Unknown Strains Research Articles

New species of aquatic chytrids from Oman

ABSTRACT Oman is a desert country in the south of the Middle East. Springs and other water sources that harbor aquatic organisms can be separated by hundreds of kilometers. In Oct 2019, we isolated four freshwater aquatic fungi (Chytridiomycota) from benthic detritus baited with pine pollen on a general nutrient medium near Salalah, Oman. Database queries of nuc 28S rRNA (28S) and internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) revealed that one of these strains was Dinochytrium kinnereticum, a recently described algal pathogen from the Sea of Galilee. The other three strains had low molecular identity to available ITS sequences. These unknown strains varied in size and released endogenously swarming zoospores through papillae from mature zoosporangia. Zoospore ultrastructure was consistent with described species in the Rhizophydiales, and molecular phylogenetic results grouped these three strains into a clade in the genus Rhizophydium. We circumscribe these three strains as a sp. nov., thereby expanding the diversity within Rhizophydium described as the new species R. jobii. In doing so, we provide the first report of Chytridiomycota from Oman.

COVID-19 diagnostic approaches: different roads to the same destination.

“SARS-CoV2”, a previously unknown strain of coronaviruses caused a severe respiratory disease called Coronavirus disease (COVID-19) which emerged from Wuhan city of China on 30 December 2019, and declared as Global health problem by World Health Organisation within a month. In less than two and half months (11 March, 2020) it was declared as a pandemic disease due to its rapid spreading ability, it covered more than 211 countries infecting around 1.7 million persons and claiming around 1.1 lakhs lives within merely 100 days of its emergence. Containment of the infection of this virus is the only available measure to control the disease as no vaccine or specific antiviral treatment is available. Confirmed detection of the virus followed by isolation of the infected person at the earliest possible is the only measure to prevent this disease. Although there are number of methods available for detection of virus and to combat this disease in the present pandemic situation, but these available diagnostic methods have their own limitations. The speedy and exponential global spread of this disease strongly urges the fast and economic diagnostics tools. Additional to the available diagnostic methods, there is a sudden surge for development of various of methods and platforms to diagnose the COVID-19. The review summarized the advantage and disadvantage of various diagnostic approaches being used presently for COVID-19, newer detection methods in developmental stage and the feasibility of advanced platforms like newer nano-sensor based on-the-spot detection technologies.

The polarization process of ferroelectric materials in the framework of variational inequalities

AbstractWe are concerned with the mathematical modeling of the polarization process in ferroelectric media. We assume that this dissipative process is governed by two constitutive functions, which are the free energy function and the dissipation function. The dissipation function, which is closely connected to the dissipated energy, is usually non‐differentiable. Thus, a minimization condition for the overall energy includes the subdifferential of the dissipation function. This condition can also be formulated by way of a variational inequality in the unknown fields strain, dielectric displacement, remanent polarization and remanent strain. We analyze the mathematical well‐posedness of this problem. We provide an existence and uniqueness result for the time‐discrete update equation. Under stronger assumptions, we can prove existence of a solution to the time‐dependent variational inequality. To solve the discretized variational inequality, we use mixed finite elements, where mechanical displacement and dielectric displacement are unknowns, as well as polarization (and, if included in the model, remanent strain). It is then possible to satisfy Gauss' law of zero free charges exactly. We propose to regularize the dissipation function and solve for all unknowns at once in a single Newton iteration. We present numerical examples gained in the open source software package Netgen/NGSolve.

Genome sequences of two Salmonella enterica strains (MEZSAL74 and MEZSAL81) harbouring multiple antimicrobial resistance genes isolated from livestock in South Africa

ObjectivesAntimicrobial-resistant livestock-associated Salmonella enterica infections pose a significant public-health threat worldwide. Here we report for the first time the draft genome sequences of two multidrug-resistant livestock-associated S. enterica strains isolated from a chicken and a cow in South Africa. MethodsGenomic DNA of S. enterica strains MEZSAL74 and MEZSAL81 was sequenced using an Illumina MiSeq platform. The generated reads were trimmed and de novo assembled. The assembled contigs were analysed for antimicrobial resistance genes, chromosomal mutations and extrachromosomal plasmids. Multilocus sequence typing (MLST) was also performed. In order to compare isolates MEZSAL74 and MEZSAL81 with other previously sequenced S. enterica isolates, raw read sequences were downloaded and all sequence files were treated identically to generate a bootstrapped maximum likelihood phylogenetic tree. ResultsExtrachromosomal plasmids and genetic determinants of antimicrobial resistance were detected in both sequenced bacterial isolates to aminoglycosides and fluoroquinolones. By MLST, strain MEZSAL74 belonged to an unknown sequence type (ST) and strain MEZSAL81 belonged to ST33. ConclusionThe genome sequences of strains MEZSAL74 and MEZSAL81 reported here will serve as a reference for molecular epidemiological studies of antimicrobial-resistant livestock-associated S. enterica in Africa.

Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study.

BackgroundHuman infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans.MethodsWe collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Wuhan Jinyintan Hospital, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed.ResultsFive patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown β-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor.ConclusionA novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.

Numerical-and-Analytical Method for Creep Investigation in Functionally Graded Complex-Shaped Bodies of Rotation

The spatial axisymmetric creep problem for complex-shaped bodies of rotation from functionally graded materials is considered. For the variational problem statement, the Lagrangian-form functional is defined for kinematically possible displacement rates. For the main unknown creep parameters, namely displacements, stresses, and strains, the Cauchy problem in time scale is formulated for the spatial discretization points. In this case, the initial conditions for the unknown functions are derived by solving the problem of elastic deformation of the body. The numerical-andanalytical method was developed for solving a nonlinear initial boundary-value creep problem based on applying the R-function, Ritz, and Runge–Kutta–Merson methods. The advantages of the proposed method include: the accurate analytical account of the boundary value problem geometry features without their approximation, representation of an approximate solution in the analytical form, and automatic selection of a time step. The creep problem solutions are derived for a hollow straight cylinder and a complex-shaped body of rotation, namely a cylinder with an elliptical notch on the outer surface, loaded by constant internal pressure. The material creep is described by the Norton law. Several variation patterns of the material creep properties along the radial coordinate are considered. The effect of the material gradient properties and geometry on the stress-strain state of bodies of rotation is analyzed. It is shown that the above geometry effect in creep strongly depends on the material properties.

Toward Predicting Gene Expression and Metabolic Functions from Label-Free Raman Imaging of Living Cells

Current label-free imaging methods do not allow to estimate or predict the gene expression and specific metabolic functions directly from living cells. We hypothesized that the spectral signatures of molecules obtained by Raman spectroscopy are linked to the gene expression through the complex metabolic/proteomic network of the cell. We developed a unique approach to predict, in an accurate manner, the gene expression of thousands of living cells observed by microscopic spectral imaging, without any labeling nor destructive treatment. Using a trained AI model, our multi-omic approach enabled the prediction of the full transcriptome of living bacteria cells from new spectral data of unknown strain with high accuracy. In addition, the model parameters highlighted metabolic functions with cell-line specificity corresponding to the phenotype of the cells, demonstrating the model does match biological information. Escherichia coli strains exhibiting antibiotic resistance are used as a case study. This work is a milestone toward a revolution of the use of Raman spectral imaging in biomedical and industrial fields.

Saezia sanguinis gen. nov., sp. nov., a Betaproteobacteria member of order Burkholderiales, isolated from human blood.

The taxonomic position of an unknown bacterial strain designated CNM695-12, isolated from the blood of an immunocompromised subject, was investigated via phenotypic, chemotaxonomic, genotypic and genomic analyses. Bacterial cells were determined to be Gram-stain-negative bacilli, aerobic, non-motile and non-spore-forming. The strain showed catalase activity but no oxidase activity. Optimal growth occurred at 37 °C, pH 7 and with 0-1 % NaCl. C16 : 0, summed feature 8 (comprising C18 : 1ω7c /C18:1 ω6c), and C18 : 1ω9c were the most abundant fatty acids, and ubiquinone 8 was the major respiratory quinone. The polar lipids present included phosphatidylglycerol, phosphatidylethanolamine and other aminophospholipids. The 16S rRNA gene sequence showed approximately 93.5 % similarity to those of different species with validly published names within the order Burkholderiales (e.g. Leptothrix mobilis Feox-1T, Aquabacterium commune B8T , Aquabacterium citratiphilum B4T and Schlegelella thermodepolymerans K14T). Phylogenetic analyses based on 16S rRNA gene sequences and concatenated alignments including the sequences for 107 essential proteins, revealed the strain to form a novel lineage close to members of the family Comamonadaceae. The highest average nucleotide identity and average amino acid identity values were obtained with Schlegelella thermodepolymerans K14T (69.6 and 55.7 % respectively). The genome, with a size of 3.35 Mb, had a DNA G+C content of 52.4 mol% and encoded 3056 predicted genes, 3 rRNA, 1 transfer-messengerRNA and 51 tRNA. Strain CNM695-12 thus represents a novel species belonging to a novel genus within the order Burkholderiales, for which the name Saezia sanguinis gen. nov., sp. nov. is proposed. The type strain is CNM695-12T (=DSM 104959T=CECT 9208T).

A Comprehensive Analysis of the Global Human Gut Archaeome from a Thousand Genome Catalogue

The human gut microbiome plays an important role in health and disease, but the archaeal diversity therein remains largely unexplored. Here we report the pioneering analysis of 1167 non-redundant archaeal genomes recovered from diverse human gastrointestinal tract microbiomes. We identified 15 novel species including 52 previously unknown archaeal strains. Based on distinct genomic features, we warrant the split of the Methanobrevibacter smithii clade into two separate species. Patterns derived from 1.8 million proteins coded in these genomes showed substantial correlation with socio-demographic characteristics such as age and lifestyle. We infer that archaea are actively replicating in the human gut and are characterized by specific genomic and functional adaptations to the host. The archaeome carries a complex virome, with some species showing unexpected host flexibility. Our work furthers our current understanding of the human archaeome, and provides a large genome catalogue for future analyses to decipher its impact on human physiology.

From Gene to Function: A Safe Laboratory Exercise Promoting the Learning of the Central Dogma of Biology for the Undergraduate Biology Curriculum.

The central dogma of biology, which explains how the information in genes flows into proteins, can be challenging to teach in the undergraduate classroom. To allow students to understand the correlation between genes and protein functions, we have developed a practical laboratory activity that complements classroom learning. By using PCR analysis targeting the flagellin gene flgA1, students will investigate the genotype of an unknown strain of Haloferax volcanii, a halophilic archaeon appropriate for activities with undergraduates as it is nonpathogenic, inexpensive, easy to grow using “grocery store ingredients,” and offers many genetic tools. This species swims by means of flagella, and its motility can be tested using modified agar plates. Motile colonies will form swimming halos; meanwhile motility mutants will show only a dot on the motility plate, at the point of inoculation. First students will extract DNA for PCR amplification. They will not be told whether the strain they are assigned is motile or not. They will learn how to design primers for the target gene and set up the PCR reaction. Subsequently they will set up a motility assay and control of the provided strain. In the next laboratory period, students perform gel electrophoresis on the products of their PCR reactions and analyze the results. They compare these to the results of the motility assay to confirm the motility of the strain. In summary, this inquiry-based lab is easy and safe to perform and allows students to follow the information flow from a gene to a protein product.

Facile Discovery and Quantification of Isonitrile Natural Products via Tetrazine-Based Click Reactions.

A facile method for the quick discovery and quantification of isonitrile compounds from microbial cultures was established based on the isonitrile-tetrazine click reaction. This method was successfully applied to the rediscovery of diisonitrile antibotic SF2768 from an unknown strain Streptomyces tsukubensis. Finally, an in situ reduction further enabled bioorthogonal ligation of primary and secondary isonitriles for the first time.

Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children.

Background: As the most frequent infectious disease among children worldwide, dental caries have a strong relationship with oral hygiene status, specifically in the development of infection. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status. Here, we studied the oral hygiene status by examining the composition and proportion of oral Veillonella species in saliva of Japanese children. Methods: Microbial samples collected from 15 Japanese children divided into three oral hygiene groups were cultured under anaerobic conditions after homogenization and dilution, and inoculated onto brain heart infusion and selective medium Veillonella agar. Genomic DNA was extracted from each isolate. Veillonella species were detected by one-step PCR using rpoB species-specific primers. To analyse the phylogenetic properties of the unknown Veillonella strains, PCR amplification and sequence analysis of rpoB were conducted for 10 representative strains. Results: Although V. rogosae was found as the predominant species among all groups, its prevalence was significantly lower in the children with poor oral hygiene than in those with good oral hygiene. V. parvula was the prevalent species in the poor oral hygiene group. Approximately 10% of the isolated Veillonella strains were not classified to any established species; the phylogenetic analysis showed that they were most closely related to V.infantiumConclusions: This study demonstrates that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status. Changes in detection ratios of V. parvula and V. rogosae can be useful indicators of oral hygiene status. Furthermore, new strains closely related to V. infantium were isolated from the saliva of Japanese children.

Molecular-genetic characterization of Avian avulavirus 20 strains isolated from wild birds.

Previously unknown paramyxovirus strains were isolated from wild birds in 2013-2014 in Kazakhstan and subsequently identified as representatives of the novel Avian avulavirus 20 species. The aims and tasks were molecular genetic characterization of novel avulaviruses and investigation of their phylogenetic relationships. Embryonated chicken eggs were inoculated with cloacal and tracheal swabs from wild birds with subsequent virus isolation. The complete nucleotide sequences of viral genomes were obtained by massive parallel sequencing with subsequent bioinformatics processing. By initial infection of chicken embryos with samples from 179 wild birds belonging to the Anatidae, Laridae, Scolopacidae and Charadriidae families, 19 hemagglutinating agents were isolated, and five of them were identified as representatives of new viral species. The study of their sequenced genomes revealed their similarity in size, but there was a significant genetic variability within the species. 2,640 nucleotide substitutions were identified and 273 of them were nonsynonymous, influencing the protein structure of viruses. It was shown that isolates Avian avulavirus 20/black-headed gull/Balkhash/5844/2013 and Avian avulavirus 20 /great black-headed gull/Atyrau/5541/2013 were 86% and 95% respectively identical to the previously described reference strain, indicating a significant evolutionary divergence within species. The authors suggest the existence of two independent lineages - the Caspian, represented by the reference strain Aktau/5976 and Atyrau/5541, as well as the second, geographically significantly distant Balkhash lineage. The study confirms the role of the birds of the Laridae family as the main reservoir of Avian avulavirus 20 in the avifauna that plays a key role in maintaining viruses of the genus Avulavirus in the biosphere and is a potential natural source for the emergence of new viral variants. Continuous surveillance of them in the wild is one of the most important tasks in ensuring the safety of the poultry industry.

Life cycle assessment of Chlorella species producing biodiesel and remediating wastewater

Constantly rising energy demands, finite fossil fuel reserves and deteriorating environmental conditions have invoked worldwide interest to explore the sustainable sources of renewable biofuels. Locally adapted photosynthetic oleaginous microalgae with rapid growth on variable temperatures could be an ideal way for bioremediating the wastewater (WW) while producing the feedstock for biodiesel. To test this notion, an unknown strain was isolated from a sewage fed lake (Neela-Hauz). It was discerned as Chlorella sorokiniana-I using the 16S rDNA and 18S rDNA barcodes. The culture conditions such as pH, illumination, different temperature ranges and growth medium were cohesively optimized prior to the assessment of C. sorokiniana-I's efficacy to remediate the WWand biodiesel production. The strain has thrived well up to 40°C when continuously grown for 15 days. The highest lipid accumulation and biomass productivity were recorded in 100% WW. Fatty acid methyl ester (FAME) content was observed to be more than twice in WW (47%), compared to control synthetic media, TAP (20%) and BG11 (10%), which indicate the importance of this new isolate for producing economically viable biodiesel. Moreover, it is highly efficient in removing the total nitrogen (77%), total phosphorous (81%), iron (67%) and calcium (42%) from the WW. The quality of WW was considerably improved by reducing the overall chemical oxygen demand (48%), biological oxygen demand (47%) and alkalinity (15%). Thus, C. sorokiniana-I could be an ideal alga for the tropical countries in the remediation of WW while producing feedstock for biodiesel in a cost-effective manner.

Genetic and phenotypic characterization of bacterial strains isolated in Japan that resemble Erwinia rhapontici and E. persicinus

The taxonomic identity of Japanese isolates of bacteria with physiological and biochemical characteristics similar to Erwinia rhapontici or E. persicinus was clarified using genetic analyses, pathogenicity tests and phenotypic characterization. In a phylogenetic analysis based on 16S rRNA and housekeeping genes gyrB, rpoD, and recA, the strains formed two independent clusters, one for E. rhapontici and another for E. persicinus, each including the type strain. The E. rhapontici cluster had three subclusters: pink-pigmented strains isolated in Japan, the type strain, and nonpigmented strains isolated from wasabi. rep-PCR banding patterns confirmed this grouping. The ability to cause rot on plants and the rot severity depended on the strain, though virulence of E. rhapontici strains isolated in Japan tended to be relatively weaker than that of the others. Pink pigment formation by E. rhapontici and E. persicinus strains was abundant on onion, Welsh onion and garlic, but E. rhapontici strains isolated from wasabi did not produce pink pigment on any plants or media tested. Thus, the unknown strains from Japan that produced pink pigment were E. rhapontici or E. persicinus. Despite being nonpigmented, E. rhapontici strains from wasabi were genetically closely related to pink-pigmented E. rhapontici strains. Based on the rpoD sequence, two specific primer sets were newly designed to discriminate E. rhapontici and E. persicinus.

Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children

Background: As the most frequent infectious disease among children worldwide, dental caries have a strong relationship with oral hygiene status, specifically in the development of infection. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status. Here, we studied the oral hygiene status by examining the composition and proportion of oral Veillonella species in saliva of Japanese children. Methods: Microbial samples collected from 15 Japanese children divided into three oral hygiene groups were cultured under anaerobic conditions after homogenization and dilution, and inoculated onto brain heart infusion and selective medium Veillonella agar. Genomic DNA was extracted from each isolate. Veillonella species were detected by one-step PCR using rpoB species-specific primers. To analyse the phylogenetic properties of the unknown Veillonella strains, PCR amplification and sequence analysis of rpoB were conducted for 10 representative strains. Results: Although V. rogosae was found as the predominant species among all groups, its prevalence was significantly lower in the children with poor oral hygiene than in those with good oral hygiene. V. parvula was the prevalent species in the poor oral hygiene group. Approximately 10% of the isolated Veillonella strains were not classified to any established species; the phylogenetic analysis showed that they were most closely related to V. infantium Conclusions: This study demonstrates that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status. Changes in detection ratios of V. parvula and V. rogosae can be useful indicators of oral hygiene status. Furthermore, new strains closely related to V. infantium were isolated from the saliva of Japanese children.

Spoligotyping analysis of Mycobacterium tuberculosis in Khyber Pakhtunkhwa area, Pakistan.

Background: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics.Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France.Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, χ2=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (χ2=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa).Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.

Analysis of antimicrobial resistance and staphylococcal cassette chromosome mec (SCCmec) in 99 Staphylococcus epidermidis strains isolated from early pregnancy cervical swabs and external environment

Objective To analyze the antimicrobial resistance, distribution of resistance genes and staphylococcal cassette chromosome mec (SCCmec) in 99 strains of mecA gene-positive Staphylococcus epidermidis strains isolated from early pregnancy cervical swabs and external environment in Beijing Chaoyang District from 2015 to 2016. Methods Kirby-Bauer disk diffusion method was performed to detect the susceptibility of the 99 Staphylococcus epidermidis strains to cefoxitin. Microbroth dilution method was used to test their susceptibility to vancomycin, daptomycin, penicillin, erythromycin, compound sulfamethoxazole, tetracycline, ciprofloxacin, clindamycin, gentamicin and chloramphenicol. PCR was used to detect drug resistance genes of ermA, ermB, ermC, msrA, norA1, norA2, sul1, sul2, sul3, aac(6′)/aph(2″), ant(4′, 4″), ant(6) and tetM and to analyze the SCCmec types ofⅠ, Ⅱ, Ⅲ, Ⅳa, Ⅳb, Ⅳc, Ⅳd and Ⅴ. The results were compared with those of capillary electrophoresis. SPSS was used for data analysis. Results All of the 99 mecA-positive Staphylococcus epidermidis strains were sensitive to vancomycin and 93.94% of them were sensitive to datomycin. The resistance rates to penicillin, erythromycin, cefoxitin, compound sulfamethoxazole, tetracycline, ciprofloxacin, clindamycin, gentamicin and chloramphenicol were 97.98%, 85.86%, 79.80%, 52.54%, 27.27%, 43.43%, 36.36%, 23.23% and 11.11%. The strains that carried the genes of norA1, norA2, ermA, ermB, ermC, msrA, sul1, sul2, sul3, aac(6′)/aph(2″), ant(4′, 4″), ant(6) and tetM accounted for 100%, 93.94%, 0.00%, 3.03%, 17.17%, 57.58%, 50.51%, 12.12%, 4.04%, 30.30%, 8.08%, 4.04% and 25.26%, respectively. Among the 99 strains, 5.05%, 0%, 43.43%, 10.10%, 0.00%, 3.03%, 3.03% and 19.19% belonged to SCCmecⅠ, Ⅱ, Ⅲ, Ⅳa, Ⅳb, Ⅳc, Ⅳd and Ⅴ, respectively, and 4.04% (4/99) were positive to two SCCmec types. The types of 12.12% (12/99) of the strains were unidentified. Conclusions All of the 99 strains of mecA-positive Staphylococcus epidermidis were sensitive to vancomycin. Among them, the strains carrying multidrug resistance genes accounted for 89.90%. The main mechanisms of resistance to macrolides, sulfonamides and aminoglycosides in local strains were related to the resistance genes of msrA, sul1 and aac(6′)/aph(2″). SCCmec Ⅲ was the prevalent type. There were 88.37% of SCCmec Ⅲ type strains and 75% of unknown type strains carrying multiple resistance genes. Apart from that, the isolated strains of other SCCmec Ⅲ types all carried multiple resistance genes. Key words: Staphylococcus epidermidis; mecA; MSSE; MRSE; Drug-resistance gene; SCCmec

Anastomosis group and subgroup identification of Rhizoctonia solani strains deposited in NARO Genebank, Japan

The Rhizoctonia solani species complex is classified by anastomosis groups (AGs)-1–13 based on the hyphal fusion reaction. Most AGs are further divided into subgroups. However, the AG and subgroup designations of many strains of Japanese R. solani, including pathogens isolated from important crops, have not been determined. We thus identified AGs and subgroups of 45 R. solani strains (38 AG unknown and seven subgroup unknown strains), which had been isolated from 30 crops and identified by depositors in the NARO Genebank, Japan, based on the number of nuclei, culture morphology, PCR amplification with specific primers, and sequences of the rDNA-ITS region. Of the 45 strains, 37 were multinucleate, however, eight were binucleate. Of the multinucleate strains, 18 were identified as R. solani AG-4 HG-I, the rest as AG-1 IA (one strain), AG-1 IB (one strain), AG-2-1 (one strain), AG-2-2 IIIB (one strain), AG-2-2 LP (five strains), AG-2-2 hybrid subgroup between IIIB and IV (newly designated in this study; one strain), AG-3 PT (one strain), AG-4 hybrid subgroup between HG-I and HG-II (one strain), AG-4 HG-III (two strains), AG-5 (two strains), and AG-6 (three strains). Six of the eight binucleate strains were identified as binucleate Rhizoctonia AG-R (one strain) and AG-U (five strains). The rDNA-ITS sequences of the other two binucleate strains did not match any from known binucleate Rhizoctonia AGs-A–W. This study revealed new findings of Rhizoctonia diseases in Japan in 28 strains obtained from 21 crops.

The Likelihood of Misidentifying Rodent Pasteurellaceae by Using Results from a Single PCR Assay.

The precise identification of rodent Pasteurellaceae is known to be highly challenging. An unknown strain of Pasteurellaceae appeared and rapidly spread throughout our animal facilities. Standard microbiology, combined with biochemical analysis, suggested that the bacteria strain was Rodentibacter pneumotropicus or R. heylii. We submitted samples of the unknown bacteria and known isolates of R. pneumotropicus, R. heylii, and Muribacter muris, to 2 service laboratories that provide animal health monitoring. Results of microbiology tests performed by both laboratories, species-specific PCR analysis performed by one laboratory, and independent 16S rRNA gene sequencing yielded identical identification of the unknown bacteria as Pasteurellaceae (Pasteurella spp.) and not R. pneumotropicus or R. heylii. In contrast, the similarly intended PCR assay performed by the other laboratory identified the bacteria as R. heylii. Careful evaluation of all of the results led us to conclude that the correct identification of the bacteria is Pasteurellaceae. From our experience, we recommend that a combination of several methods should be used to achieve correct identification of rodent Pasteurellaceae. Specifically, we advise that all primer sets used should be disclosed when reporting PCR test results, including in health reports provided by service laboratories and animal vendors. Careful, correct, and informative health monitoring reports are most beneficial to animal researchers and caretakers who might encounter the presence and effects of rodent Pasteurellaceae.