Detection of an unusual G8P[8] rotavirus in a Rotarix-vaccinated child with acute gastroenteritis using Nanopore MinION sequencing: A case report.

RATIONALE: Rotavirus is routinely diagnosed by the detection of antigens or the viral genome. However, these tests have limitations, in that they do not detect all rotavirus strains. PATIENT CONCERNS: We present a case of a 27-month-old girl who was hospitalized for 4 days with severe gastroenteritis, including high fever, vomiting, diarrhea, mild dehydration, and periumbilical pain. Notably, the patient previously received the Rotarix vaccine. DIAGNOSES: The laboratory tests were negative for rotavirus, astrovirus, adenovirus, and norovirus as well as common diarrhea-causing bacteria. Human-bovine recombinant rotavirus was detected by MinION sequencing. INTERVENTIONS: To investigate the cause agents from the unexplained severe gastroenteritis infant, the stool sample was prepared by random amplification for Nanopore MinION sequencing. OUTCOMES: Treatment through the administration of ORS solution and galtase powder with probiotics was successful after the diagnosis of unusual rotavirus infection. LESSONS: This case report is the first detection of an unusual human-bovine recombinant rotavirus in an idiopathic gastroenteritis using Nanopore MinION sequencing.

Effect of inorganic salts on bacterial omega-3 PUFA production.

The increasing demand of omega-3 in the market and the challenges facing its conventional supplies led to an increasing interest to microbial omega-3 sources. This research concentrates on the statistical role of some metal ions on the biosynthesis and productivity of eicosapentaenoic acid (essential omega-3 element) in bacterial isolate, Shewanella 717. A Plackett-Burman design was applied to screen the main effect of all metal salts entrenched in the artificial sea water medium components. Four salts, in particular, in addition to the interaction among them were highlighted as having a statistically significant effect upon the growth and/or eicosapentaenoic acid production. A subsequent central composite design was performed to determine the exact optimum concentration of each of the chosen variables which was found to be 2.5, 1.8, 1.2, and 23 g/l, for Na2HPO4, MgSO4, KCl, and NaCl, respectively. All the experiments were performed with the minimal amount of carbon and nitrogen to eliminate any potential masking effect. A bioreactor batch run was operated and the ion uptake was monitored, using EDAX® electron microscopy, concluding that the process of microbial omega-3 production could be a phosphate-limited process. Optimizing the concentration of the tested metal ions led to a remarkable increase in the omega-3 productivity resulted in a 30, 9, and 10 times increase in yield, concentration, and percentage to the total fatty acids, respectively, even though the carbon and nitrogen were kept constant all over the research work.

Kangiella chungangensis sp. nov. isolated from a marine sand.

A Gram-negative bacterium, designated CAU 1040(T), which was isolated from marine sand obtained from Jeju Island in South Korea, was characterized as an aerobic rod-shaped organism that that was non-motile, non-spore-forming and halophilic. The bacterium grew optimally at 37 °C, at pH 8, and in the presence of 2% (w/v) NaCl. The taxonomic classification of CAU 1040(T) was investigated using a polyphasic characterization approach. While phylogenetic analysis of the 16S rRNA gene sequence revealed that CAU 1040(T) belongs to the genus Kangiella, the strain exhibited only 94.4-95.4% sequence similarity to the previously described Kangiella species. Similar to other Kangiella species, Q-8 was the predominant ubiquionone and iso-C(15:0) was the major cellular fatty acid detected in strain CAU 1040(T). The predominant polar lipids identified were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The G+C content of the CAU 1040(T) genome was 45.3 mol%. The phylogenetic, physiological, biochemical and chemotaxonomic data obtained in this study indicate that strain CAU 1040(T) represents a novel species of the genus Kangiella, for which the name Kangiella chungangensis sp. nov. is hereby proposed. The type strain is CAU 1040(T) (KCTC 42299(T), NBRC 110728(T)).

Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses.

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

Screening of marine bacterial producers of polyunsaturated fatty acids and optimisation of production.

Water samples from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea were screened in order to isolate new polyunsaturated fatty acids (PUFAs) bacterial producers especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Two hundred and fifty-one isolates were screened for PUFA production and among them the highest number of producers was isolated from the Mid-Atlantic Ridge followed by the Red Sea while no producers were found in the Mediterranean Sea samples. The screening strategy included a simple colourimetric method followed by a confirmation via GC/MS. Among the tested producers, an isolate named 66 was found to be a potentially high PUFA producer producing relatively high levels of EPA in particular. A Plackett-Burman statistical design of experiments was applied to screen a wide number of media components identifying glycerol and whey as components of a production medium. The potential low-cost production medium was optimised by applying a response surface methodology to obtain the highest productivity converting industrial by-products into value-added products. The maximum achieved productivity of EPA was 20 mg/g, 45 mg/l, representing 11% of the total fatty acids, which is approximately five times more than the amount produced prior to optimisation. The production medium composition was 10.79 g/l whey and 6.87 g/l glycerol. To our knowledge, this is the first investigation of potential bacteria PUFA producers from Mediterranean and Red Seas providing an evaluation of a colourimetric screening method as means of rapid screening of a large number of isolates.

Response surface methodology for optimising the culture conditions for eicosapentaenoic acid production by marine bacteria.

Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA), are increasingly attracting scientific attention owing to their significant health-promoting role in the human body. However, the human body lacks the ability to produce them in vivo. The limitations associated with the current sources of ω-3 fatty acids from animal and plant sources have led to increased interest in microbial production. Bacterial isolate 717 was identified as a potential high EPA producer. As an important step in the process development of the microbial PUFA production, the culture conditions at the bioreactor scale were optimised for the isolate 717 using a response surface methodology exploring the significant effect of temperature, pH and dissolved oxygen and the interaction between them on the EPA production. This optimisation strategy led to a significant increase in the amount of EPA produced by the isolate under investigation, where the amount of EPA increased from 9 mg/g biomass (33 mg/l representing 7.6 % of the total fatty acids) to 45 mg/g (350 mg/l representing 25 % of the total fatty acids). To avoid additional costs associated with extreme cooling at large scale, a temperature shock experiment was carried out reducing the overall cooling time from the whole cultivation process to 4 h only prior to harvest. The ability of the organism to produce EPA under the complete absence of oxygen was tested revealing that oxygen is not critically required for the biosynthesis of EPA but the production improved in the presence of oxygen. The stability of the produced oil and the complete absence of heavy metals in the bacterial biomass are considered as an additional benefit of bacterial EPA compared to other sources of PUFA. To our knowledge this is the first report of a bacterial isolate producing EPA with such high yields making the large-scale manufacture much more economically viable.

Polyunsaturated fatty acid production by marine bacteria.

Polyunsaturated fatty acids are important in maintaining human health. Limitations associated with current sources of ω-3 fatty acids and ω-6 fatty acids, from animal and plant sources, have led to increased interest in microbial production. Marine bacteria may provide a suitable alternative, although the isolation of production strains and the identification of operating conditions must be addressed before manufacturing processes become economically viable. Marine isolate 560 was identified as an eicosapentaenoic acid (EPA) producer via GC/MS. The isolate was initially identified as Vibrio cyclitrophicus by 16S rRNA sequencing. Statistically based experimental designs were applied to the optimisation of medium components and environmental factors for the production of EPA. A Plackett-Burman design was used to screen for the effect of temperature, pH, and media components. Subsequently, the concentrations of NaCl, yeast extract, and peptone, identified as significant factors, were optimised using a central composite design. The predicted optimal combination of media components for maximum EPA production (4.8 mg/g dry weight) was determined as 7.9 g/l peptone, 16.2 g/l NaCl, and 6.2 g/l yeast extract. On transfer of this process to bioreactor cultivation, where a range of pH and DO values were tested, the maximum amount of EPA produced increased to 7.5 mg/g dry weight and 10 % of the total fatty acid.

Use of physiological information and process optimisation enhances production of extracellular nuclease by a marine strain of Bacillus licheniformis.

The extracellular nuclease, NucB, from Bacillus licheniformis, can digest extracellular DNA in biofilms, causing biofilm dispersal, and may therefore be used commercially to remove biofilms. However, producing quantities of this secreted peptide is difficult and our aim was therefore to improve its laboratory scale production. This study builds on our understanding of B. licheniformis physiology to enhance NucB production. The addition of manganese, which triggers sporulation and enhances NucB expression, lead to a 5-fold increase in NucB production. Optimisation via Placket-Burman design of experiments identified 3 significant medium components and a subsequent Central Composite Design, to determine the optimum levels of these components, resulted in a 10-fold increase to 471U/ml. The optimal phosphate concentration was less than 0.3mM as this is known to inhibit nuclease production. The use of physiologically relevant information combined with optimisation represents a promising approach to increased enzyme production, which may also be widely applicable.

Surface microbial consortia from Livarot, a French smear-ripened cheese.

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.

Mycetocola reblochoni sp. nov., isolated from the surface microbial flora of Reblochon cheese.

Four Gram-positive, aerobic, non-sporulating, rod-shaped bacteria isolated from the surface microflora of Reblochon cheese at the late stage of ripening had chemotaxonomic properties characteristic of members of the family Microbacteriaceae. The isolates had virtually identical SDS-PAGE whole-organism protein patterns, shared many chemical and phenotypic characteristics and formed an independent branch in the Microbacteriaceae 16S rRNA gene tree that was most closely related to the type strains of Mycetocola species. The new isolates had chemotaxonomic properties consistent with their classification in the genus Mycetocola but were readily distinguished from recognized members of this taxon based on DNA-DNA relatedness, whole-organism protein and phenotypic data. The combined genotypic and phenotypic data indicate that the isolates should be classified in the genus Mycetocola as members of a novel species, for which the name Mycetocola reblochoni sp. nov. is proposed. The type strain is LMG 22367(T) (=R-20377(T) =BRB-1L41(T) =DSM 18580(T)).

Individual variation in 3-methylbutanal: a putative link between human leukocyte antigen and skin microflora.

The human derma emits volatile compounds whose interaction with a receiver's olfactory sensory system may affect individual recognition and mating preferences. Studies suggest that both genes and environmental factors determine characteristic odor of an individual. We used solid-phase microextraction and gas chromatography-mass spectrometry to identify 3-methylbutanal in human axillary odor; we showed that the abundance of this volatile compound varies significantly among individuals and demonstrated that its formation in vitro may be influenced by interaction between human leukocyte antigen peptide and dermal microflora.

Characterisation of micromonosporae from aquatic environments using molecular taxonomic methods.

Large numbers of strains assigned to the genus Micromonospora on the basis of typical colonial and pigmentation features were isolated from diverse aquatic sediments using a standard selective isolation procedure. Two hundred and six isolates and eight representatives of the genus Micromonospora were assigned to 24 multimembered groups based on a numerical analysis of banding patterns generated using BOX and ERIC primers. Representatives of multimembered groups encompassing isolated micromonosporae were the subject of 16S rRNA gene sequencing analyses. Good congruence was found between the molecular fingerprinting and 16S rRNA sequence data indicating that the groups based upon the former are taxonomically meaningful. Nearly all of the isolates that were chosen for the 16S rRNA gene sequencing analyses showed that the ecosystems studied are a rich source of novel micromonosporae. These findings have implications for high throughput screening for novel micromonosporae as BOX and ERIC fingerprinting, which is rapid and reproducible, can be applied as a robust dereplication procedure to indicate which environmental isolates have been cultured previously.

Streptomyces griseus 45H, a producer of the extracellular autoregulator protein factor C, is a member of the species Streptomyces albidoflavus.

Streptomyces griseus strain 45H, isolated in 1960 during a mutagenesis programme on the industrial streptomycin producer S. griseus 52-1, encodes an extracellular, pleiotropic autoregulatory signalling protein, factor C, which stimulates sporulation of S. griseus 52-1 in submerged culture. The facC gene, which codes for factor C, is present in very few streptomycetes and is not present in S. griseus 52-1. Based on 16S rRNA gene sequencing and other molecular data, S. griseus 45H, the factor C producer, is here shown to be related to the original laboratory strain of Streptomyces flavofungini, which was being studied in the same laboratory in 1960, and to Streptomyces albidoflavus. Southern blotting revealed that three out of four independently isolated strains of S. albidoflavus possess facC. Both the original strain of S. flavofungini and S. griseus 45H are therefore identified as members of the species Streptomyces albidoflavus, and we propose that S. griseus 45H should be renamed Streptomyces albidoflavus 45H.

Diversity and geographical distribution of members of the Streptomyces violaceusniger 16S rRNA gene clade detected by clade-specific PCR primers.

The Streptomyces violaceusniger 16S rRNA gene clade contains organisms that are of ecological interest and a rich source of novel bioactive metabolites. Improvements in the classification of members of the S. violaceusniger clade made it possible to design, evaluate and use an oligonucleotide primer set to gain an insight into the presence, distribution and taxonomic diversity of members of this taxon in environmental samples. In silico testing showed that the primers had a perfect match with representatives of the S. violaceusniger clade. The primers, designated S-S-Svio-66-a-S-20 and S-S-Svio-1274-a-A-20, amplified an approximately 1190-bp stretch of 16S rRNA gene from authenticated members of the S. violaceusniger clade, but not from representatives of other actinomycete taxa. Following amplification of DNA extracted from sediment and soil samples, the sequences of cloned PCR products confirmed the specific amplification of target sequences in 87% of the clones; the use of 16S rRNA gene fragment similarity correlations indicated that the clones represented new species. The primers can be used to facilitate the isolation of novel members of the S. violaceusniger 16S rRNA gene clade by allowing prescreening of environmental samples and the subsequent detection and retrieval of targetted strains through the use of selective isolation procedures.

Prevalence and distribution of nucleotide sequences typical for pMEA-like accessory genetic elements in the genus Amycolatopsis.

The prevalence and distribution of pMEA-like elements in the genus Amycolatopsis was studied. For this purpose, a set of 95 recently isolated Amycolatopsis strains and 16 Amycolatopsis type strains were examined for the presence of two unique pMEA-sequences (repAM and traJ), encoding proteins essential for replication and conjugative transfer. Homologues of repAM and traJ were found in 10 and 26 of 111 investigated strains, respectively, a result which shows that pMEA-like sequences, though not very abundant, can be found in several Amycolatopsis strains. Phylogenetic analysis of the deduced RepAM and TraJ protein sequences revealed clustering with the protein sequences of either pMEA300 or pMEA100. Furthermore, two geographically different populations of pMEA-like elements were distinguished, one originating in Europe and the other in Australia and Asia. Linkage between the distribution of repAM and traJ and the chromosomal identifier, the 16S rRNA gene, indicated that these elements coevolved with their hosts, suggesting that they evolved in an integrated form rather than by horizontal gene transfer of the free replicating form.

Murine scent mark microbial communities are genetically determined.

Scent marking in mice allows males to communicate information such as territory ownership, male competitive ability and current reproductive, nutritional, social and health status. It has been suggested that female mice eavesdrop on these olfactory cues, using them as a means of selecting mates with dissimilar major histocompatibility complex (MHC) genes, known as H2 in mice. The mechanisms underpinning MHC-dependent olfactory communication remain unresolved. Using congenic mouse strains and molecular methods we explore the involvement of the microbial communities, a known source of odourants, in scent marks to test the hypothesis that the microbial communities and hence the olfactory signals are genetically determined. Here we show that the indigenous microbial community of murine scent marks is genetically determined. Both background genotype and H2 haplotype influence the community structure of the scent mark flora, removing the possibility that community composition is solely orchestrated by the MHC. Qualitative and quantitative components of the bacterial community associated with MHC haplotype and background genotype were identified. The analyses confirm that the four groups of congenic mice tested are distinguishable on basis of the microbiology of their scent marks alone, strengthening the role of microorganisms in the development of MHC-dependent odours.

Agrococcus casei sp. nov., isolated from the surfaces of smear-ripened cheeses.

Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4-98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2(T) (=DSM 18061(T)=LMG 22410(T)) is the type strain of Agrococcus casei sp. nov.

Dermacoccus barathri sp. nov. and Dermacoccus profundi sp. nov., novel actinomycetes isolated from deep-sea mud of the Mariana Trench.

The taxonomic positions of two actinobacterial strains isolated from Mariana Trench sediment were established using a combination of genotypic and phenotypic data. The strains, isolates MT2.1(T) and MT2.2(T), formed a distinct phyletic line in the Micrococcineae 16S rRNA gene tree together with Dermacoccus abyssi NCIMB 14084(T). The isolates had chemical and phenotypic properties typical of members of the genus Dermacoccus and could be distinguished sharply from one another and from the type strains of Dermacoccus abyssi and Dermacoccus nishinomiyaensis using DNA-DNA relatedness data. A range of phenotypic properties served to distinguish the two novel strains from one another and from the type strains of established Dermacoccus species. The G+C contents of the DNAs of strains MT2.1(T) and MT2.2(T) were 66.8 and 69.1 mol%, respectively. It is evident that the two isolates merit recognition as novel species within the genus Dermacoccus. The names proposed for these taxa are Dermacoccus barathri sp. nov. (type strain MT2.1(T)=DSM 17574(T)=NCIMB 14081(T)) and Dermacoccus profundi sp. nov. (type strain MT2.2(T)=DSM 17575(T)=NCIMB 14126(T)) [corrected]

Fluostatins C-E, novel members of the fluostatin family produced by Streptomyces strain Acta 1383.

Three new members of the fluostatin family, fluostatins C-E, were discovered in a culture filtrate extract of strain Acta 1383 during an HPLC screening program. The producing strain belongs to the genus Streptomyces and is closely related to type strains classified in the Streptomyces lavendulae 16S rRNA subclade. Fluostatins are named by their characteristic fluorenone chromophore. Fluostatin C shows moderate activity against selected human tumor cell lines.

Phosphate starvation induces the sporulation killing factor of Bacillus subtilis.

Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.