosm model presented in the current study is a robust and straightforward method of accurate estimation of amounts of different types of gases release from landfill. It can also be extrapolate for estimation of different gases release from actual landfill sites by setting the on-site experiments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00995-7.

Two-Dimensional Cell Separation: a High-Throughput Approach to Enhance the Culturability of Bacterial Cells from Environmental Samples.

Culture-independent sequence data from various environmental samples have revealed an immense microbial diversity of environmental, clinical, and industrial importance that has not yet been cultured. Cultivation is imperative to validate findings emerging from cultivation-independent molecular data and exploit the isolated organisms for biotechnological purposes. Efforts have been made to boost the cultivability of microbes from environmental samples by use of a range of techniques and instrumentation. The manuscript presents a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. By employing gradient centrifugation combined with serial dilution ("two-dimensional cell separation"), significantly higher numbers of genera (>2-fold higher) and species (>3-fold higher) were isolated from environmental samples, including soil, anaerobic sludge, and landfill leachate, than from using serial dilution alone. This simple and robust protocol can be modified for any environment and culture medium and provides access to untapped microbial diversity. IMPORTANCE In the manuscript, we have developed a novel yet simple and innovative approach to improving the cultivability of natural microorganisms without sophisticated instrumentation. The method used gradient centrifugation combined with serial dilution (two-dimensional cell separation) to improve taxum recovery from samples. This simple and robust protocol can be modified for any environment and culture medium and provides access to untapped microbial diversity. This approach can be incorporated with less labor and complexity in laboratories with minimal instrumentation. As cultivation is a workflow that is well suited to lower-resource microbiology labs, we believe improvements in cultivability can increase opportunities for scientific collaborations between low-resource labs and groups focused on high-resource cultivation-independent methodologies.

Antibiotic susceptibility of human gut-derived facultative anaerobic bacteria is different under aerobic versus anaerobic test conditions.

Facultative anaerobes are the most common cause of infections in anoxic parts of the human body, including deep wound, vagina, periodontal pockets, gastrointestinal tract, genitourinary tract and lungs. Generally, antibiotic susceptibility tests (AST) for facultative anaerobes are performed under aerobic conditions due to ease of handling and rapid growth. However, variation in susceptibility of facultative anaerobes to antibiotics under aerobic and anaerobic conditions can lead to failure of antibiotic treatment. Our study evaluated the susceptibility of facultative anaerobic microorganisms to antibiotics during growth under anaerobic or aerobic conditions. We compared the resistance patterns of representatives from 15 bacterial genera isolated from the human-gastrointestinal tract against 22 different antibiotics from six classes under aerobic and anaerobic conditions. Preliminary results obtained by a disc diffusion method were verified using minimum inhibitory concentration (MIC) testing. The results demonstrated that 7-strains had a similar pattern of drug resistance under both conditions, while the remaining ten strains had significant differences in resistance patterns between aerobic and anaerobic conditions for at least one antibiotic. We conclude that successful antibiotic therapy for host-associated pathogens requires proper assessment of the oxygen condition of the growth environment and MIC testing of each pathogen under anaerobic and aerobic conditions.

A Recent Overview of Microbes and Microbiome Preservation.

Microbes are mediators in almost all ecosystem processes and act as a pivotal game changer in various ecological activities, globally. Therefore, understanding of microbial community structure and related functions in different environmental and micro-environmental niches is not only critical, but also a matter of greatest importance. Due to our inability to cultivate and preserve all sorts of microorganisms, we are losing some ecologically and industrially relevant components of microbial community, due to extinction caused by environmental and climatic variations with time. Intact sample and microbiome preservation are crucial for future cultivation as well as to study the effects of ecological and climatic variations on community functionality and shift with time, using OMICS. Although, methods for pure culture preservation are almost optimized, the techniques of microbiome preservation still remain as an unsolved challenge for microbiologists due to technical and physiological constraints. Present article discusses, recent approaches of microbial preservation with special reference to intact sample, mixed culture and microbiome preservation. It also incorporates recent practices used to achieve the highest viability and metabolic activities in long-term preserved microbiome.

Assessment of the Role of Wastewater Treatment Plant in Spread of Antibiotic Resistance and Bacterial Pathogens.

In current study, we performed a comparative study on bacterial load, total coliform counts and type of organisms present in pre- and post-treated wastewater samples from municipal wastewater treatment plant of Pune, India. In addition, we also studied the antibiotic resistance profiling and role of the selected treatment plant in spread of antibiotic resistance in the environment. Data showed that total 30 different bacterial species from 18-different genera were present in untreated wastewater while only 9 species from 6-different genera were present in post-treated effluent. Furthermore, pre-treated wastewater sample contains wide range of organisms with high levels of antibiotic resistance while bacterial load reduced drastically and pathogens were absent from post-treated effluent.

Comparative genome analysis reveals key genetic factors associated with probiotic property in Enterococcus faecium strains.

BACKGROUND: Enterococcus faecium though commensal in the human gut, few strains provide a beneficial effect to humans as probiotics while few are responsible for the nosocomial infection. Comparative genomics of E. faecium can decipher the genomic differences responsible for probiotic, pathogenic and non-pathogenic properties. In this study, we compared E. faecium strain 17OM39 with a marketed probiotic, non-pathogenic non-probiotic (NPNP) and pathogenic strains. RESULTS: E. faecium 17OM39 was found to be closely related with marketed probiotic strain T110 based on core genome analysis. Strain 17OM39 was devoid of known vancomycin, tetracycline resistance and functional virulence genes. Moreover, E. faecium 17OM39 genome was found to be more stable due to the absence of frequently found transposable elements. Genes imparting beneficial functional properties were observed to be present in marketed probiotic T110 and 17OM39 strains. Genes associated with colonization and survival within gastrointestinal tract was also detected across all the strains. CONCLUSIONS: Beyond shared genetic features; this study particularly identified genes that are responsible for imparting probiotic, non-pathogenic and pathogenic features to the strains of E. faecium. Higher genomic stability, absence of known virulence factors and antibiotic resistance genes and close genomic relatedness with marketed probiotics makes E. faecium 17OM39 a potential probiotic candidate. The work presented here demonstrates that comparative genome analyses can be applied to large numbers of genomes, to find potential probiotic candidates.

Genomic and physiological analyses of an indigenous strain, Enterococcus faecium 17OM39.

The human gut microbiome plays a crucial role in human health and efforts need to be done for cultivation and characterisation of bacteria with potential health benefits. Here, we isolated a bacterium from a healthy Indian adult faeces and investigated its potential as probiotic. The cultured bacterial strain 17OM39 was identified as Enterococcus faecium by 16S rRNA gene sequencing. The strain 17OM39 exhibited tolerance to acidic pH, showed antimicrobial activity and displayed strong cell surface traits such as hydrophobicity and autoaggregation capacity. The strain was able to tolerate bile salts and showed bile salt hydrolytic (BSH) activity, exopolysaccharide production and adherence to human HT-29 cell line. Importantly, partial haemolytic activity was detected and the strain was susceptible to the human serum. Genomics investigation of strain 17OM39 revealed the presence of diverse genes encoding for proteolytic enzymes, stress response systems and the ability to produce essential amino acids, vitamins and antimicrobial compound Bacteriocin-A. No virulence factors and plasmids were found in this genome of the strain 17OM39. Collectively, these physiological and genomic features of 17OM39 confirm the potential of this strain as a candidate probiotic.

Optimization of Nutrients and Culture Conditions for Alkaline Protease Production Using Two Endophytic Micrococci: Micrococcus aloeverae and Micrococcus yunnanensis.

An endophytic species of Micrococcus was isolated from Aloe vera leaf (syn. Aloe barbadensis) and screened for protease production with five other species of Micrococcus. Data indicated that endophytic Micrococcus aloeverae AE-6 MCC 2184T and Micrococcus yunnanensis DSM 21948T showed efficient protease production potential and secreted active protease at high salt (10%), temperature (40 °C) and in wide range of pH 8-10. Unlike M. yunnanensis DSM 21948T, protease production by M. aloeverae AE-6 MCC 2184T was stringently controlled by pH. Protease induction study using different group of peptides, peptide carbohydrates and peptide macronutrient combinations showed variable response with both the organisms. Result indicated that the amount of protease was not directly related to cell biomass but it depends on nature of inducible peptides. In this study we also developed a modified agar-well assay for semi-quantitative data from large number of replicates.

Description of Auricoccus indicus gen. nov., sp. nov., isolated from skin of human ear.

A Gram-stain-positive, non-motile, non-spore-forming, small spherical bacterium, strain S31T, was isolated from skin surface (external ear lobe) of a healthy human subject and characterized using a polyphasic approach. On the basis of 1507 bp 16S rRNA gene sequence comparison, S31T showed highest (92.8 %, AY119686) sequence similarity with Macrococcus brunensis CCUG 47200T followed by Macrococcus caseolyticus DSM 20597T (92.7 % AP009484) and formed a separate clade with 65 % bootstrap support. The DNA G+C content was found to be 34 mol%. Anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0 are the predominant fatty acids in fatty acid methyl ester (FAME) profile of strain S31T. It contained A3α type peptidoglycan with l-Lys-Gly3-l-Ala peptide. Comparative study of morphological and physiological traits indicated that S31T has phenetically diverged from its closest relatives. On the basis of morphological, chemotaxonomic and genotypic data, S31T showed marked distinctions from its closest relatives of the family Staphylococcaceae and is proposed to represent a novel genus Auricoccus with Auricoccus indicus as type species of the genus. S31T (CCUG 69858T=KCTC 33611T=MCC 3027T) is the type strain of the species.

Diversity and Distribution of Phenol Oxidase Producing Fungi from Soda Lake and Description of Curvularia lonarensis sp. nov.

Soda lake is hyper alkaline and saline habitat located in closed craters with high evaporation rate. In current study fungal diversity from water and sediment samples of a soda lake (Lonar lake) located in Buldhana district of Maharashtra, India was investigated using extensive culturomics approach and mimicking the natural conditions of Lonar lake in culture media. A total of 104 diverse isolates of extremophilic fungi were recovered from this study and phylogenetically characterized by internal transcribed spacer (ITS) region sequencing. In addition, due to important role of phenol oxidase, and peroxidase in degradation of toxic phenol, lignin, etc., all isolated pure cultures were also screened for extracellular phenol oxidase and peroxidase production potential. Diversity analysis indicated that different groups of extremophilic fungi are present in the water and sediment samples of Lonar lake. A total of 38 species of fungi belonging to 18-different genera were recovered. Out of 104 isolates 32 showed ≤97% sequences similarity, which were morphologically different and could be potential novel isolates of extremophilic fungi. However, out of 104 isolates only 14 showed the extracellular phenol oxidase production potentials at alkaline pH. Curvularia sp. strain MEF018 showed highest phenol oxidase production at alkaline condition and had low sequence similarity with previously characterized species (96% with Curvularia pseudorobusta). Taxonomic characterization (morphological and physiological) and multi locus sequence analysis (MLSA) using combined alignment of ITS-LSU-gpd of strain MEF018 showed that it is a novel species of the genus Curvularia and hence proposed as Curvularia lonarensis sp. nov.

Pantoea intestinalis sp. nov., isolated from the human gut.

A novel bacterial strain, 29Y89BT, was isolated from a faecal sample of a healthy human subject. Cells were Gram-stain-negative, motile, non-spore-forming and rod-shaped. Strain 29Y89BT formed cream-coloured colonies 2 mm in diameter on trypticase soy agar and showed optimum growth at 35 °C. Strain 29Y89BT showed highest 16S rRNA gene sequence similarity to Pantoea gaviniae A18/07T (98.4 %) followed by Pantoea calida 1400/07T (97.2 %). Multi-locus sequence analysis using atpD (ATP synthase ß subunit), gyrB (DNA gyrase), infB (initiation translation factor 2) and rpoB (RNA polymerase ß subunit) genes also supported the result of 16S rRNA gene sequence based phylogeny. Strain 29Y89BT showed 62 and 40.7 % DNA-DNA relatedness with P. calida DSM 22759T and P. gaviniae DSM 22758T. Strain 29Y89BT contained C17 : 0 cyclo, C19 : 0 cyclo ω8c, C16 : 0, C14 : 0 and C12 : 0 as predominant fatty acids. In addition, strain 29Y89BT showed physiological and phenotypic differences from its closest relatives P. gaviniae DSM 22758T and P. calida DSM 22759T. The polar lipid profile mainly comprised phospholipids. The DNA G+C content was 59.1 mol%. Thus, based on the findings of the current study, strain 29Y89BT showed clear delineations from its closest relatives P. gaviniae DSM 22758T and P. calida DSM 22759T, and is thus considered to represent a novel species of the genus Pantoea, for which the name Pantoea intestinalis sp. nov. is proposed. The type strain is 29Y89BT ( = DSM 28113T = MCC 2554T).

Proposal for creation of a new genus Neomicrococcus gen. nov. to accommodate Zhihengliuella aestuarii Baik et al. 2011 and Micrococcus lactis Chittpurna et al. 2011 as Neomicrococcus aestuarii comb. nov. and Neomicrococcus lactis comb. nov.

Micrococcus lactis and Zhihengliuella aestuarii were described independently in 2011. Their type strains showed high levels of 16S rRNA gene sequence similarity (99.3%). Phylogenetic analysis revealed that M. lactis MCC 2278T and Z. aestuarii JCM 16166T formed a monophyletic group and showed distant relationships to other members of closely related genera such as Micrococcus, Zhihengliuella, Arthrobacter and Citricoccus. The presence of large proportions of iso-C14:0 and iso-C16:0 with small amounts of iso-C15:0 distinguished M. lactis MCC 2278T and Z. aestuarii JCM 16166T from other members of the genera Micrococcus and Zhihengliuella. Unlike other members of the genera Zhihengliuella and Micrococcus, M. lactis MCC 2278T and Z. aestuarii JCM 16166T showed growth at low concentrations of NaCl. Thus, based on distinctive phylogenetic, chemotaxonomic and physiological features of these two organisms in comparison with other members of the genera Micrococcus and Zhihengliuella, it is clear that they do not fit within the existing classification and deserve separate status. DNA-DNA hybridization between the two type strains was 63%, indicating that they represent separate species. In this study, we propose the creation of a novel genus, Neomicrococcus gen. nov., to accommodate the two species with Neomicrococcus aestuarii gen. nov., comb. nov. (type strain JCM 16166T = KCTC 19557T) as the type species. Neomicrococcus lactis comb. nov. (type strain MCC 2278T = DSM 23694T) is also proposed.