16S-23S rRNA Intergenic Spacer Research Articles

Mesorhizobium jarvisii is a dominant and widespread species symbiotically efficient on Astragalus sinicus L. in the Southwest of China

In order to identify rhizobia of Astragalus sinicus L. and estimate their geographic distribution in the Southwest China, native rhizobia nodulating A. sinicus were isolated and their genetic diversity were studied at 13 sites cultivated in four Chinese provinces. A total of 451 rhizobial isolates were trapped with A. sinicus plants from soils and classified into 8 different genotypes defined by PCR-based restriction fragment length polymorphism (RFLP) of 16S–23S rRNA intergenic spacer (IGS). Twenty-one representative strains were further identified into three defined Mesorhizobium species by phylogenetic analyses of 16S rRNA genes and housekeeping genes (glnII and atpD). M. jarvisii was dominant accounting for 76.3% of the total isolates, 22.8% of the isolates were identified as M. huakuii and five strains belonged to M. qingshengii. All representatives were assigned to the symbiovar astragali by sharing high nodC sequence similarities of more than 99%. Furthermore, the biogeography distribution of these rhizobial genotypes and species was mainly affected by contents of available phosphorus, available potassium, total salts and pH in soils. The most remarkable point was the identification of M. jarvisii as a widespread and predominant species of A. sinicus in southwest of China. These results revealed a novel geographic pattern of rhizobia associated with A. sinicus in China.

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy)

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Genomic Insights Into the Mycobacterium kansasii Complex: An Update.

Only very recently, has it been proposed that the hitherto existing Mycobacterium kansasii subtypes (I–VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, a monophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.

Are Cowpea-Nodulating Bradyrhizobial Communities Influenced by Biochar Amendments in Soils? Genetic Diversity and Symbiotic Effectiveness Assessment of Two Agricultural Soils of Brazilian Drylands

The objective of this study was to evaluate the influence of biochar amendment on the bacterial genetic diversity and symbiosis in cowpea-nodulating rhizobial communities in two agricultural soils of Brazilian drylands. Vertisol and Ultisol surface layer soil samples were collected in the Brazilian semiarid region. The soils were mixed with biochar and cowpea seeds (BRS Marataoa) and sown in pots. Forty-five days after plant emergence, the nodule bacteria were isolated. The genetic variability of the nodule bacteria was evaluated by means of restriction fragment length polymorphism of 16S-23S rRNA intergenic spacer region (IGS-RFLP), and those that shared identical IGS-RFLP profiles were fingerprinted by BOX-PCR. By selecting of representative isolates in the genetic clusters, 27 bacteria were identified by 16S-23S IGS sequencing and symbiotically assessed. The IGS-RFLP dendrogram of the 73 isolates had 20 clusters; the multivariate correspondence analysis grouped both Vertisol treatments, and the Ultisol treatments were placed far away in the biplot. Ultisol showed higher bradyrhizobial diversity than the Vertisol. The fingerprinting indicated the presence of only three clonal colonies, showing high intraspecific diversity. The 16S-23S IGS sequences indicated the prevalence of bacteria related to Bradyrhizobium guangxiense in the Vertisol and Bradyrhizobium zhanjiangense in the Ultisol, in addition to the presence of three putative Microvirga spp. in the Ultisol. The symbiotic efficiency of 22 out of 27 bacteria was comparable with that of the inoculant strain Bradyrhizobium yuanmingense BR 3267. The genetic diversity of the cowpea-nodulating bradyrhizobia was not primarily affected by the biochar application in two agricultural soils of Brazilian drylands. Both soils harbored symbiotically efficient bacteria, without the influence of biochar amendment .

Mycoplasma nasistruthionis sp. nov. and Mycoplasma struthionis sp. nov. isolated from ostriches with respiratory disease

Twelve Mycoplasma (M.) strains isolated from the nose, the trachea, and the lung of ostriches (Struthio camelus) displaying respiratory disease were investigated. Analysis of 16S rRNA gene sequences placed five of these strains within the M. synoviae cluster, and seven strains within the M. hominis cluster of genus Mycoplasma, which was further confirmed by analyses of the 16S-23S rRNA intergenic spacer region, and partial rpoB gene and amino acid sequences. Genomic information as well as phenotypic features obtained by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry analysis and serological reactions indicated that the strains examined are representatives of two hitherto unclassified species of genus Mycoplasma, for which the names Mycoplasma nasistruthionis sp. nov., with type strain 2F1AT (= ATCC BAA-1893T = DSM 22456T), and Mycoplasma struthionis sp. nov., with type strain 237IAT (= ATCC BAA-1890T = DSM 22453T), are proposed.

Molecular Detection and Characterization of Borrelia garinii (Spirochaetales: Borreliaceae) in Ixodes nipponensis (Ixodida: Ixodidae) Parasitizing a Dog in Korea

The present study aimed to detect and characterize Borrelia spp. in ticks attached to dogs in Korea. Overall, 562 ticks (276 pools) attached to dogs were collected and tested for Borrelia infection by PCR targeting the 5S-23S rRNA intergenic spacer region (rrf-rrl). One tick larva (pool level, 0.4%; individual level, 0.2%) was confirmed by sequencing Borrelia garinii, a zoonotic pathogen. For molecular characterization, the outer surface protein A (ospA) and flagellin genes were analyzed. Phylogenetic ospA analysis distinguished B. garinii from B. bavariensis, which has been recently identified as a novel Borrelia species. On the other hand, phylogenetic analysis showed that single gene analysis involving rrf-rrl or flagellin was not sufficient to differentiate B. garinii from B. bavariensis. In addition, the B. garinii-infected tick was identified as Ixodes nipponensis by sequencing according to mitochondrial 16S rRNA and the second transcribed spacer region. To our knowledge, this is the first study to report the molecular detection of B. garinii in I. nipponensis parasitizing a dog in Korea. Continuous monitoring of tick-borne pathogens in ticks attached to animals is required to avoid disease distribution and possible transmission to humans.

Soymilk bio-enrichment by indigenously isolated riboflavin-producing strains of Lactobacillus plantarum

Lactobacilli (n = 68) isolated from human feces and fermented milk products were screened for the production of riboflavin (vitamin B2) by culturing into riboflavin assay medium (RAM). Cell-free culture supernatants from positive isolates (BBC32A, BBC32B, BBC33 and BIF43) were transferred onto RAM agar pre-seeded with Enterococcus faecalis MTCC2729 (a riboflavin auxotroph). The enhanced growth of B2-auxotroph confirmed the bioavailability of produced vitamin. Isolate BBC32B produced the highest riboflavin (319 ± 36 μg/l), followed by BBC33 (304 ± 91 μg/l), BBC32A (276 ± 8 μg/l) and BIF43 (257 ± 91 μg/l). All four isolates contained riboflavin genes ribG, ribB, ribA and ribH. Sequencing of DNA fragments amplified from the 16S–23S rRNA intergenic spacer region and areas flanking the 23S rRNA gene grouped these isolates within the species Lactobacillus plantarum. Identifications were confirmed by sequencing 1300-bp of amplified 16S rDNA fragments. Fermentation of soymilk by single cultures of L. plantarum BBC32B, BBC33 and BIF43 yielded 49.05%, 38.97% and 35.94% riboflavin enrichment respectively, which is more than 18.75% recorded in literature for Lactobacillus fermentum MTCC8711. Maximum levels of riboflavin were obtained within 12 h of fermentation in soymilk. Lactobacillus plantarum BBC32B may be used as starter culture for developing of riboflavin-enriched soymilk.

Rhizobium sophorae, Rhizobium laguerreae, and two novel Rhizobium genospecies associated with Vicia sativa L. in Northwest China

To identify the rhizobia nodulating Vicia sativa in Northwestern China and to estimate their geographic distribution. Rhizobia trapped with V. sativa plants from soils at six sites in the northwest of China were classified into genotypes by PCR-based restriction fragment length polymorphism (RFLP) of 16S–23S rRNA intergenic spacer (IGS) and 16S rRNA genes, and phylogenetic analyses of housekeeping (16S rRNA, recA, atpD) and symbiotic genes were performed for the representative strains. Soil physicochemical characteristics were recorded and canonical correlation analysis was performed to examine the correlations between soil features and distribution of rhizobial genotypes. A total of 202 rhizobial isolates were discriminated by RFLP analyses into 15 IGS types and a single 16S rRNA type, which were identified as Rhizobium by 16S rRNA gene phylogeny and as four clusters by multilocus sequence analysis (MLSA). Cluster 1 covering 86 strains and 7 IGS types prevalent in all sites was identified as Rhizobium laguerreae; cluster 2 was R. sophorae with 18 strains in 2 IGS types found in the site Q-GD. Each of cluster 3 and cluster 4 contained three IGS types representing two novel Rhizobium genospecies specific to Gansu Province and Shanxi Province, respectively. Four nodC phylogenetic clades were defined among the isolates. R. sophorae, R. laguerreae, and two novel Rhizobium genospecies with diverse symbiotic genotypes are associated with Vicia sativa L. in Northwest China. Their biogeographic patterns are mainly directed by soil pH and salinity. This is the first study on the diversity of rhizobia nodulating Vicia sativa in China.

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.

Prevalence of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in ixodid ticks in the Far East of Russia.

Borrelia burgdorferi sensu lato (s.l.) DNA was detected by PCR in Ixodes persulcatus Schulze, 1930, Haemaphysalis concinna Koch, 1844, Haemaphysalis japonica douglasi Nuttall et Warburton, 1915 and Dermacentor silvarum Olenev, 1932 ticks collected in the Amur region, the Jewish Autonomous region, the Sakhalin region and on the Khabarovsk territory. Infection rate of I. persulcatus with B. burgdorferi s.l. 10–69% exceeded the corresponding values of three other tick species in all examined regions during 1999–2014 despite different tick abundance and dominance structure. Bacterial loads estimated on the base of quantitative real time PCR varied from 102 to 109 genome-equivalents per a tick with maximal values for I. persulcatus and H. japonica. Phylogenetic analysis of 16S rRNA gene and 5S—23S rRNA intergenic spacer nucleotide sequences revealed two species: 1) Borrelia garinii of Asian type NT29 with several isolates of European type 20047; 2) Borrelia afzelii with identical sequences of the majority of studied isolates and VS461 reference strain in all regions except the Sakhalin Island where B. afzelii was not found. Borrelia miyamotoi of the relapsing fever group was detected as monoinfection or in combination with B. burgdorferi s.l. in 4.0 ± 0.9% and 4.8 ± 0.9% I. persulcatus ticks, respectively. Multiple locus sequence analysis of three fragments of 16S rRNA, glpQ and p66 genes proved that all the Far Eastern B. miyamotoi isolates belonged to the Asian type identical to FR64b strain (GenBank CP004217) from Japan. Wide distribution of Borrelia DNA in ticks, relative genetic homogeneity with similar sequences of the coding regions and the intergenic spacer of Borrelia wild isolates and temporal stability with high homology levels of the Far Eastern isolates of B. garinii, B. afzelii and B. miyamotoi with previously described spirochetes from the surrounding regions of Russia, China and Japan allowed us to suggest multiple ecological niches as the stability factor of the parasitic system.

Molecular epidemiology and genetic characterisation of carbapenem-resistant Acinetobacter baumannii isolates from Guangdong Province, South China.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a worldwide issue. This study aimed to characterise the epidemiology and genetic relationships of A. baumannii isolates in Guangdong Province, China. CRAB isolates were collected from five municipal hospitals from June-December 2017. The 16S-23S rRNA intergenic spacer region was used for confirmation of strain identity. Antimicrobial susceptibility testing and the CarbAcineto NP test were performed to analyse the resistance spectrum and carbapenemase production of the isolates. PCR-based assays were used to detect β-lactamase genes and related mobile genetic elements. Genetic diversity among the isolates was analysed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, multilocus sequence typing (MLST) and multiplex PCR. A total of 122 isolates were confirmed as A. baumannii; all were resistant to the tested antibiotics except for tigecycline and colistin. The CarbAcineto NP test showed that 93.4% of the isolates produced a carbapenemase. blaOXA-23-like and extended-spectrum β-lactamase-encoding genes were found by PCR in 94.3% and 91.8% of the isolates, respectively. Furthermore, the genetic environment of blaOXA-23-like was mainly associated with transposons Tn2008 (46.1%), Tn2006 (27.0%) and Tn2009 (20.9%). MLST identified six existing sequence types (STs) and three novel STs, of which ST195 (35.7%) and ST208 (32.1%) were the most common, belonging to clonal group 92 and European clone II. This study suggests that co-production of β-lactamases was the major resistance mechanism of CRAB isolates. Dissemination of blaOXA-23-like may be facilitated by transposable elements. ST195 and ST208 were the predominant epidemic types of A. baumannii in Guangdong Province.

Multilocus sequence analysis of Borrelia burgdorferi sensu lato isolates from Western Siberia, Russia and Northern Mongolia

Lyme borreliosis (LB) is the most frequently recorded tick-transmitted disease in Eurasia. Tomsk Province, Western Siberia in Russia and Selenge Aimag in Northern Mongolia are leading regions in the LB incidence rate in these countries. Spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex isolated from Ixodes ticks from Tomsk Province (n = 56) and Ixodes persulcatus ticks from Selenge Aimag (n = 5) were genetically characterized using Multi Locus Sequence Typing (MLST), analysis of the 5S23S rRNA intergenic spacer (IGS) amplicons, and p83/100 gene sequencing. According to MLST, B. afzelii (n = 26), B. bavariensis (n = 23), B. garinii (n = 11), and B. valaisiana (n = 1) isolates were detected in Tomsk Province, while B. afzelii and B. bavariensis isolates were identified in Selenge Aimag. Of the 32 revealed sequence types (ST), 21 STs were new and 14 of the new STs belonged to B. afzelii. Several STs of B. afzelii, B. garinii and B. valaisiana identified in this study clustered with European STs found in I. ricinus ticks. Analysis of the 5S23S IGS demonstrated that the studied Borrelia strains showed RFLP pattern characteristic for the following 5S23S IGS types: VS461 (B. afselii), NT29 (B. bavariensis), 20047 (B. bavariensis and B. garinii), VS116 (B. valaisiana), and three new groups (B. afzelii and B. bavariensis). Notably, this is the first report of Asian B. bavariensis possessing a 5S23S IGS RFLP pattern identical to 20047, and analysis of the 5S23S IGS did not provide correct determination of Borrelia species occurring in Asia. Genotyping of Borrelia strains using the clpA, pepX, and p83/100 genes demonstrated the same result as genotyping based on MLST; and further investigations are required to confirm that these three genetic loci could be used for determination of bacterial species from the B. burgdorferi s.l. complex because data based on single loci may be misleading.

First report of Coxiella burnetii and Borrelia burgdorferi sensu lato in poultry red mites, Dermanyssus gallinae (Mesostigmata, Acari), related to urban outbreaks of dermatitis in Italy.

The poultry red mite (PRM), Dermanyssus gallinae, is a nonburrowing haematophagous nest-dwelling ectoparasite of birds; occasionally it bites humans, inducing dermatitis. The possibility that this parasite may also be involved in transmission of pathogens is an additional concern. We investigated the presence of zoonotic agents in PRMs from bird nests and pets, and related them to urban outbreaks of dermatitis. A total of 98 PRMs from 12 outbreaks of PRM dermatitis that occurred in Italian cities from 2001 to 2017 were molecularly investigated for detection of Coxiella spp. (16S rRNA), Chlamydophila spp. (16S rRNA), Rickettsia spp. (17 kDa protein-encoding gene), Borrelia burgdorferi sensu lato (groEL gene) and Bartonella spp. (16S–23S rRNA intergenic spacer). Of the 12 tested mite pools, one was positive for Coxiella burnetii (100% identity) and two for B. burgdorferi sensu lato (99% with Borrelia afzelii). For the first time, the presence of B. burgdorferi sensu lato and C. burnetii is reported in PRMs from urban areas. Birds, mainly pigeons, can harbour both pathogens. Therefore, birds and their nest-dwelling PRMs may play a role in the epidemiology of these infections.

Examination of 16S-23S rRNA intergenic spacer region (ISR) heterogeneity in a population of clinical Streptococcus pneumoniae- a new laboratory epidemiological genotyping tool to aid outbreak analysis

Streptococcus pneumoniae (pneumococci) are the most frequent cause of bacterial pneumonia of people of all ages, as well as an important cause of meningitis and otitis media. Although enormous effo...

Vicia faba L. in the Bejaia region of Algeria is nodulated by Rhizobium leguminosarum sv. viciae, Rhizobium laguerreae and two new genospecies

Fifty-eight rhizobial strains were isolated from root nodules of Vicia faba cv. Equina and Vicia faba cv. Minor by the host-trapping method in soils collected from eleven sites in Bejaia, Eastern Algeria. Eleven genotypic groups were distinguished based on the combined PCR/RFLP of 16S rRNA, 16S–23S rRNA intergenic spacer and symbiotic (nodC and nodD-F) genes and further confirmed by multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and the nodulation genes nodC and nodD. Of the 11 genotypes, 5 were dominant and 2 were the most represented. Most of the strains shared high nodD gene sequence similarity with Rhizobium leguminosarum sv. viciae; their nodC sequences were similar to both Rhizobium leguminosarum and Rhizobium laguerreae. Sequence analyses of the 16S–23S rRNA intergenic spacer showed that all the new strains were phylogenetically related to those described from Vicia sativa and V. faba in several African, European, American and Asian countries, with which they form a group related to Rhizobium leguminosarum. Phylogenetic analysis based on MLSA of 16S rRNA, recA, atpD and rpoB genes allowed the affiliations of strain AM11R to Rhizobium leguminosarum sv. viciae and of strains EB1 and ES8 to Rhizobium laguerreae. In addition, two separate clades with <97% similarity may represent two novel genospecies within the genus Rhizobium.

Diversity and prevalence of Bartonella species in small mammals from Slovakia, Central Europe.

Wild-living rodents are important hosts for zoonotic pathogens. Bartonella infections are widespread in rodents; however, in Slovakia, knowledge on the prevalence of these bacteria in small mammals is limited. We investigated the prevalence and diversity of Bartonella species in the spleens of 640 rodents of six species (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus, and Micromys minutus) and in the European mole (Talpa europaea) from three different habitat types in south-western and central Slovakia. Overall, the prevalence of Bartonella spp. in rodents was 64.8%; a rate of 73.8% was found in natural habitat (deciduous forest), 56.0% in suburban forest park and 64.9% in rural habitat. Bartonella spp. were detected in 63.0% of A. flavicollis, 69% of My. glareolus and 61.1% of M. arvalis and in T. europaea. However, Bartonella were not found in the other examined rodents. Molecular analyses of the 16S-23S rRNA intergenic spacer region revealed the presence of four different Bartonella spp. clusters. We identified B. taylorii, B. rochalimae, B. elizabethae, B. grahamii and Bartonella sp. wbs11 in A. flavicollis and My. glareolus. Bartonella genotypes ascribed to B. taylorii and B. rochalimae were found in M. arvalis. B. taylorii was identified in T. europaea. Questing Ixodes ricinus ticks that were collected at the study sites were not infected with Bartonella. This study improves our understanding of the ecoepidemiology of Bartonella spp. in Europe and underlines the necessity for further research on Bartonella-host-vector associations and their consequences on animal and human health in Slovakia.

Mycoplasma tullyi sp. nov., isolated from penguins of the genus Spheniscus.

A mycoplasma isolated from the liver of a dead Humboldt penguin (Spheniscus humboldti) and designated strain 56A97T, was investigated to determine its taxonomic status. Complete 16S rRNA gene sequence analysis indicated that the organism was most closely related to Mycoplasma gallisepticum and Mycoplasma imitans(99.7 and 99.9 % similarity, respectively). The average DNA-DNA hybridization values between strain 56A97T and M. gallisepticum and M. imitans were 39.5 and 30 %, respectively and the Genome to Genome Distance Calculator gave results of 29.10 and 23.50 %, respectively. The 16S-23S rRNA intergenic spacer was 72-73 % similar to M. gallisepticum strains and 52.2 % to M. imitans. A partial sequence of rpoB was 91.1-92 % similar to M. gallisepticum strains and 84.7 % to M. imitans. Colonies possessed a typical fried-egg appearance and electron micrographs revealed the lack of a cell wall and a nearly spherical morphology, with an electron-dense tip-like structure on some flask-shaped cells. The isolate required sterol for growth, fermented glucose, adsorbed and haemolysed erythrocytes, but did not hydrolyse arginine or urea. The strain was compared serologically against 110 previously described Mycoplasma reference strains, showing that, except for M. gallisepticum, strain 56A97T is not related to any of the previously described species, although weak cross-reactions were evident. Genomic information, serological reactions and phenotypic properties demonstrate that this organism represents a novel species of the genus Mycoplasma, for which the name Mycoplasma tullyi sp. nov. is proposed; the type strain is 56A97T (ATCC BAA-1432T, DSM 21909T, NCTC 11747T).

Impact of Transgenic Brassica napus Harboring the Antifungal Synthetic Chitinase (NiC) Gene on Rhizosphere Microbial Diversity and Enzyme Activities.

Transgenic Brassica napus harboring the synthetic chitinase (NiC) gene exhibits broad-spectrum antifungal resistance. As the rhizosphere microorganisms play an important role in element cycling and nutrient transformation, therefore, biosafety assessment of NiC containing transgenic plants on soil ecosystem is a regulatory requirement. The current study is designed to evaluate the impact of NiC gene on the rhizosphere enzyme activities and microbial community structure. The transgenic lines with the synthetic chitinase gene (NiC) showed resistance to Alternaria brassicicola, a common disease causing fungal pathogen. The rhizosphere enzyme analysis showed no significant difference in the activities of fivesoil enzymes: alkalyine phosphomonoestarase, arylsulphatase, β-glucosidase, urease and sucrase between the transgenic and non-transgenic lines of B. napus varieties, Durr-e-NIFA (DN) and Abasyne-95 (AB-95). However, varietal differences were observed based on the analysis of molecular variance. Some individual enzymes were significantly different in the transgenic lines from those of non-transgenic but the results were not reproducible in the second trail and thus were considered as environmental effect. Genotypic diversity of soil microbes through 16S–23S rRNA intergenic spacer region amplification was conducted to evaluate the potential impact of the transgene. No significant diversity (4% for bacteria and 12% for fungal) between soil microbes of NiC B. napus and the non-transgenic lines was found. However, significant varietal differences were observed between DN and AB-95 with 79% for bacterial and 54% for fungal diversity. We conclude that the NiC B. napus lines may not affect the microbial enzyme activities and community structure of the rhizosphere soil. Varietal differences might be responsible for minor changes in the tested parameters.

Trueperella pyogenes isolated from a brain abscess of an adult roebuck (Capreolus capreolus).

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S-23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.

Involvement of multiple Chlamydia suis genotypes in porcine conjunctivitis.

Chlamydia suis has been detected in numerous disease conditions of pigs, particularly in eye infections. This study examined recurring conjunctivitis cases in five commercial pig farms in Japan. 40.5% of the cases were identified as Chlamydia positive using impression cytology of ocular smears and a genus-specific direct fluorescent antibody. C.suis was detected in 59.5% of the samples using PCR tests targeting 16S-23S rRNA intergenic spacer region (ISR) and ompA gene. Genetic analysis of PCR amplicons revealed nine sequence variants of 16S-23S rRNA ISR and 20 sequence variants within ompA gene. Among C.suis-positive conjunctivitis cases, 36.4% showed concurrent infection with 2-4 varied ompA sequence types and 9.1% showed multiple 16S-23S rRNA ISR sequence types of C.suis. Multiple genotypes were found circulating in four of five farms. All 20 detected strains and 25 previously reported C.suis strains were grouped into four clusters. Japanese C.suis strains were closely related to American and European strains indicating wide distribution of these genetically variant strains. This study is the first to show multiple and genetically diverse C.suis strain associations in pig conjunctivitis.