Allobaculum mucilyticum sp. nov. and Allobaculum fili sp. nov., isolated from the human intestinal tract.

As part of a culturomics study to identify bacterial species associated with inflammatory bowel disease, a large collection of bacteria was isolated from patients with ulcerative colitis. Two of these isolates were tentatively identified as members of the family Erysipelotrichaceae. Following phylogenetic analysis based on 16S rRNA gene sequence and genome sequences, both strain 128T and 539T were found to be most closely related to Allobaculum stercoricanis, with G+C contents of 48.6 and 50.5 mol%, respectively, and the genome sizes of 2 864 314 and 2 580 362 base pairs, respectively. Strains 128T and 539T were strict anaerobe rods that grew in long chains between 37 and 42 °C. Scanning electron microscopy did not reveal flagella, fimbriae or visible endospores. Biochemical analysis showed nearly identical results for both strains with enzymatic activity of C4 and C8 esterases, acid phosphatase, naphthol-AS-BI-phosphohydrolase, ß-glucuronidase, N-acetyl-ß-glucosaminidase and arginine arylamidase. In addition, both strains produced indole and reduced nitrate. Major fatty acids were identified as C18:1 ω9c (oleic acid, 64.06% in 128T and 74.35% in 539T), C18:1 ω7c/C18:1 ω9t/C18:1 ω12t/UN17.834 (16.18 % in 128T and 6.22% in 539T) and C16:0 (6.23% in 128T and 7.37% in 538T). Based on these analyses two novel species are proposed, Allobaculum mucilyticum sp. nov. with the type strain 128T (=NCTC 14626T=DSM 112815T) and Allobaculum fili sp. nov. with the type strain 539T (=NCTC 14627T=DSM 112814T).

Isolation and identification of a human intestinal bacterium capable of daidzein conversion.

Equol, which produced from daidzein (one of the principal isoflavones), is recognized to be the most resultful in stimulating an estrogenic and antioxidant response. The daidzein transformation was studied during fermentation of five growth media inoculated with feces from a healthy human, and a daidzein conversion strain was isolated. To enrich the bacterial population involved in daidzein metabolism in a complex mixture, fecal samples were treated with antibiotics. The improved propidium monoazide combined with the quantitative polymerase chain reaction (PMAxx-qPCR) assay showed that the ampicillin treatment of samples did result in a reduction of the total visible bacteria counts by 52.2% compared to the treatment without antibiotics. On this basis, the newly isolated rod-shaped, Gram-positive anaerobic bacterium, named strain Y11 (MN560033), was able to metabolize daidzein to equol under anaerobic conditions, with a conversion ratio (equol ratio: the amount of equol produced/amount of supplemented daizein) of 0.56 over 120 h. The 16S rRNA partial sequence of the strain Y11 exhibited 99.8% identity to that of Slackia equolifaciens strain DZE (NR116295). This study will provide new insights into the biotransformation of equol from daidzein by intestinal microbiota from the strain-level and explore the possibility of probiotic interventions.

Reclassification of Catabacter hongkongensis as Christensenella hongkongensis comb. nov. based on whole genome analysis.

The genera Catabacter (family 'Catabacteraceae') and Christensenella (family Christensenellaceae) are close relatives within the phylum Firmicutes. Members of these genera are strictly anaerobic, non-spore-forming and short straight rods with diverse phenotypes. Phylogenetic analysis of 16S rRNA genes suggest that Catabacter splits Christensenella into a polyphyletic clade. In an effort to ensure that family/genus names represent monophyletic clades, we performed a whole-genome based analysis of the genomes available for the cultured representatives of these genera: four species of Christensenella and two strains of Catabacter hongkongensis. A concatenated alignment of 135 shared protein sequences of single-copy core genes present in the included strains indicates that C. hongkongensis is indeed nested within the Christensenella clade. Based on their evolutionary relationship, we propose the transfer of Catabacter hongkongensis to the genus Christensenella as Christensenella hongkongensis comb. nov.

Isachenkonia alkalipeptolytica gen. nov. sp. nov., a new anaerobic, alkaliphilic proteolytic bacterium capable of reducing Fe(III) and sulfur.

An obligately alkaliphilic, anaerobic, proteolytic bacterium was isolated from a sample of Tanatar III soda lake sediment (Altai region, Russia) and designated as strain Z-1701T. Cells of strain Z-1701T were short, straight, motile Gram-stain-positive rods. Growth of Z-1701T obligately depended on the presence of sodium carbonate. Strain Z-1701T could utilize various peptides mixtures, such as beef and yeast extracts, peptone, soytone, trypticase and tryptone, as well as such proteins as albumin, gelatin and sodium caseinate. It was able to grow oligotrophically with 0.02 g l-1 yeast extract as the sole energy and carbon source. Carbohydrates did not support the growth of strain Z-1701T. The main products released during the growth of strain Z-1701T on tryptone were formate, acetate and ammonium. Strain Z-1701T was able to reduce ferrihydrite, Fe(III)-EDTA, anthraquinone-2,6-disulfonate and elemental sulfur, using proteinaceous substrates as electron donors. In all cases the presence of the electron acceptor in the medium stimulated growth. The main cellular fatty acids were iso-C15 : 0, iso-C15 : 0 aldehyde, iso-C15 : 1 ω6, C16 : 0, iso-C17 : 0 aldehyde, C16 : 0 aldehyde and C14 : 0. The DNA G+C content of the isolate was 43.9 mol%. Phylogenetic analysis based on the concatenated alignment of 120 protein-marker sequences revealed that strain Z-1701T falls into a cluster with the genus Tindallia, family Clostridiaceae. 16S rRNA gene sequence identity between strain Z-1701T and Tindallia species were 88.3-89.75 %. On the basis of its phenotypic characteristics and phylogenetic position, the novel isolate is considered to be a representative of a novel genus and species for which the name Isachenkonia alkalipeptolytica gen. nov., sp. nov. is proposed, with Z-1701T (=JCM 32929Т=DSM 109060Т=VKM B-3261Т) as its type strain.

Evaluation of Three Bacterial Identification Systems for Species Identification of Bacteria Isolated from Bovine Mastitis and Bulk Tank Milk Samples.

A study was conducted to evaluate Sensititre® Automated Reading and Incubation System 2x System (ARIS), API® (API), and Bruker MALDI-TOF MS (MALDI) bacterial species identification systems using 132 diverse bacterial isolates from bovine milk samples and bulk tank milk received at the Penn State Animal Diagnostic Laboratory. The results were compared with 16S rRNA gene sequence analysis, which served as the reference method for species identification. The ARIS, API, and MALDI identified 0%, 40%, and 33.4% of species classified as Gram-positive rod isolates belonging to genera Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, and Corynebacterium, respectively. It was observed that 76.5%, 93.9%, and 96.9% of catalase-negative, Gram-positive cocci (n = 33; Aerococcus, Enterococcus, Lactococcus, Streptococcus) were correctly identified to the species level by ARIS, API, and MALDI, respectively, while 33.4%, 84.5%, and 97.7% of catalase-positive, Gram-positive cocci (n = 45; Kocuria, Staphylococcus) were correctly identified to their species by ARIS, API, and MALDI, respectively. A total of 48 isolates (Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Pasteurella, Providencia, Pseduomonas, Serratia) of Gram-negative bacteria were examined, of which 85.4%, 93.7%, and 95.8% of the isolates were correctly identified to the species level by ARIS, API, and MALDI, respectively. In our laboratory, the MALDI had the least costs associated with consumables and reagents compared to ARIS, API, and 16S rRNA identification methods. Identification of bacterial species was accomplished in <2 h using MALDI and 24 h for ARIS, API, and 16S rRNA identification systems.

Clinical Significance of Commensal Gram-Positive Rods Routinely Isolated from Patient Samples.

Commensal bacteria from the skin and mucosal surfaces are routinely isolated from patient samples and considered contaminants. The majority of these isolates are catalase-positive Gram-positive rods from multiple genera routinely classified as diphtheroids. These organisms can be seen upon Gram staining of clinical specimens or can be isolated as the predominant or pure species in culture, raising a priori suspicion of a possible involvement in infection. With the development and adoption of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), suspicious isolates are now routinely identified to the species level. In this study, we performed a retrospective data review (2012 to 2015) and utilized site-specific laboratory criteria and chart reviews to identify species within the diphtheroid classification representative of true infection versus contamination. Our data set included 762 isolates from 13 genera constituting 41 bacterial species. Only 18% represented true infection, and 82% were deemed contaminants. Clinically significant isolates were identified in anaerobic wounds (18%), aerobic wounds (30%), blood (5.5%), urine (22%), cerebrospinal fluid (24%), ophthalmologic cultures (8%), and sterile sites (20%). Organisms deemed clinically significant included multiple Actinomyces species in wounds, Propionibacterium species in joints and cerebrospinal fluid associated with central nervous system hardware, Corynebacterium kroppenstedtii (100%) in breast, and Corynebacterium striatum in multiple sites. Novel findings include clinically significant urinary tract infections by Actinomyces neuii (21%) and Corynebacterium aurimucosum (21%). Taken together, these findings indicate that species-level identification of diphtheroids isolated with a priori suspicion of infection is essential to accurately determine whether an isolate belongs to a species associated with specific types of infection.

Aliifodinibius halophilus sp. nov., a moderately halophilic member of the genus Aliifodinibius, and proposal of Balneolaceae fam. nov.

A novel Gram-stain-negative, rod-shaped, facultatively anaerobic, oxidase-negative and catalase-positive bacterium, designated 2W32T, was isolated from a marine solar saltern on the coast of Weihai, Shandong Province, China. Strain 2W32T was tolerant to moderate salt conditions. Optimal growth occurred at 33-37 °C (range 20-45 °C) and pH 7.5-8.0 (range pH 7.0-8.5) with 6-10 % (w/v) NaCl (range 2-18 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2W32T shared highest similarity with Aliifodinibius sediminis YIM J21T (94.6 %), Aliifodinibius roseus YIM D15T (94.4 %), Fodinibius salinus YIM C003T (93.6 %), Gracilimonas tropica CL-CB462T (88.6 %) and Balneola vulgaris 13IX/A01/164T (86.4 %) and less than 83.0 % similarity with other species of the phylum Bacteroidetes. The isolate and closely related species formed a novel family-level clade in the phylum Bacteroidetes. The polar lipid profile of the novel isolate consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminolipid, an unidentified glycolipid and an unidentified lipid. The dominant cellular fatty acids (>10 %) were iso-C15 : 0, iso-C17 : 1ω9c and summed feature 3 (C16:1ω7c and/or iso-C15 : 0 2-OH) and the sole respiratory quinone was menaquinone 7 (MK-7). The DNA G+C content of strain 2W32T was 47.5 mol %. Comparative analysis of 16S rRNA gene sequences and characterization indicated that strain 2W32T represents a novel species within the genus Aliifodinibius, for which the name Aliifodinibius halophilus sp. nov. is proposed. The type strain is 2W32T (=KCTC 42497T=CICC 23869T). In addition, a novel family, Balneolaceae fam. nov., is proposed to accommodate the genera Fodinibius, Aliifodinibius, Gracilimonas and Balneola.

Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils.

The diversity of thermophilic bacteria was investigated in four hot springs, three salt marshes and 12 desert sites in Morocco. Two hundred and forty (240) thermophilic bacteria were recovered, identified and characterized. All isolates were Gram positive, rod-shaped, spore forming and halotolerant. Based on BOXA1R-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by the genus Bacillus (97.5%) represented by B. licheniformis (119), B. aerius (44), B. sonorensis (33), B. subtilis (subsp. spizizenii (2) and subsp. inaquosurum (6)), B. amyloliquefaciens (subsp. amyloliquefaciens (4) and subsp. plantarum (4)), B. tequilensis (3), B. pumilus (3) and Bacillus sp. (19). Only six isolates (2.5%) belonged to the genus Aeribacillus represented by A. pallidus (4) and Aeribacillus sp. (2). In this study, B. aerius and B. tequilensis are described for the first time as thermophilic bacteria. Moreover, 71.25%, 50.41% and 5.41% of total strains exhibited high amylolytic, proteolytic or cellulolytic activity respectively.

Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils

The diversity of thermophilic bacteria was investigated in four hot springs, three salt marshes and 12 desert sites in Morocco. Two hundred and forty (240) thermophilic bacteria were recovered, identified and characterized. All isolates were Gram positive, rod-shaped, spore forming and halotolerant. Based on BOXA1R-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by the genus Bacillus (97.5%) represented by B. licheniformis (119), B. aerius (44), B. sonorensis (33), B. subtilis (subsp. spizizenii (2) and subsp. inaquosurum (6)), B. amyloliquefaciens (subsp. amyloliquefaciens (4) and subsp. plantarum (4)), B. tequilensis (3), B. pumilus (3) and Bacillus sp. (19). Only six isolates (2.5%) belonged to the genus Aeribacillus represented by A. pallidus (4) and Aeribacillus sp. (2). In this study, B. aerius and B. tequilensis are described for the first time as thermophilic bacteria. Moreover, 71.25%, 50.41% and 5.41% of total strains exhibited high amylolytic, proteolytic or cellulolytic activity respectively.

Caldicellulosiruptor changbaiensis sp. nov., a cellulolytic and hydrogen-producing bacterium from a hot spring.

A novel thermophilic bacterial strain, CBS-Z(T), was isolated from a terrestrial hot spring in the Changbai Mountains, PR China. Cells of strain CBS-Z(T) were short straight rods without flagella and had Gram-positive cell walls. Growth was observed at 40-90 °C (optimum 75 °C) and at pH 5.6-8.6 (optimum pH 7.8). The primary end-products from the fermentation of filter paper by strain CBS-Z(T) were acetate, lactate, H2, and CO2. The main cellular fatty acids were iso-C17:0, iso-C14:0 3-OH and C16:0. The G+C content of the genomic DNA was 36.08 mol%. Multiple sequence alignment of the 16S rRNA gene sequence and phylogenetic analyses indicated that strain CBS-Z(T) belongs to the genus Caldicellulosiruptor and the most similar micro-organism was Caldicellulosiruptor saccharolyticus DSM 8903(T) (96.36% 16S rRNA gene sequence similarity); the 16S rRNA gene sequence similarity of strain CBS-Z(T) to other species was below 95%. Based on its phylogenetic and phenotypic characteristics, strain CBS-Z(T) represents a novel species of the genus Caldicellulosiruptor, for which the name Caldicellulosiruptor changbaiensis sp. nov. is proposed. The type strain is CBS-Z(T) ( =DSM 26941(T) =CGMCC 1.5180(T)).

Catenisphaera adipataccumulans gen. nov., sp. nov., a member of the family Erysipelotrichaceae isolated from an anaerobic digester.

An obligately anaerobic bacterium, designated strain GK12(T), was isolated from an anaerobic digester in Fukagawa, Hokkaido Prefecture, Japan. The cells of strain GK12(T) were non-motile, non-spore-forming cocci that commonly occurred in chains. 16S rRNA gene sequence analysis revealed that strain GK12(T) was affiliated with the family Erysipelotrichaceae in the phylum Firmicutes and showed 91.8 % sequence similarity to the most closely related species, Faecalicoccus acidiformans. The strain grew at 30-50 °C (optimally at 40 °C) and at pH 5.5-8.5 (optimally at pH 7.5). The main end product of glucose fermentation was lactate. Yeast extract was required for growth. The strain contained C14 : 0, C14 : 0 1,1-dimethoxyalkane (DMA), C16 : 0 DMA and C18 : 0 DMA as the major cellular fatty acids (>10 % of the total). The polar lipid profile was composed of phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. The whole-cell sugars were galactose, rhamnose and ribose. The cell-wall murein contained alanine, glutamic acid, lysine, serine and threonine, but not diaminopimelic acid. The G+C content of the genomic DNA was 47.7 mol%. Based on phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Catenisphaera adipataccumulans gen. nov., sp. nov., is proposed to accommodate strain GK12(T) ( = NBRC 108915(T) = DSM 25799(T)).

Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Nocardia, Rhodococcus, Kocuria, Gordonia, Tsukamurella, and Listeria species.

We evaluated whether the Bruker Biotyper matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system provides accurate species-level identifications of 147 isolates of aerobically growing Gram-positive rods (GPRs). The bacterial isolates included Nocardia (n = 74), Listeria (n = 39), Kocuria (n = 15), Rhodococcus (n = 10), Gordonia (n = 7), and Tsukamurella (n = 2) species, which had all been identified by conventional methods, molecular methods, or both. In total, 89.7% of Listeria monocytogenes, 80% of Rhodococcus species, 26.7% of Kocuria species, and 14.9% of Nocardia species (n = 11, all N. nova and N. otitidiscaviarum) were correctly identified to the species level (score values, ≥ 2.0). A clustering analysis of spectra generated by the Bruker Biotyper identified six clusters of Nocardia species, i.e., cluster 1 (N. cyriacigeorgica), cluster 2 (N. brasiliensis), cluster 3 (N. farcinica), cluster 4 (N. puris), cluster 5 (N. asiatica), and cluster 6 (N. beijingensis), based on the six peaks generated by ClinProTools with the genetic algorithm, i.e., m/z 2,774.477 (cluster 1), m/z 5,389.792 (cluster 2), m/z 6,505.720 (cluster 3), m/z 5,428.795 (cluster 4), m/z 6,525.326 (cluster 5), and m/z 16,085.216 (cluster 6). Two clusters of L. monocytogenes spectra were also found according to the five peaks, i.e., m/z 5,594.85, m/z 6,184.39, and m/z 11,187.31, for cluster 1 (serotype 1/2a) and m/z 5,601.21 and m/z 11,199.33 for cluster 2 (serotypes 1/2b and 4b). The Bruker Biotyper system was unable to accurately identify Nocardia (except for N. nova and N. otitidiscaviarum), Tsukamurella, or Gordonia species. Continuous expansion of the MALDI-TOF MS databases to include more GPRs is necessary.

Tumebacillus flagellatus sp. nov., an α-amylase/pullulanase-producing bacterium isolated from cassava wastewater.

A novel α-amylase/pullulanase-producing bacterium, designated strain GST4(T), was isolated from samples collected from the wastewater of a cassava starch factory in Nanning, Guangxi Autonomous Region, southern China. Cells of strain GST4(T) were rod-shaped bacilli containing ellipsoidal terminal spores and found to be Gram-reaction-positive, aerobic, motile, oxidase-positive, catalase-negative and formed light yellow colonies on agar plates. Strain GST4(T) was able to grow at pH 4.5-8.5 (optimum at pH 5.5), temperatures ranging from 20 to 42 °C (optimum at 37 °C) and salt concentrations of 0-1% (w/v) NaCl (optimum at 0.5%, w/v) on R2A medium. Strain GST4(T) grew heterotrophically on complex carbon substrates and chemolithoautotrophically on inorganic sulfur compounds, as demonstrated by growth on sodium thiosulfate and sulfite as sole electron donors. It can reduce nitrate and nitrite. Strain GST4(T) contained iso-C(15:0) and anteiso-C(15:0) as the major cellular fatty acids and menaquinone 7 (MK-7) as the major respiratory quinone. The cell-wall peptidoglycan was of type A1γ. The genomic DNA G+C content of strain GST4(T) was 53.7 mol%. Physiological and chemotaxonomic characteristics combined with phylogenetic analysis based on 16S rRNA gene sequences revealed that strain GST4(T) was a member of the genus Tumebacillus and most closely related to Tumebacillus permanentifrigoris DSM 18773(T) and Tumebacillus ginsengisoli DSM 18389(T) with 97.3 and 94.5% sequence similarity, respectively. The DNA-DNA relatedness values between strain GST4(T) and T. permanentifrigoris DSM 18773(T), and strain GST4(T) and T. ginsengisoli DSM 18389(T) were 44.0 and 60.4%, respectively. The new isolate differed from those species of the genus Tumebacillus in that it has peritrichous flagella for motility. Based on the evidence obtained from this study, strain GST4(T) represents a novel species of the genus Tumebacillus, for which the name Tumebacillus flagellatus sp. nov. is proposed. The type strain is GST4(T) ( =CGMCC 1.12170(T) =DSM 25748(T)).

Halanaerobium sehlinense sp. nov., an extremely halophilic, fermentative, strictly anaerobic bacterium from sediments of the hypersaline lake Sehline Sebkha.

A strictly anaerobic, extremely halophilic, Gram-positive, rod-shaped bacterium was isolated from the hypersaline (>20% NaCl) surface sediments of Sehline Sebkha in Tunisia. The strain, designated 1Sehel(T), was strictly halophilic and proliferated at NaCl concentrations of between 5% and 30% (saturation), with optimal growth at 20% NaCl. Strain 1Sehel(T) was non-spore-forming, non-motile, appearing singly or in pairs, or occasionally as long chains and measured 0.5-0.8 µm by 3-10 µm. Strain 1Sehel(T) grew optimally at pH values of 7.4 but had a very broad pH range for growth (pH 5.2-9.4). It grew at temperatures between 20 and 50 °C with an optimum at 43 °C. Strain 1Sehel(T) required yeast extract for growth. The isolate fermented glucose, galactose, fructose, glycerol, mannose, maltose, ribose, pyruvate and sucrose. The fermentation products from glucose utilization were lactate, acetate, formate, ethanol, CO2 and H2. The G+C ratio of the DNA was 32.7 mol%. The major fatty acids were C15:1ω6c/7c, C16:1ω7c, C16:0 and C15:0. On the basis of phylogenetic and physiological properties, strain 1Sehel(T) (=DSM 25582(T)=JCM 18213(T)) is proposed as the type strain of Halanaerobium sehlinense sp. nov., within the family Halanaerobiaceae.

Exiguobacterium aquaticum sp. nov., a member of the genus Exiguobacterium.

A Gram-positive, motile, short rod-shaped, orange pigmented bacterium, designated strain IMTB-3094(T), was isolated from a water sample collected from Tikkar Tal Lake, Haryana, and subjected to detailed polyphasic taxonomic analysis. Strain IMTB-3094(T) possessed most of the phenotypic and chemotaxonomic properties of the genus Exiguobacterium and, based on 16S rRNA gene sequence analysis, was assigned to this genus. Strain IMTB-3094(T) exhibited the highest 16S rRNA gene sequence similarity to Exiguobacterium mexicanum MTCC 7759(T) (99.5 %) followed by Exiguobacterium aurantiacum MTCC 6414(T) (99.1 %), Exiguobacterium aestuarii MTCC 7750(T) (98.0 %), Exiguobacterium profundum MTCC 10851(T) (98.0 %) and Exiguobacterium marinum MTCC 7751(T) (98.0 %). The G+C content of the genomic DNA of strain IMTB-3094(T) was 53.2 mol% and a DNA-DNA relatedness study confirmed that it represents a novel species. The major fatty acids of strain IMTB-3094(T) were iso-C(17 : 0) (16.1 %), anteiso-C(13 : 0) (19.0 %), iso-C(13 : 0) (11.9 %), iso-C(15 : 0) (9.8 %) and iso-C(17 : 1) (12.7 %). The predominant quinones were MK-7 (55.0 %) and MK-6 (26.0 %) with minor amounts of MK-8 (12.0 %). Based on phenotypic, chemotaxonomic and phylogenetic analyses, strain IMTB-3094(T) represents a novel species of the genus Exiguobacterium, for which the name Exiguobacterium aquaticum sp. nov. is proposed. The type strain is IMTB-3094(T) (= MTCC 10958(T) = JCM 17977(T)).

A pilot study: microbiological conditions of the oral cavity in minipigs for peri-implantitis models.

As peri-implantitis is an emerging problem, the development of validated animal models is mandatory. The aim of this pilot study was to provide a first step in describing the normal oral flora of minipigs. In five minipigs, samples of the oral flora were collected with sterile cotton swabs from the buccal gingiva of the lower jaw. Two swabs per animal were collected, followed by bacterial isolation under both aerobe and anaerobe conditions. Microbiological analyses included biochemical tests, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rDNA gene sequence analysis. A total of 61 taxa were detected, 14-21 different bacterial taxa from each minipig. Among the Gram-positive cocci, mainly staphylococcal and streptococcal species were identified. Different Actinomyces species were the most abundant taxa in the group of Gram-positive rods. Among the anaerobic bacteria, the Gram-negative genera Fusobacterium, Bacteroides and Prevotella were the most often observed taxa. This is the first study which begins to describe the normal oral flora in minipigs in cultures to allow for the detection of a broad spectrum. Several bacterial species identified are different from human ones. No specific species for peri-implantitis could be detected in that healthy sample.

Salsuginibacillus halophilus sp. nov., a halophilic bacterium isolated from a soda lake.

A Gram-stain-positive, rod-shaped, endospore-forming, halophilic, alkalitolerant bacterium, designated halo-1(T), was isolated from sediment of Xiarinaoer soda lake, located in the Inner Mongolia Autonomous Region of China. Strain halo-1(T) grew in the presence of 9-30 % (w/v) NaCl (optimum 19 %) and at pH 5-10 (optimum pH 9). The cell-wall peptidoglycan contained meso-diaminopimelic acid and the major respiratory isoprenoid quinone was MK-7. The predominant cellular fatty acids of the isolate were anteiso-C(15 : 0) (58.35 %), anteiso-C(17 : 0) (12.89 %) and C(16 : 0) (6.52 %). The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, glycolipid and a phospholipid of unknown structure. The DNA G+C content of the strain was 46.4 mol%. On the basis of 16S rRNA gene sequence similarity, strain halo-1(T) showed the highest similarity (93.9 %) to Salsuginibacillus kocurii CH9d(T). Strain halo-1(T) could be clearly differentiated from its closest phylogenetic relative on the basis of several phenotypic, genotypic and chemotaxonomic features. Therefore, strain halo-1(T) represents a novel species, for which the name Salsuginibacillus halophilus sp. nov. is proposed, with the type strain halo-1(T) (=CGMCC 1.7653(T) =NBRC 104934(T)).

The conundrum of the gram-positive rod: are we missing important pathogens in complicated skin and soft-tissue infections? A case report and review of the literature.

BACKGROUND: The designation "gram-positive bacillus" includes a variety of pleomorphic microorganisms, including diphtheroids, coryneform species, coccobacilli, and other small rods. Despite differing greatly in their virulence, sources, and even genus, these microscopically similar organisms are often difficult to differentiate without genetic testing. METHODS: We present a patient with necrotizing fasciitis and a review of the literature to exemplify and assess the scope of this diagnostic conundrum. Cultures taken intra-operatively during surgical debridement grew Morganella morganii and "diphtheroids." Given the low virulence of both organisms, the diphtheroids were reexamined microscopically and assayed for enzymatic activity. Genetic sequence analysis of 16S ribosomal ribonucleic acid (rRNA) was required for species identification. RESULTS: Microscopic inspection identified small, non-spore-forming, gram-positive rods, arranged in clusters, that formed circular, smooth colonies. These were facultatively anaerobic, catalase-negative, non-hemolytic, and unable to reduce nitrates. Standard techniques and assays were unable to identify our organism to species. Ultimately, 16S rRNA gene sequencing of 833 base pairs achieved a 99.04% species match to Arcanobacterium bernardiae. CONCLUSION: At our facility, diphtheroids are generally considered non-pathogenic contaminants in skin and soft tissue infections. The finding of A. bernardiae in necrotizing fasciitis is unusual and clinically important but would have been missed using conventional methods. As the "gram-positive bacillus" comes to include an ever-increasing number of organisms, genetic sequencing will probably be required more regularly for species identification. Furthermore, given that these genera are similar, often mistaken as contaminants, and difficult to differentiate using standard assays, they may often be missed and are possibly a more-frequent cause of complicated skin and soft tissue infections than the literature would suggest.

Bhargavaea cecembensis gen. nov., sp. nov., isolated from the Chagos-Laccadive ridge system in the Indian Ocean.

A novel Gram-positive, rod-shaped, non-motile, non-spore-forming bacterium, strain DSE10(T), was isolated from a deep-sea sediment sample collected at a depth of 5904 m from the Chagos-Laccadive ridge system in the Indian Ocean. Cells of strain DSE10(T) were positive for catalase, oxidase, urease and lipase activities and contained iso-C(14 : 0), iso-C(15 : 0), iso-C(16 : 0) and anteiso-C(15 : 0) as the major fatty acids. The major respiratory quinones were MK-6 and MK-8 and the major lipids were phosphatidylglycerol and diphosphatidylglycerol. The cell-wall peptidoglycan contained diaminopimelic acid as the diagnostic diamino acid. A blast sequence similarity search based on 16S rRNA gene sequences indicated that the genera Planococcus, Planomicrobium, Bacillus and Geobacillus were the nearest phylogenetic neighbours to the novel isolate with gene sequence similarities ranging from 94.9 to 95.2 %. Phylogenetic analyses using neighbour-joining, minimum-evolution and maximum-parsimony methods indicated that strain DSE10(T) formed a deeply rooted lineage distinct from the clades represented by the genera Planococcus, Planomicrobium, Bacillus and Geobacillus. Further, strain DSE10(T) could be distinguished from the above-mentioned genera based on the presence of signature nucleotides G, A, C, T, C, A, G, C and T at positions 182, 444, 480, 492, 563, 931, 1253, 1300 and 1391, respectively, in the 16S rRNA gene sequence. Based on the phenotypic and phylogenetic characteristics determined in this study, strain DSE10(T) was assigned as the type species of a new genus, Bhargavaea gen. nov., as Bhargavaea cecembensis sp. nov. The type strain of Bhargavaea cecembensis gen. nov., sp. nov. is DSE10(T) (=LMG 24411(T)=JCM 14375(T)). The genomic DNA G+C content of strain DSE10(T) is 59.5+/-2.5 mol%.