16S-23S rDNA Intergenic Spacer Region Research Articles

Plant Growth-Promoting (PGP) Traits of Endophytic Bacteria from In Vitro Cultivated Tectona grandis L.f

Tectona grandis L.f. (teak) is a tropical tree cultivated mainly due to its resistance, valuable wood and tolerance to biotic and abiotic factors. An abundant bacteria community exists in teak tissues and knowledge of the functional roles of teak endophytic bacteria, from in vitro tissue culture, is essential for improving micropropagation techniques. In this study, we isolated endophytic bacteria with plant growth-promoting (PGP) traits from two teak clones (Proteca® A3 and E4) in calli, leaves, and stems segments in a culture medium. We analyzed colony pigmentation, gram reaction, and evaluated PGP traits (phosphorous solubilization, nitrogen fixation and indol-acetic acid production) of 54 colonies from clone A3 and 50 colonies from clone E4. A total of 35 colonies of clone A3 and 42 colonies of clone E4 were capable of fixing nitrogen. Four isolated bacteria from clone A3 were capable of solubilizing phosphorous (P-Ca3(PO4)2), while no strain of E4 clone showed that capacity. Furthermore, 49 endophytic bacteria from clone A3 showed capacity to synthesize indol-acetic acid, while only 4 bacteria from the E4 clone presented that characteristic. We also identified six teak endophytic bacteria, by analysis of the 16S-23S rDNA intergenic spacer region, showing high identity with the genera Curtobacterium sp., Ochrobactrum sp., and Bacillus spp. Therefore, we demonstrate here that the abundant bacterial community existing in teak tissues, including those of in vitro cultivated plants, have PGP traits that can be further harnessed for preparation of bioformulations, for example. Our findings open the possibility for studying these isolated endophytic bacteria more closely in order to understand their association with teak growth.

Phenotypic and genotypic approach to characterize a Trueperella pecoris strain isolated from necrotic vestibulitis of a camel (Camelus dromedarius).

The present study was designed to characterize phenotypically and genotypically a Trueperella (T.) pecoris strain isolated from necrotic vestibulitis of a 10-year-old camel (Camelus dromedarius). The species identity of T. pecoris 203/7 investigated in the present study could be confirmed by phenotypic properties and by phylogenetic analyses based on partial sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, elongation factor Tu encoding gene tuf and the target gene rpoB encoding the β-subunit of bacterial RNA polymerase. T. pecoris strain 203/7 was grouped within the genus Trueperella in the family Arcanobacteriaceae. The 16S rRNA gene analysis showed a sequence identity of 99·9% to reference strain T. pecoris DSM 111392T . The present isolate was clearly identified as T. pecoris, the most recently described species of the genus Trueperella. Strain T. pecoris 203/7 was isolated in moderate numbers from necrotic vestibulitis of the camel and could be of some importance for the infectious process. However, the investigated strain represents the first isolation of T. pecoris from a camel.

Prevalence, Enterotoxigenic Potential and Antimicrobial Resistance of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Algerian Ready to Eat Foods.

Staphylococcus aureus causes a foodborne intoxication due to the production of enterotoxins and shows antimicrobial resistance, as in the case of methicillin-resistant strains (MRSA). Herein, we analyzed 207 ready-to-eat foods collected in Algeria, reporting a S. aureus prevalence of 23.2% (48/207) and respective loads of coagulase positive staphylococci (CPS) ranging from 1.00 ± 0.5 to 5.11 ± 0.24 Log CFU/g. The 48 S. aureus isolates were widely characterized by staphylococcal enterotoxin gene (SEg)-typing and 16S-23S rDNA intergenic spacer region (ISR)-PCR, as well as by detecting tst and mecA genes, genetic determinants of toxic shock syndrome toxin-1 and methicillin resistance, respectively. We found that the S. aureus isolates belonged to seven different SEg-types harboring the following combinations of genes: (1) selW, selX; (2) egc (seG, seI, seM, seN, seO), selW, selX; (3) seA, seH, seK, seQ, selW, selX; (4) seB, selW, selX; (5) seD, selJ, seR, selW, selX; (6) seH, selW, selX, selY; and (7) seA, egc, selW, selX, while among these, 2.1% and 4.2% were tst- and mecA- (staphylococcal chromosomal cassette mec-type IV) positive, respectively. Selected strains belonging to the 12 detected ISR-types were resistant towards antimicrobials including benzylpenicillin, ofloxacin, erythromycin, lincomycin, tetracyclin, kanamycin, oxacillin, and cefoxitin; 8.3% (1/12) were confirmed as MRSA and 16.7% (2/12) were multidrug resistant. The present study shows the heterogeneity of the S. aureus population in Algerian ready-to-eat foods as for their toxigenic potential and antimicrobial resistance, shedding the light on the quality and safety related to the consume of ready-to-eat foods in Algeria.

A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee

An isolated bacterium TBE-8, was identified as Leuconostoc mesenteroides according to the sequences of 16S rDNA and the 16S–23S rDNA intergenic spacer region. The probiotic properties of the L. mesenteroides TBE-8 strain were characterized and revealed that TBE-8 could utilize various carbohydrates, exhibited high tolerance to sucrose’s osmotic pressure and acidic conditions, and could mitigate the impact of the bee pathogen Paenibacillus larvae. In addition, we found that the TBE-8 broth increased the expression of the nutrition-related genes major royal jelly protein 1 and vitellogenin in bees by approximately 1400- and 20-fold, respectively. The expression of genes encoding two antibacterial peptides, hymenoptaecin and apidaecin, in the bee abdomen was significantly increased by 17- and 7-fold in bees fed with the TBE-8 fermented broth. Furthermore, we fed four-frame bee colonies with 50% sucrose syrup containing TBE-8 and can detect the presence of approximately 2 × 106 16S rDNA copies of TBE-8 in the guts of all bees in 24 h, and the retention of TBE-8 in the bee gut for at least 5 days. These findings indicate that the L. mesenteroides TBE-8 has high potential as a bee probiotic and could enhance the health of bee colonies.

Studies on Trueperella pyogenes isolated from an okapi (Okapia johnstoni) and a royal python (Python regius)

BackgroundThe present study was designed to characterize phenotypically and genotypically two Trueperella pyogenes strains isolated from an okapi (Okapia johnstoni) and a royal python (Python regius).Case presentationThe species identity could be confirmed by phenotypic properties, by MALDI-TOF MS analysis and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. Furthermore, sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region (ISR), the target genes rpoB encoding the β-subunit of bacterial RNA polymerase, tuf encoding elongation factor tu and plo encoding the putative virulence factor pyolysin allowed the identification of both T. pyogenes isolates at species level.ConclusionsBoth strains could be clearly identified as T. pyogenes. The T. pyogenes strain isolated in high number from the vaginal discharge of an okapi seems to be of importance for the infectious process; the T. pyogenes strain from the royal python could be isolated from an apparently non-infectious process. However, both strains represent the first isolation of T. pyogenes from these animal species.

Potential probiotic of Lactobacillus strains isolated from the intestinal tracts of pigs and feces of dogs with antibacterial activity against multidrug-resistant pathogenic bacteria.

The occurrence of multidrug-resistant pathogenic bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MDRAB), extended-spectrum β-lactamase (ESBL) Escherichia coli, and Pseudomonas aeruginosa, has become a serious problem in animals and public. The objective of this study was to identify and isolate lactic acid bacterial (LAB) strains from the intestinal tracts of pigs and feces of dogs and then characterize them as potential probiotics with antimicrobial activity against multidrug-resistant pathogenic bacteria. In a preliminary isolation screening, 45 of 1167 isolated LAB strains were found to have anti-S. aureus ATCC 27,735 activity. Using 16S rDNA and 16S-23S rDNA intergenic spacer region (ISR) sequences, five of these isolates were further identified as Lactobacillus animalis 30a-2, Lactobacillus reuteri 4-12E, Weissella cibaria C34, Lactococcus lactis 5-12H, and Lactococcus lactis 6-3H. Antimicrobial substance assays suggest that the L. lactis 5-12H, L. lactis 6-3H, L. animalis 30a-2, L. reuteri 4-12E, and W. cibaria C34 strains might produce bacteriocins and hydrogen peroxide (H2O2) as antimicrobial substances. The L. animalis 30a-2 and W. cibaria C34 strains were further characterized for probiotic properties and shown to have high acid and bile salt tolerance. Additionally, they have broad antimicrobial spectra, and can significantly repress the growth of all of the tested strains of MRSA isolates, some MDRAB, ESBL E. coli, and P. aeruginosa isolates, along with food-borne pathogenic bacteria such as Bacillus cereus ATCC 11778, Listeria monocytogens ATCC 19111, Salmonella spp., Shigella spp., and Yersinia enterocolitica BCRC 12986. This is the first report of H2O2-producing L. animalis 30a-2 and W. cibaria C34 isolated from the intestinal tracts of pigs and feces of dogs that have good antimicrobial activity against multidrug-resistant and food-borne pathogenic bacteria and have excellent probiotic properties.

Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk

In this study, 53 Staphylococcus (S.) aureus strains were typed by 16S–23S rDNA intergenic spacer region (ISR) typing and staphylococcal enterotoxin gene (SEg) typing for all the staphylococcal enterotoxin (se) and staphylococcal enterotoxin-like toxin (sel) genes known to date, revealing a higher discriminatory power than that of multi locus sequence typing. Six strains, one of each ISR- and SEg-type, were genome sequenced and the ability to produce some classical and new SEs when growing in milk was investigated.The manual analysis of the six genomes allowed us to confirm, correct and expand the results of common available genomic data pipelines such as VirulenceFinder. Moreover, it enabled us to (i) investigate the actual location of se and sel genes, even for genes such as selY, whose location (in the core genome) was so far unknown, (ii) find novel allelic variants of se and sel genes and pseudogenes, (iii) correctly annotate se and sel genes and pseudogenes, and (iv) discover a novel type of enterotoxin gene cluster (egc), i.e. the egc type 5 in strains 356P and 364P, while S. argenteus MSHR1132 harbored the egc type 6.Four of the six S. aureus strains produced sufficient amounts of SEA, SEC, SED and SEH in milk to cause staphylococcal food poisoning (SFP), with S. aureus 372 P being the highest producer of SED in milk found to date, producing as much as ca. 47,300 ng/mL and 49,200 ng/mL of SED, after 24 and 48 h of incubation in milk at 37 °C, respectively. S. aureus 372 P released a low amount of SER in milk, most likely because the seR gene was present as a pseudogene, putatively encoding only 51 amino acids. These findings confirm that not only the classical SEs, but also the new ones can represent a potential hazard for the consumers’ health if produced in foods in sufficient amounts. Therefore, the detection of SEs in foods, especially if involved in SFP cases, should focus not only on classical, but also on all the new SEs and SEls known to date. Where reference methods are unavailable, the presence of the relevant genes, by using the conventional and real time PCR protocols we exhaustively provided herein, and their nucleotide sequences, should be investigated.

Characterization and Differentiation of Chicken Mycoplasma Isolates Using 16S-23S Intergenic Spacer Region Sequencing

ABSTRACT The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: Sao Paulo, Minas Gerais, Rio de Janeiro and Espirito Santo. The isolates were recovered from the Sao Paulo, Rio de Janeiro and Espirito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

Further characteristics of Arcanobacterium pinnipediorum DSM 28752T and Arcanobacterium wilhelmae DSM 102162T, two novel species of genus Arcanobacterium.

The newly described type strains Arcanobacterium pinnipediorum DSM 28752T and Arcanobacterium wilhelmae DSM 102162T, initially isolated from an anal swab of a harbor seal (Sammra et al. Int J Syst Evol Microbiol 65:4539-4543, 2015) and the genital tract of a rhinoceros (Sammra et al. Int J Syst Evol Microbiol 67:2093-2097, 2017), could be further characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Fourier transform infrared (FT-IR) spectroscopy and by sequencing the genomic targets 16S-23S rDNA intergenic spacer region (ISR) and the genes rpoB, gap, and tuf. The two strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of both species remained unclear. However, the detection of specific spectra by MALDI-TOF MS and by FT-IR spectroscopy and the presented genotypic approaches might help to identify A. pinnipediorum and A. wilhelmae in the future and might elucidate the role these two species play in infections of animals.

Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs.

Aim:This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog’s clinical samples.Materials and Methods:A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711.Results:The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89).Conclusion:We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples.

VARIASI GENETIK Lactobacillus fermentum Beijerink ASAL SAYUR ASIN BERDASARKAN ANALISIS RFLP 16S-23S rDNA ISR, RAPD-PCR DAN ERIC-PCR

Molecular analysis of Lactobacillus fermentum isolates is essential to understand their genetic variation in relations to their roles in sayur asin fermentation process. Combination of three molecular techniques which is restriction fragment length polymorphism (RFLP) of 16S23S rDNA intergenic spacer region (ISR), random amplified polymorphic DNA (RAPD-PCR) and an enterobacterial repetitive intergenic consensus (ERIC-PCR) analysis were performed to discriminate 19 representative isolates of L. fermentum isolated from sayur asin. The result showed that L. fermentum strain D11 is distantly related to other isolates based on RFLP using HhaI restriction enzyme and RAPDPCR analyses. In addition, both of RAPD-PCR and ERIC-PCR successfully determined the genetic variation among L. fermentum strains by exhibiting distinct 4-8 bands (800-2080 bp) and 4-10 bands (280-3050 bp), respectively. A dendogram generated from UPGMA cluster analysis of both RAPD-PCR and ERIC-PCR data showed two distinct genotypic groups exist among L. fermentum isolated from sayur asin in Indonesia.

Identification of Arcanobacterium hippocoleae by MALDI-TOF MS analysis and by various genotypical properties

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the β subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses.

Phenotypic and genotypic approach to characterize Arcanobacterium pluranimalium isolated from bovine milk samples.

In the present study, three Arcanobacterium pluranimalium strains isolated from bovine milk samples of three cows of three farms (two cows with subclinical mastitis) could successfully be identified by phenotypical investigations, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis and genotypically by sequencing the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR), the β subunit of bacterial RNA polymerase encoding gene rpoB, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, the elongation factor tu encoding gene tuf, and the pluranimaliumlysin encoding gene pla. The latter could also be identified by a loop-mediated isothermal amplification (LAMP) assay. The presented phenotypic and genotypic approaches might support the identification of A. pluranimalium in future and might help to understand the role this species plays in bovine mastitis.

Rhizobial strains isolated from nodules of Medicago marina in southwest Spain are abiotic-stress tolerant and symbiotically diverse

The isolation and characterisation of nitrogen-fixing root nodule bacteria from Medicago marina, a tolerant legume species, were studied in two areas from southwest Spain. A total of 30 out of 82 isolates with distinct ERIC-PCR fingerprints were analysed on the basis of molecular (PCR-RFLP of the 16S-23S rDNA intergenic spacer region (IGS) with two endonucleases, analysis of the 16S rDNA and symbiotic nodC gene sequences, plasmid profiles and SDS-PAGE of LPS, including the partial sequence of the housekeeping gene glnII and the symbiotic gene nodA of some representatives), physiological (utilisation of sole carbon sources, tolerance to antibiotics, NaCl, heavy metals, temperature and pH) and symbiotic parameters (efficacy on M. marina, M. minima, M. murex, M. orbicularis, M. polymorpha, M. sativa and M. truncatula). All the bacteria isolated from M. marina nodules belonged to Ensifer meliloti, except for one strain that belonged to E. medicae. To determine the nodulation range of M. marina, 10 different Ensifer species were tested for their ability to nodulate on this plant. E. kummerowiae CCBAU 71714 and the E. medicae control strain M19.1 were the only Ensifer species tested that developed nitrogen-fixing nodules on this plant. Most of the M. marina-nodulating strains showed tolerance to stress factors and all of them shared the presence of a gene similar to cadA, a gene that encodes for a PIB-type ATPase, which is a transporter belonging to the large superfamily of ATP-driven pumps involved in the transport of metals across cell membranes.

Genetic characteristics of Streptococcus dysgalactiae isolated from cage cultured cobia, Rachycentron canadum (L.).

Disease outbreaks occurred during 2007-2013 in Taiwan with 2.5-10% mortality among the cage cultured cobia, Rachycentron canadum (L.), characterized by the presence of polyserositis, pericarditis and peritonitis. The micro-organisms isolated from internal organs were Gram-positive cocci. The isolates were confirmed as Streptococcus dysgalactiae by a polymerase chain reaction assay that yielded the expected specific 259 bp amplicon. Additionally, partial sequence of the 16S-23S rDNA intergenic spacer region of the GCS strain isolates from fish was also compared and produced 100% sequence identity with S. dysgalactiae (GenBank accession number AB252398). The genetic characterization was then determined by pulsed-field gel electrophoresis (PFGE) analysis. Based on PFGE, the Apa I or Sma I digestion patterns of chromosomal DNA of these isolates were grouped into three main clusters. Taiwanese strains were divided into two clusters, and the tet(M) gene was detected in cluster 1 (pulsotypes: A1-A2 and S1-S3), but not in cluster 2 strains (pulsotypes: A3-A4 and S4-S5). Three Japanese strains from amberjack, Seriola dumerili (Risso), were grouped into cluster 3 (pulsotypes: A5-A7 and S6-S8) and displayed no mortality to cobia in the challenge experiment. Conversely, Taiwanese strains from cobia and snubnose pompano, Trachinotus blochii (L.), displayed a mortality rate of 50-87.5% in cobia.

Identification of hypervariable regions within the 16S–23S rRNA intergenic spacer region of Flavobacterium columnare and its application in assigning genomovar group to an individual strain

Flavobacterium columnare is an important bacterial pathogen of fish with wide ge- netic variability within species. This intraspecies diversity has been termed as genomovars and genomovar groups on the basis of Restriction Fragment Length Polymorphisms of 16S rDNA and 16S-23S rDNA Intergenic Spacer Region (ISR), respectively. In this study, we demonstrate the source of genetic heterogeneity in the F. columnare by sequence analysis of ISR. Length of ISR sequences of different genomovars varied from 553 to 592 nucleotides, while the similarity among sequences ranged from 76.1 to 92.6%. A common ISR structure with tRNAAa and tRNAne embedded within the sequence was identified in all the genomovars ofF. columnare. The results show that strains of F. columnare can be categorized into five genomovar groups based on the heterogeneity in the ISR sequences. Of these, strains belonging to Genomovar I and II can be sub-divided into two groups each; while strains of Genomovar III belonged to one group. Sequence similarity between genomovar groups was lower for ISR (76.1-92.6%) as compared to 16S rDNA (96.1-99.4%) indicating its ability to resolve closely related groups within the genomovars of F. columnare. The main source of variation between genomovar groups is the presence of three hyper variable regions (V1, V2 & V3) in the ISR Of the three, V3 was found to be the most heterogeneous region and was found to be useful in assigning genomovar group to an individual strain of F. columnare.

Phenotypic and genotypic characteristics of Arcanobacterium haemolyticum isolated from clinical samples in a Danish hospital

Six Arcanobacterium haemolyticum strains isolated from six patients of two hospitals in Denmark were identified phenotypically, also including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and by genotypic methods. The latter were performed by sequencing 16S rDNA and glyceraldehyde 3-phosphate dehydrogenase encoding gene gap and by amplification of an A. haemolyticum specific region of 16S-23S rDNA intergenic spacer region and 23S rDNA. The six A. haemolyticum strains were further investigated for the presence of seven potential virulence genes encoding arcanolysin, phospholipase D, hemolysin A, CAMP factor family protein, collagen binding protein, neuraminidase A and neuraminidase H which appeared to be present in two (seven virulence genes), two (six virulence genes) and two strains (four virulence genes), respectively. The phenotypic and genotypic properties described in the present study might help to reliably identify and further characterize A. haemolyticum isolated from human patients, a species which seems to be of increasing importance.

Further Studies on Arcanobacterium phocisimile: a Novel Species of Genus Arcanobacterium.

Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698T isolated from a vaginal swab of a harbour seal and four additional A. phocisimile strains also isolated from four harbour seals could reliably be identified by phenotypic properties, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and by sequencing the genomic targets 16S rDNA and 16S-23S rDNA intergenic spacer region and the genes rpoB and gap. The A. phocisimile strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of A. phocisimile remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approach might help to identify A. phocisimile in future.

Eco-geographical diversity of cowpea bradyrhizobia in Senegal is marked by dominance of two genetic types

The genetic diversity of native cowpea rhizobia originating from 60 sites across four eco-geographic zones in Senegal was studied. More than 300 cowpea nodules were analyzed by PCR-RFLP of the 16S–23S rDNA InterGenic Spacer region (IGS). Alignments of IGS sequences indicated that all genotypes were grouping within the Bradyrhizobium genus. The geographical distribution showed that apart from five IGS types, the others were specifically found in only one region. The diversity was significantly higher in the Senegal River valley zone, which presents lower mean annual rainfalls and slightly alkaline soils. Interestingly, two IGS types dominated the Senegalese rhizobial collection, one IGS type (VI) was found on more than half of the nodules collected in the northern Senegal River valley while another IGS type (I) was recovered from the great majority of nodules in the three other regions sampled. Two representative strains from each of these two dominant types were isolated and further analyzed. Multi Locus Sequence Analyses using 6 housekeeping genes indicate that they belong to a new Bradyrhizobium species closely related to B. yuanmingense. Phylogenetic analyses of 2 symbiotic genes nodC and nifH show that they are clustered with B. arachidis. Physiological tests on these strains have shown that under laboratory conditions, the growth of the IGS type VI strains was slightly less affected by a higher osmotic strength in the medium and to alkaline pH, which corroborates the soil physico-chemical parameters.

Further characteristics of Arcanobacterium canis, a novel species of genus Arcanobacterium

Comparable to previously conducted phenotypical and genotypical investigations characterizing Arcanobacterium canis, a newly described species with the type strain A. canis DSM 25104 isolated from an otitis externa of a dog, four additional A. canis strains isolated from infections of three dogs and one cat could reliably be identified by phenotypic properties, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and by sequencing the genomic targets 16S rDNA, 16S-23S rDNA intergenic spacer region, 23S rDNA, and the genes rpoB and gap. All four A. canis investigated in the present study were isolated from the infected animals together with several other bacterial species indicating that the pathogenic importance of A. canis remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approaches might help to identify A. canis in future and might elucidate the role this species plays in infections of dogs and cats.