Mycobacterium species identification--a new approach via dnaJ gene sequencing

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.     

Prevalence of Cryptosporidium genotypes in pre and post-weaned pigs in Australia

A total of 289 pig faecal samples were collected from pre-weaned and post-weaned piglets and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia and screened at the 18S rRNA locus for the presence of Cryptosporidium. An overall prevalence of 22.1% (64/289) was identified. Cryptosporidium was more prevalent in post-weaned animals (p < 0.05); 32.7% (51/156) versus 10.6% (13/123) for pre-weaned animals. Sequence analysis identified Cryptosporidium suis in all pre-weaned isolates genotyped (7/13). In post-weaned pigs that were genotyped (n = 38), the non-zoonotic Cryptosporidium species, pig genotype II was identified in 32 samples and C. suis in 6 samples. The zoonotic species Cryptosporidium parvum was not detected, suggesting that domestic pigs do not pose a significant public health risk. Pig genotype II was significantly (p < 0.05) associated with ‘normal’ stools, indicating an asymptomatic nature in the porcine host.     

First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central USA

Nosema ceranae is an emerging microsporidian parasite of European honey bees, Apis mellifera, but its distribution is not well known. Six Nosema-positive samples (determined from light microscopy of spores) of adult worker bees from Canada (two each from Nova Scotia, New Brunswick, and Prince Edward Island) and two from USA (Minnesota) were tested to determine Nosema species using previously-developed PCR primers of the 16S rRNA gene. We detected for the first time N. ceranae in Canada and central USA. One haplotype of N. ceranae was identified; its virulence may differ from that of other haplotypes.     

A combined cultivation and cultivation-independent approach shows high bacterial diversity in water-miscible metalworking fluids

Ten metalworking fluids (MWF) and seven water preparation basis samples (WPB) were taken from five industrial plants in Germany. Total cells (TCC) and colony forming units (CFU) were counted, strains were isolated and their 16S rRNA gene was sequenced. Additionally, DNA was extracted directly from the samples, and clone libraries of 16S rRNA genes were built and gene sequenced. TCC ranged from 7.6×10(4) TCC/mL MWF to 1.6×10(8) TCC/mL MWF, and from 4.6×10(2) TCC/mL WPB to 7.8×10(7) TCC/mL WPB. The CFU showed similar but often lower results. A total of 70 isolates and 732 clones were 16S rRNA gene sequenced and all isolates, as well as 183 of the nearly full length 16S rRNA of these clones, were gene sequenced. A total of 98 different genera were detected in all 17 samples. The number of genera within each sample varied highly, with 1-22 genera per sample. The dominant genera in MWF were Leucobacter, Desemzia, Sphingomonas and Wautersiella. From these, only Sphingomonas was detected in WPB as well. This study showed that MWF can harbour a high bacterial diversity, which differs significantly from the bacterial flora of the corresponding WPB.     

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.     

First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens)

Faecal samples taken from eight underweight, approximately 5-week-old broiler chickens in a poultry abattoir were investigated for microsporidial infections by light microscopy, electron microscopy, and PCR. In two of six chickens, which were suspected of being infected with microsporidia by light microscopy, Enterocytozoon bieneusi (genotype 'J') was detected by PCR and DNA sequencing, and in one of the two PCR-positive samples by extensive electron microscopy. This is the first time that E. bieneusi has been detected in chickens, i.e. in a non-mammalian species.     

Molecular identification and prevalence of Isospora sp. in pigs in Western Australia using a PCR-RFLP assay

Little is known about the prevalence of Isospora in domestic pigs in Western Australia. A total of 289 pig faecal samples were collected from pre- and post-weaned pigs and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia. Faecal samples were screened using a PCR-RFLP assay based on the ITS-1 rDNA locus. An overall prevalence of 10.4% (30/289) was identified. Isospora was detected in 16.3% (20/123) of pre-weaned animals and 6.4% (10/156) of post-weaned animals. PCR-RFLP analysis confirmed the presence of Isospora suis in 86.7% of the positive Isospora isolates. Isospora was significantly associated with diarrhea and the findings of this study suggest that management factors such as cleaning practices, flooring types and stocking densities need to be investigated in the porcine host to find new and improved measures for control.     

Neoscardovia arbecensis gen. nov., sp. nov., isolated from porcine slurries

Three Gram-positive, anaerobic, pleomorphic strains (PG10(T), PG18 and PG22), were selected among five strains isolated from pig slurries while searching for host specific bifidobacteria to track the source of fecal pollution in water. Analysis of the 16S rRNA gene sequence showed a maximum identity of 94% to various species of the family Bifidobacteriaceae. However, phylogenetic analyses of 16S rRNA and HSP60 gene sequences revealed a closer relationship of these strains to members of the recently described Aeriscardovia, Parascardovia and Scardovia genera, than to other Bifidobacterium species. The names Neoscardovia gen. nov. and Neoscardovia arbecensis sp. nov. are proposed for a new genus and for the first species belonging to this genus, respectively, and for which PG10(T) (CECT 8111(T), DSM 25737(T)) was designated as the type strain. This new species should be placed in the Bifidobacteriaceae family within the class Actinobacteria, with Aeriscardovia aeriphila being the closest relative. The prevailing cellular fatty acids were C(16:0) and C(18:1)ω9c, and the major polar lipids consisted of a variety of glycolipids, diphosphatidyl glycerol, two unidentified phospholipids, and phosphatidyl glycerol. The peptidoglycan structure was A1γmeso-Dpm-direct. The GenBank accession numbers for the 16S rRNA gene and HSP60 gene sequences of strains PG10(T), PG18 and PG22 are JF519691, JF519693, JQ767128 and JQ767130, JQ767131, JQ767133, respectively.     

Detection of Thelohania solenopsae (Microsporidia: Thelohaniidae) in Solenopsis invicta (Hymenoptera: Formicidae) by multiplex PCR

Oligonucleotide primer pairs were designed to unique areas of the small subunit (16S) rRNA gene of Thelohania solenopsae and a region of the Gp-9 gene of Solenopsis invicta. Multiplex PCR resulted in sensitive and specific detection of T. solenopsae infection of S. invicta. The T. solenopsae-specific primer pair only amplified DNA from T. solenopsae and T. solenopsae-infected S. invicta. This primer pair did not produce any amplification products from DNA preparations from uninfected S. invicta, seven additional species of microsporidia (including Vairimorpha invictae), or Mattesia spp. The Gp-9-specific primers recognized and amplified DNA from Solenopsis xyloni, Solenopsis richteri, Solenopsis geminata, the invicta/richteri hybrid, and monogyne and polygyne S. invicta, but not from T. solenopsae, and, as such, served as a positive control verifying successful DNA preparation. Multiplex PCR detected T. solenopsae in worker fire ants infected with as few as 5000 spores. Furthermore, multiplex PCR detected T. solenopsae in all developmental stages of S. invicta. However, detection could be made more sensitive by using only the T. solenopsae-specific primer pair; ants infected with as few as 10 spores were able to be discerned. Multiplex PCR detection of T. solenopsae offers the advantages of a positive control, a single PCR amplification, detection of all developmental stages, and increased sensitivity and specificity compared with microscopy.     

Hymenobacter perfusus sp. nov., Hymenobacter flocculans sp. nov. and Hymenobacter metalli sp. nov. three new species isolated from an uranium mine waste water treatment system

Three red-pink pigmented strains, designated A1-12(T), A2-50A(T) and A2-91(T), were recovered from two different sites in a uranium mine. For all strains, the optimum growth temperature was 25°C, the optimum pH was 6.0-6.5 and the DNA G+C contents were between 60 and 63.4 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the fatty acid profiles contained iso- and anteiso-branched C15 fatty acids, summed feature 3 (16:1 ω6c and/or ω7c and/or 15:0 iso 2-OH), summed feature 4 (17:1 anteiso B and/or iso I) and the unsaturated fatty acid 16:1 ω5c as the major components. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented three distinct branches within the family Flexibacteraceae most closely related to the members of the genus Hymenobacter. Strain A1-12(T) formed a distinct phylogenetic line along with H. rigui KCTC 12533(T) and they shared approximately 98.9% 16S rRNA gene sequence similarity. However, these two strains shared only 14.7% pairwise similarity in their genomic DNA. Strains A2-50A(T) and A2-91(T) formed two distinct lineages, related to the species H. soli KCTC 12607(T), sharing about 95.5% 16S rRNA gene sequence similarity between themselves, and 88.3 and 92.0% with other members of the genus Hymenobacter. Based on the phylogenetic analysis and physiological and biochemical characteristics, these isolates were considered to represent three novel species for which we propose the names Hymenobacter perfusus for strain A1-12(T) (=CIP 110166=LMG 26000), Hymenobacter flocculans for strain A2-50A(T) (=CIP 110139=LMG 25699) and Hymenobacter metalli for strain A2-91(T) (=CIP 110140=LMG 25700).     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different legume species

The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.     

Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine (Tenerife, Canary Islands) are two symbiovars of the same species, Mesorhizobium tamadayense sp. nov

Barranco de Tamadaya is a deep ravine located in southern Tenerife, which is included within a protected area where several endemic plants grow. Among them, two legumes are catalogued as critically endangered, Anagyris latifolia and Lotus berthelotii. Rhizobial strains isolated from their root nodules grown in soil samples from this ravine harboured symbiotic genes belonging to two distant symbiovars, but they shared identical 16S rRNA gene sequences (rrs). The phylogeny based on the rrs sequences placed these isolates in a separate subbranch that did not include any of the currently recognised Mesorhizobium species, but the resolution of the ribosomal tree did not permit further taxonomic conclusions. Nevertheless, multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, recA, glnII and dnaK) and the rrs gene generated a highly supported Bayesian phylogeny, identifying these isolates as a new Mesorhizobium lineage. DNA-DNA hybridisation homology percentages were lower than 30% compared to type strains of the closest related species, and supported the phylogenetic data. Phenotypic characterisation also distinguished this lineage from the other closest Mesorhizobium species. The polyphasic approach thus confirmed that the isolates represented a novel species for which we propose the name Mesorhizobium tamadayense sp. nov. The type strain is Ala-3(T) (CECT 8040(T), LMG 26736(T)).     

Distribution and diversity of members of the bacterial phylum Fibrobacteres in environments where cellulose degradation occurs

The Fibrobacteres phylum contains two described species, Fibrobacter succinogenes and Fibrobacter intestinalis, both of which are prolific degraders of cellulosic plant biomass in the herbivore gut. However, recent 16S rRNA gene sequencing studies have identified novel Fibrobacteres in landfill sites, freshwater lakes and the termite hindgut, suggesting that members of the Fibrobacteres occupy a broader ecological range than previously appreciated. In this study, the ecology and diversity of Fibrobacteres was evaluated in 64 samples from contrasting environments where cellulose degradation occurred. Fibrobacters were detected in 23 of the 64 samples using Fibrobacter genus-specific 16S rRNA gene PCR, which provided their first targeted detection in marine and estuarine sediments, cryoconite from Arctic glaciers, as well as a broader range of environmental samples. To determine the phylogenetic diversity of the Fibrobacteres phylum, Fibrobacter-specific 16S rRNA gene clone libraries derived from 17 samples were sequenced (384 clones) and compared with all available Fibrobacteres sequences in the Ribosomal Database Project repository. Phylogenetic analysis revealed 63 lineages of Fibrobacteres (95% OTUs), with many representing as yet unclassified species. Of these, 24 OTUs were exclusively comprised of fibrobacters derived from environmental (non-gut) samples, 17 were exclusive to the mammalian gut, 15 to the termite hindgut, and 7 comprised both environmental and mammalian strains, thus establishing Fibrobacter spp. as indigenous members of microbial communities beyond the gut ecosystem. The data highlighted significant taxonomic and ecological diversity within the Fibrobacteres, a phylum circumscribed by potent cellulolytic activity, suggesting considerable functional importance in the conversion of lignocellulosic biomass in the biosphere.     

Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain

The genetic diversity and prevalence of virtually all Theileria and Babesia species in a sheep population were studied using a specifically designed reverse line blot macroarray. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against generic and species-specific probes. In a first screening (Study I), 320 apparently healthy animals corresponding to 32 flocks located in the Basque Country (Northern Spain) were analysed. The survey demonstrated a high prevalence of subclinical infections (64.7%). Three Theileria genotypes were identified, sharing 96.7-97.0% similarity between their 18S rRNA gene sequences: Theileria ovis, Theileria sp. OT1 (99.6% similarity with the recently described pathogenic piroplasm Theileria sp. China 1), and Theileria sp. OT3. Two Babesia species sharing 91.5% similarity were also detected: Babesia ovis and Babesia motasi. The complete 18S rRNA gene sequences of these and other piroplasm species were phylogenetically analysed. Prevalence of piroplasms was also investigated in a second group of 80 sheep from 16 flocks reared in mountain areas that had been heavily exposed to ticks and had suffered a recent abortion episode (Study II). The screening revealed a significantly higher (P < 0.05) prevalence (78.7%) of piroplasm infections compared to Study I. Although the prevalence rates for some piroplasm species were significantly related to abortion (e.g. Theileria sp. OT3), decreases in the red cell parameters were not significant. The widespread distribution of Theileria spp. in the studied sheep population suggests that the parasites involved are of relatively low pathogenicity, in contrast to what has been reported for Theileria sp. China 1 in other countries.     

Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated from contaminated buildings were screened for MVOC production on malt extract agar by means of headspace solid-phase microextraction followed by gas chromatography-mass spectrometry (GC-MS) analysis. Some sesquiterpenes, specifically derived from fungal growth, were detected in the sampled environments and the corresponding fungal producers were identified. Statistical analysis of the detected MVOC profiles allowed the identification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum spp. were clearly differentiated by their volatile production from a group of 76 fungal strains belonging to different genera. These results are useful in the chemotaxonomic discrimination of fungal species, in aid to the classical morphological and molecular identification techniques.     

Identification of zoonotic Cryptosporidium and Giardia genotypes infecting animals in Sydney's water catchments

To identify the animal sources for Cryptosporidium and Giardia contamination, we genotyped Cryptosporidium and Giardia spp. in wildlife from Sydney’s water catchments using sequence analysis at the 18S rRNA locus for Cryptosporidium and 18S rRNA and glutamate dehydrogenase (gdh) for Giardia. A total of 564 faecal samples from 16 different host species were analysed. Cryptosporidium was identified in 8.5% (48/564) samples from eight host species and Giardia was identified in 13.8% (78/564) from seven host species. Eight species/genotypes of Cryptosporidium were identified. Five G. duodenalis assemblages were detected including the zoonotic assemblages A and B.     

Polyphasic taxonomic characterization of Lactobacillus rossiae isolates from Belgian and Italian sourdoughs reveals intraspecific heterogeneity

(GTG)₅-PCR fingerprinting and pheS sequence analysis of 18 Lactobacillus rossiae isolates, mainly originating from Belgian and Italian artisan sourdoughs, revealed intraspecies grouping as evidenced by the delineation of three and two subgroups, respectively. On the other hand, 16S rRNA and rpoA gene sequence analysis and DNA–DNA hybridizations supported the accommodation of all isolates in a single species. No correlation between genetic and phenotypic heterogeneity was observed. Collectively, these data do not warrant taxonomic division of L. rossiae. On the other hand, the considerable differences in intraspecies sequence variation of L. rossiae isolates displayed by the pheS (9.8%) and rpoA (1.1%) genes highlight that the discriminatory power of housekeeping genes as alternative genomic markers for the 16S rRNA gene in the identification of Lactobacillus species may significantly differ from gene to gene. In conclusion, this study has demonstrated that a polyphasic approach remains highly useful for identification of isolates belonging to genotypically heterogeneous species such as L. rossiae.     

Cultivation-dependent characterization of bacterial diversity from British Columbia forest soils subjected to disturbance

Bacteria from forest surface organic matter and mineral soil horizons were cultivated using four methods and characterized by fatty acid methyl ester (FAME) analysis. Soil samples from a British Columbia Ministry of Forests Long-Term Soil Productivity (LTSP) installation were collected during winter and summer from two disturbance treatments (whole-tree harvesting with no soil compaction (plot N) and whole-tree harvesting plus complete surface organic matter removal with heavy soil compaction (plot S)) and from an unlogged reference plot (REF). Seventy-five percent of 1795 bacterial isolates were affiliated with 42 genera representing beta- and gamma-Proteobacteria, Actinobacteria, the Bacillus/Clostridium group, and the Cytophaga-Flexibacter-Bacteroides group. Approximately half of the culture collection represented genetic diversity confined to four bacterial genera: Pseudomonas, Bacillus, Paenibacillus, and Arthrobacter. A significantly higher proportion of bacterial isolates belonging to Actinobacteria, and the member genus Arthrobacter, were isolated from plot S soil samples compared with soil samples from plots N and REF. Twenty-five percent of bacterial isolates were not conclusively identified to genus with FAME analysis. Sherlock Tracker cluster analysis and partial 16S rRNA gene sequence analysis enabled classification of a subset of these isolates.