Shotgun metagenomic sequencing of bulk tank milk filters reveals the role of Moraxellaceae and Enterobacteriaceae as carriers of antimicrobial resistance genes.

In the present context of growing antimicrobial resistance (AMR) concern, understanding the distribution of AMR determinants in food matrices such as milk is crucial to protect consumers and maintain high food safety standards. Herein, the resistome of different dairy farms was investigated through a shotgun metagenomic sequencing approach, taking advantage of in-line milk filters as promising tools. The application of both the reads-based and the assembly-based approaches has allowed the identification of numerous AMR determinants, enabling a comprehensive resolution of the resistome. Notably most of the species harboring AMR genes were predicted to be Gram-negative genera, namely Enterobacter, Acinetobacter, Escherichia, and Pseudomonas, pointing out the role of these bacteria as reservoirs of AMR determinants. In this context, the use of de novo assembly has allowed a more holistic AMR detection strategy, while the reads-based approach has enabled the detection of AMR genes from low abundance bacteria, usually undetectable by assembly-based methods. The application of both reads-based and assembly-based approaches, despite being computationally demanding, has facilitated the comprehensive characterization of a food chain resistome, while also allowing the construction of complete metagenome assembled genomes and the investigation of mobile genetic elements. Our findings suggest that milk filters can successfully be used to investigate the resistome of bulk tank milk through the application of the shotgun metagenomic sequencing. In accordance with our results, raw milk can be considered a source of AMR bacteria and genes; this points out the importance of properly informing food business operators about the risk associated with poor hygiene practices in the dairy production environment and consumers of the potential microbial food safety risks derived from raw milk products consumption. Translating these findings as risk assessment outputs heralds the next generation of food safety controls.

Laryngotracheal Microbiota in Adult Laryngotracheal Stenosis.

Laryngotracheal stenosis is an obstructive respiratory disease that leads to voicing difficulties and dyspnea with potential life-threatening consequences. The majority of incidences are due to iatrogenic etiology from endotracheal tube intubation; however, airway scarring also has idiopathic causes. While recent evidence suggests a microbial contribution to mucosal inflammation, the microbiota associated with different types of stenosis has not been characterized. High-throughput sequencing of the V4 region of the16S rRNA gene was performed to characterize the microbial communities of 61 swab samples from 17 iatrogenic and 10 adult idiopathic stenosis patients. Nonscar swabs from stenosis patients were internal controls, and eight swabs from four patients without stenosis represented external controls. Significant differences in diversity were observed between scar and nonscar samples and among sample sites, with decreased diversity detected in scar samples and the glottis region. Permutational analysis of variance (PERMANOVA) results revealed significant differences in community composition for scar versus nonscar samples, etiology type, sample site, groups (iatrogenic, idiopathic, and internal and external controls), and individual patients. Pairwise Spearman's correlation revealed a strong inverse correlation between Prevotella and Streptococcus among all samples. Finally, bacteria in the family Moraxellaceae were found to be distinctly associated with idiopathic stenosis samples in comparison with external controls. Our findings suggest that specific microbiota and community shifts are present with laryngotracheal stenosis in adults, with members of the family Moraxellaceae, including the known pathogens Moraxella and Acinetobacter, identified in idiopathic scar. Further work is warranted to elucidate the contributing role of bacteria on the pathogenesis of laryngotracheal stenosis.IMPORTANCE The laryngotracheal region resides at the intersection between the heavily studied nasal cavity and lungs; however, examination of the microbiome in chronic inflammatory conditions of the subglottis and trachea remains scarce. To date, studies have focused on the microbiota of the vocal folds, or the glottis, for laryngeal carcinoma, as well as healthy larynges, benign vocal fold lesions, and larynges exposed to smoking and refluxate. In this study, we seek to examine the structure and composition of the microbial community in adult laryngotracheal stenosis of various etiologies. Due to the heterogeneity among the underlying pathogenesis mechanisms and clinical outcomes seen in laryngotracheal stenosis disease, we hypothesized that different microbial profiles will be detected among various stenosis etiology types. Understanding differences in the microbiota for subglottic stenosis subtypes may shed light upon etiology-specific biomarker identification and offer novel insights into management approaches for this debilitating disease.

Amnimonas aquatica gen. nov., sp. nov., Isolated from a Freshwater River.

A gram-stain-negative and strictly aerobic bacterium, designated strain HR-ET, was isolated from a water sample of the Han River in South Korea. Cells were catalase-negative and oxidase-positive motile rods with a flagellum. The strain grew at 10-37 °C and pH 7-8 and in the presence of 0-2% (w/v) NaCl. Ubiquinone-8 and summed features 3 (comprising C16:1ω7c and/or C16:1ω6c) and 8 (comprising C18:1ω7c and/or C18:1ω6c), C16:0, iso-C10:0 and C12:0 3-OH were identified as the major respiratory quinone and fatty acids (>5%), respectively. The 16S rRNA gene sequence of strain HR-ET shared the highest similarities with Perlucidibaca piscinae IMCC 1704T (98.1%), Perlucidibaca aquatica BK296T (96.8%), Paraperlucidibaca baekdonensis RL-2T (95.8%) and Paraperlucidibaca wandonensis WT-RY4T (95.7%). However, strain HR-ET formed a phylogenetic lineage distinct from members of the family Moraxellaceae, and a taxonomic analysis by RDP Naïve Bayesian rRNA Classifier classified strain HR-ET as a new genus of the family Moraxellaceae. In addition, analyses based on rpoD, secA and gyrB gene sequences also showed that strain HR-ET formed a lineage distinct from those of the genera Perlucidibaca and Paraperlucidibaca. Average nucleotide identity and in silico DNA-DNA hybridization values between strain HR-ET and the type strains of P. piscinae and P. aquatica were very low with 80.1 and 23.6% and 75.7 and 21.2%, respectively. The DNA G+C content was 67.0 mol%. Based on the phenotypic, genotypic and chemotaxonomic analyses, strain HR-ET represents a novel genus of the family Moraxellaceae, for which the name Amnimonas aquatica gen. nov., sp. nov. is proposed. The type strain of the type species is HR-ET (=KACC 19408T=JCM 32266T).

MITE Aba12 , a Novel Mobile Miniature Inverted-Repeat Transposable Element Identified in Acinetobacter baumannii ATCC 17978 and Its Prevalence across the Moraxellaceae Family.

Insertion sequences (IS) are fundamental mediators of genome plasticity with the potential to generate phenotypic variation with significant evolutionary outcomes. Here, a recently active miniature inverted-repeat transposon element (MITE) was identified in a derivative of Acinetobacter baumannii ATCC 17978 after being subjected to stress conditions. Transposition of the novel element led to the disruption of the hns gene, resulting in a characteristic hypermotile phenotype. DNA identity shared between the terminal inverted repeats of this MITE and coresident ISAba12 elements, together with the generation of 9-bp target site duplications, provides strong evidence that ISAba12 elements were responsible for mobilization of the MITE (designated MITE Aba12 ) within this strain. A wider genome-level survey identified MITE Aba12 in 30 additional Acinetobacter genomes at various frequencies and one Moraxella osloensis genome. Ninety MITE Aba12 copies could be identified, of which 40% had target site duplications, indicating recent transposition events. Elements ranged between 111 and 114 bp; 90% were 113 bp in length. Using the MITE Aba12 consensus sequence, putative outward-facing Escherichia coli σ70 promoter sequences in both orientations were identified. The identification of transcripts originating from the promoter in one direction supports the proposal that the element can influence neighboring host gene transcription. The location of MITE Aba12 varied significantly between and within genomes, preferentially integrating into AT-rich regions. Additionally, a copy of MITE Aba12 was identified in a novel 8.5-kb composite transposon, Tn6645, in the M. osloensis CCUG 350 chromosome. Overall, this study shows that MITE Aba12 is the most abundant nonautonomous element currently found in AcinetobacterIMPORTANCE One of the most important weapons in the armory of Acinetobacter is its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from the Moraxellaceae family and its recent translocation into the hns locus in the A. baumannii ATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented examples of MITEs in the literature and underscores the plastic nature of the hns locus in A. baumannii MITE Aba12 , and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.

A longitudinal study of the infant nasopharyngeal microbiota: The effects of age, illness and antibiotic use in a cohort of South East Asian children.

A longitudinal study was undertaken in infants living in the Maela refugee camp on the Thailand-Myanmar border between 2007 and 2010. Nasopharyngeal swabs were collected monthly, from birth to 24 months of age, with additional swabs taken if the infant was diagnosed with pneumonia according to WHO clinical criteria. At the time of collection, swabs were cultured for Streptococcus pneumoniae and multiple serotype carriage was assessed. The bacterial 16S rRNA gene profiles of 544 swabs from 21 infants were analysed to see how the microbiota changes with age, respiratory infection, antibiotic consumption and pneumococcal acquisition. The nasopharyngeal microbiota is a somewhat homogenous community compared to that of other body sites. In this cohort it is dominated by five taxa: Moraxella, Streptococcus, Haemophilus, Corynebacterium and an uncharacterized Flavobacteriaceae taxon of 93% nucleotide similarity to Ornithobacterium. Infant age correlates with certain changes in the microbiota across the cohort: Staphylococcus and Corynebacterium are associated with the first few months of life while Moraxella and the uncharacterised Flavobacteriaceae increase in proportional abundance with age. Respiratory illness and antibiotic use often coincide with an unpredictable perturbation of the microbiota that differs from infant to infant and in different illness episodes. The previously described interaction between Dolosigranulum and Streptococcus was observed in these data. Monthly sampling demonstrates that the nasopharyngeal microbiota is in flux throughout the first two years of life, and that in this refugee camp population the pool of potential bacterial colonisers may be limited.

Perlucidibaca aquatica sp. nov., isolated from fresh water.

A Gram-staining-negative, non-motile, non-pigmented, strictly aerobic and rod-shape bacterium, designated BK296T, was isolated from stream water originating from a limestone cave in Samcheok, Korea. Optimal growth of strain BK296T was observed at 30 °C, pH 7.0-8.0 and without NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BK296T belonged to the genus Perlucidibaca, forming a robust clade with a member of the genus, and was most closely related to Perlucidibaca piscinae (97.8 %). The average nucleotide identity value between strain BK296T and Perlucidibacapiscinae IMCC1704T was 79.8 %, and the genome-to-genome distance was 17.5 % on mean. The G+C content of the DNA of strain BK296T was 55.7 mol%. The major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0, C12 : 0 3-OH and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The major isoprenoid quinone was ubiquinone Q-8. On the basis of phenotypic, genotypic and phylogenetic analyses, strain BK296T (=KCTC 52162T=JCM 31377T) represents a novel species of the genus Perlucidibaca, for which the name Perlucidibaca aquatica sp. nov. is proposed.

Fluviicoccus keumensis gen. nov., sp. nov., isolated from freshwater.

A Gram-stain-negative, non-spore-forming and coccus-shaped bacterial strain, designated 4DR5T, was isolated from freshwater and its taxonomic position was investigated using a polyphasic approach. Growth occurred at 10-40 °C (optimum 30 °C), at pH 6-9 (optimum pH 7) and in the presence of 0-0.4 % (w/v) NaCl (optimum 0 %) on R2A agar. On the basis of 16S rRNA gene sequence similarity, strain 4DR5T was assigned to the family Moraxellaceae of the class Gammaproteobacteria, and its closest related taxa were species of the genera Perlucidibaca (93.67 % sequence similarity), Agitococcus (93.07 %), Paraperlucidibaca (92.31-92.38 %), Alkanindiges (91.79 %) and Acinetobacter (90.24-91.23 %). The predominant isoprenoid quinone detected in strain 4DR5T was Q-10. The major cellular fatty acids were a summed feature consisting of C16 : 1ω7c and/or C16 : 1ω6c, one consisting of C18 : 1ω7c and/or C18 : 1ω6c, and C16 : 0. The major polar lipid was phosphatidylethanolamine. The genomic DNA G+C content of the strain was 61.2 mol%. The phylogenetic, chemotaxonomic and biochemical data not only supported the affiliation of strain 4DR5T to the family Moraxellaceae, but also separated it from other established genera within the family. Therefore, the novel isolate evidently represents a novel species of a new genus of Moraxellaceae, for which the name Fluviicoccus keumensis gen. nov., sp. nov. is proposed. The type strain of Fluviicoccus keumensis is 4DR5T ( = KCTC 32475T = JCM 19370T).

Faucicola mancuniensis gen. nov., sp. nov., isolated from the human oropharynx.

An aerobic, Gram-stain-negative, non-motile coccus, designated strain GVCNT2(T), was isolated from the tonsils of a healthy adult female. Cells were oxidase- and catalase-positive, positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase, and weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells were also capable of hydrolysing DNA. Growth was observed at 20-37 °C and in the presence of up to 1.5% NaCl. Phylogenetic analysis of near full-length 16S rRNA gene sequences indicated that the strain exhibited closest sequence similarity to Moraxella boevrei ATCC 700022(T) (94.68%) and an uncultured, unspeciated bacterial clone (strain S12-08; 99%). The major fatty acids were C18:1ω9c, C18 : 0, C16:0 and C16:1ω6c/C16:1ω7c. The DNA G+C content of strain GVCNT2(T) was 40.7 mol%. The major respiratory quinone identified was Q-8. Strain GVCNT2(T) exhibited a comparable phenotypic profile to other members of the genus Moraxella but could be distinguished based on its ability to produce acid (weakly) from d-glucose, melibiose, l-arabinose and rhamnose and on its ability to hydrolyse DNA. On the basis of phenotypic and phylogenetic differences from other members of the family Moraxellaceae, strain GVCNT2(T) is considered to represent a novel species of a new genus, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. The type strain of Faucicola mancuniensis is GVCNT2(T) ( =DSM 28411(T) =NCIMB 14946(T)).

scnRCA: a novel method to detect consistent patterns of translational selection in mutationally-biased genomes.

Codon usage bias (CUB) results from the complex interplay between translational selection and mutational biases. Current methods for CUB analysis apply heuristics to integrate both components, limiting the depth and scope of CUB analysis as a technique to probe into the evolution and optimization of protein-coding genes. Here we introduce a self-consistent CUB index (scnRCA) that incorporates implicit correction for mutational biases, facilitating exploration of the translational selection component of CUB. We validate this technique using gene expression data and we apply it to a detailed analysis of CUB in the Pseudomonadales. Our results illustrate how the selective enrichment of specific codons among highly expressed genes is preserved in the context of genome-wide shifts in codon frequencies, and how the balance between mutational and translational biases leads to varying definitions of codon optimality. We extend this analysis to other moderate and fast growing bacteria and we provide unified support for the hypothesis that C- and A-ending codons of two-box amino acids, and the U-ending codons of four-box amino acids, are systematically enriched among highly expressed genes across bacteria. The use of an unbiased estimator of CUB allows us to report for the first time that the signature of translational selection is strongly conserved in the Pseudomonadales in spite of drastic changes in genome composition, and extends well beyond the core set of highly optimized genes in each genome. We generalize these results to other moderate and fast growing bacteria, hinting at selection for a universal pattern of gene expression that is conserved and detectable in conserved patterns of codon usage bias.

A new subgroup of the IS3 family and properties of its representative member ISPpy1.

Recently, we described a novel insertion element, ISPpy1, isolated from a permafrost strain of Psychrobacter maritimus. In this work, we demonstrated that ISPpy1 is a member of a novel subgroup of the IS3 family of insertion sequences (ISs) that was not identified and characterized previously. IS elements of this subgroup termed the ISPpy1 subgroup are broadly distributed among different taxa of Eubacteria, including Geobacteraceae, Chlorobiaceae, Desulfobacteraceae, Methylobacteriaceae, Nitrosomonadaceae and Cyanobacteria. While displaying characteristic features of the IS3-family elements, ISPpy1 subgroup elements exhibit some unusual features. In particular, most of them have longer terminal repeats with unconventional ends and frameshifting box with an atypical organization, and, unlike many other IS3-family elements, do not exhibit any distinct IS specificity. We studied the transposition and mutagenic properties of a representative member of this subgroup, ISPpy1 and showed that in contrast to the original P. maritimus host, in a heterologous host, Escherichia coli K-12, it is able to translocate with extremely high efficiency into the chromosome, either by itself or as a part of a composite transposon containing two ISPpy1 copies. The majority of transposants carry multiple chromosomal copies (up to 12) of ISPpy1. It was discovered that ISPpy1 is characterized by a marked mutagenic activity in E. coli: its chromosomal insertions generate various types of mutations, including auxotrophic, pleiotropic and rifampicin-resistance mutations. The distribution of IS elements of the novel subgroup among different bacteria, their role in the formation of composite transposons and the horizontal transfer of genes are examined and discussed.

Paraperlucidibaca wandonensis sp. nov., isolated from seawater, and emended description of the genus Paraperlucidibaca Oh et al. 2011.

A Gram-stain-negative, aerobic, motile and rod-shaped bacterial strain, designated WT-RY4(T), was isolated from wood falls in the South Sea, South Korea, and its taxonomic position was investigated using a polyphasic approach. Strain WT-RY4(T) grew optimally at 25 °C, at pH 7.0-7.5 and in the absence of NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain WT-RY4(T) clustered with the type strain of Paraperlucidibaca baekdonensis with a bootstrap resampling value of 100 %. Strain WT-RY4(T) exhibited 16S rRNA gene sequence similarity values of 98.8 % and 96.3 % to Paraperlucidibaca baekdonensis RL-2(T) and Perlucidibaca piscinae IMCC 1704(T), respectively and less than 91.5 % to the type strains of other species used in the phylogenetic analysis. The DNA G+C content of strain WT-RY4(T) was 52.4 mol% and the mean DNA-DNA relatedness value with Paraperlucidibaca baekdonensis RL-2(T) was 25 %. Strain WT-RY4(T) contained Q-11 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C12 : 0 3-OH as the major fatty acids. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, demonstrated that strain WT-RY4(T) was distinguishable from Paraperlucidibaca baekdonensis RL-2(T). On the basis of the data presented, strain WT-RY4(T) is considered to represent a novel species of the genus Paraperlucidibaca, for which the name Paraperlucidibaca wandonensis sp. nov. is proposed. The type strain is WT-RY4(T) ( = KCTC 32216(T) = CCUG 63419(T)). An emended description of the genus Paraperlucidibaca is also provided.

Ultrastructural analysis of the rugose cell envelope of a member of the Pasteurellaceae family.

Bacterial membranes serve as selective environmental barriers and contain determinants required for bacterial colonization and survival. Cell envelopes of Gram-negative bacteria consist of an outer and an inner membrane separated by a periplasmic space. Most Gram-negative bacteria display a smooth outer surface (e.g., Enterobacteriaceae), whereas members of the Pasteurellaceae and Moraxellaceae families show convoluted surfaces. Aggregatibacter actinomycetemcomitans, an oral pathogen representative of the Pasteurellaceae family, displays a convoluted membrane morphology. This phenotype is associated with the presence of morphogenesis protein C (MorC). Inactivation of the morC gene results in a smooth membrane appearance when visualized by two-dimensional (2D) electron microscopy. In this study, 3D electron microscopy and atomic force microscopy of whole-mount bacterial preparations as well as 3D electron microscopy of ultrathin sections of high-pressure frozen and freeze-substituted specimens were used to characterize the membranes of both wild-type and morC mutant strains of A. actinomycetemcomitans. Our results show that the mutant strain contains fewer convolutions than the wild-type bacterium, which exhibits a higher curvature of the outer membrane and a periplasmic space with 2-fold larger volume/area ratio than the mutant bacterium. The inner membrane of both strains has a smooth appearance and shows connections with the outer membrane, as revealed by visualization and segmentation of 3D tomograms. The present studies and the availability of genetically modified organisms with altered outer membrane morphology make A. actinomycetemcomitans a model organism for examining membrane remodeling and its implications in antibiotic resistance and virulence in the Pasteurellaceae and Moraxellaceae bacterial families.

Genus Enhydrobacter Staley et al. 1987 should be recognized as a member of the family Rhodospirillaceae within the class Alphaproteobacteria.

The genus Enhydrobacter, first reported as a member of the family Vibrionaceae, has been placed in the family Moraxellaceae, but as a genus incertae sedis in Bergey's Manual of Systematic Bacteriology 2nd edition. During our taxonomic investigation of Enhydrobacter-like organisms, we observed that the 16S rRNA sequences of E. aerosaccus-type strain versions NCIMB 12535(T) , ATCC 27094( T) and CCUG 58314(T) were very different from the accessible data (accession no. AJ550856). Phylogenetic analysis of our 16S rRNA sequence data revealed that these organisms were located within the family Rhodospirillaceae. The genera Inquilinus, Oceanibaculum, Skermanella and Nisaea were closely related (sequence similarities were 88.3~87.0%), but Enhydrobacter could be distinguished from these genera by growth characteristics, fatty acid profiles (C(19:0) cyclo ω8c; 38.4% C(18:1) ω7c; 32.2%, and C(16:0) ; 8.9% were major components), in being non-flagellated, and differing in enzymatic activities, including trypsin and ß-glucosidase. From these data, we conclude that the genus Enhydrobacter should be recognized as an independent genus of the family Rhodospirillaceae within the class Alphaproteobacteria.

16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

Paraperlucidibaca baekdonensis gen. nov., sp. nov., isolated from seawater.

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, RL-2(T), was isolated from seawater of the East Sea in Korea and was subjected to a polyphasic taxonomic study. Strain RL-2(T) grew optimally at pH 7.5-8.0, at 20 °C and in the absence of NaCl. Phylogenetic trees based on 16S rRNA gene sequence analysis showed that strain RL-2(T) forms a cluster with Perlucidibaca piscinae IMCC1704(T) and various uncultured and unidentified gammaproteobacteria. Strain RL-2(T) exhibited 16S rRNA gene sequence similarity values of 96.1 % to Perlucidibaca piscinae IMCC1704(T) and 93.7-99.7 % to the uncultured bacterial clones belonging to the cluster and an unidentified gammaproteobacterium. The fatty acid profile of strain RL-2(T) was similar to that of Perlucidibaca piscinae IMCC1704(T), but the predominant ubiquinone type (Q-11) of strain RL-2(T) was different from that (Q-8) of Perlucidibaca piscinae IMCC1704(T). The DNA G+C content of strain RL-2(T) was 61.3 mol%. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain RL-2(T) is considered to represent a novel species of a new genus in the family Moraxellaceae, for which the name Paraperlucidibaca baekdonensis gen. nov., sp. nov. is proposed. The type strain of Paraperlucidibaca baekdonensis is RL-2(T) ( = KCTC 23145(T)  = CCUG 59307(T)).

[Novel halotolerant bacterium from cryopeg in permafrost: description of Psychrobacter muriicola sp. nov].

A novel halotolerant psychrotrophic gram-negative bacterium, strain 2pS, was isolated from lenses of water brine in Arctic permafrost (cryopeg). The optimal growth of the new strain was observed at 16-18 degrees C; the maximal and minimal growth temperatures were 37 degrees C and -2 degrees C, respectively. The pH growth range was 5.8 to 8.5 (optimum 6.5-7.5) and the range of medium salinity was 0 to 100 g/l (optimum 3-8 g/l NaCl). The strain 2pS did not produce acid from carbohydrates and utilized acetate, yeast extract, pyruvate, glutarate, fumarate, caproate, heptanoate, butyrate, malate, DL-lactate, citrate, L-proline, L-tyrosine, butanol, and dulcitol as the sole carbon and energy sources. The major fatty acids of the cell wall at optimal growth temperature were C18:1(omega 7) and C18:1(omega 9). The G + C DNA content was 46.0 mol.%. Phylogenetic analysis of the 16S rRNA gene sequences showed that the studied strain was the closest (97% similarity) to Psychrobacter nivimaris DSM 16093T, a halotolerant psychrotrophic bacterium isolated from the Arctic sea's ice. Genotypic and phenotypic differences of the new bacterium from closely related species lead to the conclusion that strain 2pS belongs to a novel species of the genus Psychrobacter: Psychrobacter muriicola sp. nov.

Exploration of Csp genes from temperate and glacier soils of the Indian Himalayas and in silico analysis of encoding proteins.

The metagenomic Csp library was constructed from the temperate and glacier soils of central Himalaya, India followed by polymerase chain reaction (PCR) amplification. The library was further screened for low-temperature adaptation, and the positive recombinants were sorted out by determining changes in the melting temperature (Tm). A homology search of cloned sequence showed their identity with the Csp genes of Pseudomonas fluorescens, Psychrobacter cryohalolentis K5, and Shewanella spp MR-4. Amino acid sequence analysis annotated the presence of conserved aromatic and basic amino acids as well as RNA binding motifs from the cold shock domain. Furthermore, a PROSITE scan showed a moderate identity of less than 60% with the known cold shock-inducible proteins (ribosomal proteins, rbfA, DEAD-box helicases), cold acclimation protein, and temperature-induced protein (SRP1/TIP1). This study highlighted the prevalence of Csp genes from cold Himalayan environments that can be explored for tailor-made crop constructions in future.

Perlucidibaca piscinae gen. nov., sp. nov., a freshwater bacterium belonging to the family Moraxellaceae.

A freshwater bacterium, designated IMCC1704(T), was isolated from a eutrophic pond. The strain was Gram-negative, oxidase-positive, catalase-negative, chemoheterotrophic and facultatively aerobic with cells that were motile rods with a single polar flagellum. Based on 16S rRNA gene sequence similarity analyses, the novel strain was most closely related to the genera Alkanindiges (91.7%), Acinetobacter (89.0-91.2%), Moraxella (87.9-90.1%), Psychrobacter (87.2-89.5%) and Enhydrobacter (87.8%). Phylogenetic trees generated using 16S rRNA gene sequences showed that the novel isolate belonged to the family Moraxellaceae of the class Gammaproteobacteria and formed a distinct phyletic lineage within the family. The DNA G+C content of the strain was 63.1 mol% and the predominant constituents of the cellular fatty acids were C(16:1)omega7c and/or iso-C(15:0) 2-OH (21.2%), C(18:1)omega7c (12.8%) and C(12:0) 3-OH (12.3%). These chemotaxonomic properties, together with several phenotypic characteristics, differentiated the novel strain from other members of the family Moraxellaceae. From the taxonomic data, which revealed the distant relationship of the new strain to the related genera, the strain should be classified as a novel genus and species in the family Moraxellaceae, for which the name Perlucidibaca piscinae gen. nov., sp. nov. is proposed. The type strain of Perlucidibaca piscinae sp. nov. is IMCC1704(T) (=KCCM 42363(T)=NBRC 102354(T)).

Psychrobacter nivimaris sp. nov., a heterotrophic bacterium attached to organic particles isolated from the South Atlantic (Antarctica).

An aggregate-attached bacterium, strain 88/2-7, was isolated from samples of the Southern Ocean and investigated in a polyphasic approach. The novel marine isolate is an aerobic, Gram-negative, oxidase- and catalase-positive, non-motile short rod and grows in form of cream-colored colonies. Growth was observed at 5-35 degrees C. The bacterium tolerated concentrations of 0-13% (w/v) NaCl and utilized a relatively restricted spectrum of carbon sources. The analysis of the fatty acids revealed 18:1 cis 9 (18:1omega9c) as main fatty acid. The G+C content of the DNA was approximately 42 mol%. The sequence of the 16S rDNA assigned strain 88/2-7 to the gamma-subclass of Proteobacteria with a similarity of 99.65% to Psychrobacter proteolyticus (DSM 13887T). A DNA-DNA-hybridization study showed only 26.8% renaturation to the respective strain. Based on the morphological, physiological and molecular properties of the new isolate, the name Psychrobacter nivimaris sp. nov. (type strain 88/2-7T) is proposed.

Psychrobacter okhotskensis sp. nov., a lipase-producing facultative psychrophile isolated from the coast of the Okhotsk Sea.

A facultatively psychrophilic bacterium, strain MD17(T), which hydrolyses lipids at 5 degrees C, was isolated from the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan, when ice carried by the cold current came to the area. The isolate is an aerobic, non-motile coccobacillus that reduces nitrate to nitrite and hydrolyses Tweens 20, 40, 60 and 80, but not gelatin, DNA or alginic acid. The isolate grows at 0 degrees C, but not at temperatures higher than 36 degrees C; its optimum growth temperature is 25 degrees C. It grows in the presence of 0-10 % NaCl. Its major isoprenoid quinone is ubiquinone-8 (Q-8) and its DNA G+C content is 46.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD17(T) is closely related to Psychrobacter glacincola DSM 12194(T) (99.0 % similarity) and Psychrobacter immobilis DSM 7229(T) (98.7 % similarity). DNA-DNA hybridization revealed 45.9 % relatedness between strain MD17(T) and P. immobilis ATCC 43116(T) and 33.4 % between strain MD17(T) and P. glacincola ATCC 700754(T). Based on physiological and biochemical characteristics, phylogenetic position (as determined by 16S rRNA gene sequence analysis) and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Psychrobacter okhotskensis sp. nov. is proposed. The type strain is MD17(T) (=NCIMB 13931(T)=JCM 11840(T)).