BOX PCR Research Articles

Occurrence, identification and characterization of diazotrophic bacteria from aerial roots of Rhynchostylis retusa (L.) Blume for plant growth-promoting activity.

In this study, the diversity of diazotrophic bacteria of orchid Rhynchostylis retusa (L.) Blume and its potential application in plant growth promotion were evaluated. About 183 nitrogen-fixing bacteria were isolated to screen various plant growth-promoting traits viz. phosphate solubilization,IAA, siderophore, HCN, biofilm and ammonia production. Based on 16S rRNA gene sequencing analysis Achromobacter, Arthrobacter, Acinetobacter, Bacillus, Brevibacterium, Curtobacterium, Erwinia, Kosakonia, Lysinibacillus, Klebseilla, Microbacterium, Mixta, Pantoea, Pseudomonas and Stenotrophomonas isolates were selected and showed positive results for PGP traits. Overall, genus Pantoea, Brevibacterium, Achromobacter, Arthrobacter, Klebsiella, Mixta, Bacillus, and Pseudomonas had the most pronounced PGP characteristics and acetylene reduction among the screened isolates. BOX PCR fingerprinting analysis showed variation in polymorphic banding patterns among diazotrophic strains. PCR amplification of nifH gene and the presence of 37kDa nitrogenase reductase enzyme band in western blot indicated presence of nitrogenase activity. Our study showed that orchid R. retusa diazotroph interaction helps orchid plant to fix nitrogen, essential nutrients, and control pathogen entry. To the best of our knowledge, this is the first report on characterization of diazotrophic bacterial community from aerial roots of R. retusa.

Effects of Phenotypic Variation on Biological Properties of Endophytic Bacteria Bacillus mojavensis PS17.

Simple SummaryMicroorganisms play an important role in agriculture by protecting and stimulating the growth of plants. The phenotypic activities of microbial biological agents (MBA) can change under different environmental conditions. However, to adapt to these harsh conditions, genetic mutations take place in bacteria that are seen phenotypically, which might not be beneficial or less beneficial to the plants. Some adaptative mechanisms used by microorganisms, especially bacteria, to face these environmental factors lead to the appearance of subpopulations with different morphotypes that may be more adapted to survive in stressful conditions. Moreover, in favorable conditions, these subpopulations may become dominant among the overall bacterial population. In this study, Bacillus mojavensis undergoes phase variation when grown in a minimal medium, in which two colonies, opaque (morphotype I) and translucent (morphotype II), were generated. The characteristics of the generated morphotypes were determined and compared with those of their original strain. Overall, the results obtained showed that the phenotypic characteristics of morphotype I statistically differed from morphotype II. This phenomenon may be one of the factors behind the dissimilarities in the results between the laboratory and field data on the application of MBA.The use of microorganism-based products in agricultural practices is gaining more interest as an alternative to chemical methods due to their non-toxic bactericidal and fungicidal properties. Various factors influence the efficacy of the microorganisms used as biological control agents in infield conditions as compared to laboratory conditions due to ecological and physiological aspects. Abiotic factors have been shown to trigger phase variations in bacterial microorganisms as a mechanism for adapting to hostile environments. In this study, we investigated the stability of the morphotype and the effects of phenotypic variation on the biological properties of Bacillus mojavensis strain PS17. B. mojavensis PS17 generated two variants (opaque and translucent) that were given the names morphotype I and II, respectively. The partial sequence of the 16S rRNA gene revealed that both morphotypes belonged to B. mojavensis. BOX and ERIC fingerprinting PCR also showed the same DNA profiles in both morphotypes. The characteristics of morphotype I did not differ from the original strain, while morphotype II showed a lower hydrolytic enzyme activity, phytohormone production, and antagonistic ability against phytopathogenic fungi. Both morphotypes demonstrated endophytic ability in tomato plants. A low growth rate of the strain PS17(II) in a minimal medium was observed in comparison to the PS17(I) strain. Furthermore, the capacity for biocontrol of B. mojavensis PS17(II) was not effective in the suppression of root rot disease in the tomato plants caused by Fusarium oxysporum f. sp. radices-lycopersici stain ZUM2407, compared to B. mojavensis PS17(I), whose inhibition was almost 47.9 ± 1.03% effective.

Molecular genotyping of enterococcus faecalis isolated from clinical sources by BOX-PCR and ERIC-PCR

Gram-positive bacteria, Enterococcus faecalis, cause serious nosocomial infections such as UTI, bloodstream infections, and endocarditis. The study's aimed to isolate and identify Enterococcus faecalis from various clinical specimens in Al-Diwanyiah province hospitals ,testing the sensitivity of isolates to antibiotics as well as to genotyping of E.faecalis isolates by BOX –PCR or ERIC-PCR Analysis. The study extended from October 2021 to January 2022. Biochemical tests, Vitek 2 and 16S rRNA analysis identified 37 bacteria as Enterococcus faecalis. Five clusters (I-V) and about 13 polymorphic variants showed among 20 isolates by ERIC PCR. Whereas, four cluster (I-IV) and 14 polymorphic variants were showed by BOX PCR. The results showed that that there is a slight difference between ERIC and BOX-PCR analysis for Enterococcus faecalis isolated from clinical samples.

Genotyping of Acintobacter baumannii isolated from different clinical samples

A total of ( 150 ) clinical specimens were collected from patients suffering from different infections such as burns, wounds infections, urinary tract infections, and respiratory tract infection. All clinical specimens were cultured on different media. The results indicated that 22 (14.6%) of the isolates belonged to A. baumannii It was found that twelve isolates (54.5%) from sputum, five isolates ( 22.72%) from burns ,three isolates (13.63%) were detected from wounds, , and two isolate ( 9%) from urine., In this study, used BOX and ERIC-PCR for the detection of phylogenetic diversity of A. baumannii isolated from different samples, In general, 20 isolates characterized using the two molecular techniques had comparable number of bands with some degree of polymorphism. K. pneumoniae isolates from the same source were clustered in to different groups. The two molecular techniques generated 2 main clusters and the results of dendrogram of these techniques reveals that 20 polymorphic variants between A. baumannii clinical isolates detect by ERIC and BOX PCR.

BOX- Genotyping of Carbapenem Resistant Acinetobacter baumannii Clinical Isolates from Diyala/Iraq

The aim of study is to determine the typing of Carbapenem-resistant A. baumannii clinical isolates fromsome hospitals in Diyala by the BOX-PCR technique. This study was carried out during the period fromFebruary 2020 to the end of September 2020. Out of 400 specimens, 53 isolates of Acinetobacter baumanniiwere recovered. All isolates were tested toward the different class of clinically important antibiotics (12)by using agar diffusion method. The results of resistance were as following: piperacillin 53%, Ticarcillin/clavulanic acid 75%, cefotaxime 94% and ceftazidime 66%, cefepime 53%, gentamicin 84%, amikacin49%, ciprofloxacin 47%, levofloxacin 38%, Aztreonam 87%, Imipenem and Meropenem 38%. Twenty (20)isolates of Acinetobacter baumannii were found to be resistant to carbapenems. The BOX–PCR methodwas used to detect the relationship among these carbapenem resistant isolates, the fingerprinting patterns ofthe isolates were shown 15 bands on gel electrophoresis, with molecular weight ranging between (100-3000bp) among the 20 carbapenem resistant Acinetobacter baumannii isolates. Genetic relationship of differentisolates of Acinetobacter baumannii was done by using BOX-PCR method and Dendogram analysis.The results showed the genetic relationship between Acinetobacter baumannii 4 clones, while 12 isolatescontained different genotyping. In conclusion, the present study showed that the BOX-PCR technique isreproducible, easy, fast and cost effectiveness tool for investigating the genetic diversity of A. baumanniiisolates.

Genetic and metabolic diversities of rhizobacteria isolated from degraded soil of Ethiopia

Genetic and metabolic diversities of rhizobacteria are the fundamental sources for their adaptation to cope with abiotic and biotic stresses in order to enhance growth and health of plants in the soil. Thus, this study was initiated to assess the genetic and metabolic diversities of rhizobacteria isolated from plants grown in degraded soil through BOX-PCR and partial sequencing of 16S rRNA genes. A total of eighty isolates were recovered and subjected to phenotypic profiling of carbohydrate and amino acid utilization, BOX PCR and 16S rRNA profiling. The phenotypic profiling showed remarkable metabolic versatility with Ochrobactrum spp, Pseudomonas spp and Klebsiella spp, and BOX-PCR showed greater discriminatory power for fingerprinting of rhizobacterial isolates with high degree of polymorphism. Bacillus spp showed the highest Simpson's diversity Index. The 16S rRNA genes sequence assigned the rhizobacteria to phyla Proteobacteria with Gammaproteobacteria and Alphaproteobacteria classes and Firmicutes with Bacilli class. The data also showed that the most dominant species were Pseudomonas and Ochrobactrum. Genetic and metabolic diversities of the rhizobacterial isolates reveal the potential of these microbes for plant growth improvement under water deficient soil after testing other inoculant traits.

Selection of sponge-associated bacteria with high potential for the production of antibacterial compounds

The potential of sponge-associated bacteria for the biosynthesis of natural products with antibacterial activity was evaluated. In a preliminary screening 108 of 835 axenic isolates showed antibacterial activity. Active isolates were identified by 16S rRNA gene sequencing and selection of the most promising strains was done in a championship like approach, which can be done in every lab and field station without expensive equipment. In a competition assay, strains that inhibited most of the other strains were selected. In a second round, the strongest competitors from each host sponge competed against each other. To rule out that the best competitors selected in that way represent similar strains with the same metabolic profile, BOX PCR experiments were performed, and extracts of these strains were analysed using metabolic fingerprinting. This proved that the strains are different and have various metabolic profiles, even though belonging to the same genus, i.e. Bacillus. Furthermore, it was shown that co-culture experiments triggered the production of compounds with antibiotic activity, i.e. surfactins and macrolactin A. Since many members of the genus Bacillus possess the genetic equipment for the biosynthesis of these compounds, a potential synergism was analysed, showing synergistic effects between C14-surfactin and macrolactin A against methicillin-resistant Staphylococcus aureus (MRSA).

Characterization and genetic diversity of Pantoea agglomerans isolates having dual potentiality to suppress growth of Ralstonia solanacearum and plant growth promoting ability

Twenty six isolates of genus Pantoea collected from leaves of rice and carrot (Experimental Farm, IARI, New Delhi), potato tuber (Hapur, Uttar Pradesh) and tomato leaves (Bengaluru, Karnataka) were characterized as Pantoea agglomerans by using morphological, physiological, biochemical methods and 16S rRNA nucleotide sequence analysis. Twenty six isolates of P. agglomerans, two reference strains of P. agglomerans (DWM-1and DWM-2) and three out group bacteria viz., Bacillus subtilis DTBS-5, B. cereus JHTBS-7, and Pseudomonas fluorescens DTPF-3 were taken to study genetic diversity by using BOX–PCR primers. The BOX primer produced 8–26 bands with mol. size 0.25 kb to 10 kb. DRP-16 and UPP-21 isolates had maximum number of fragments (15 fragments) followed by DRP-11, DRP-13 and DRP-14, UPP-18, UPP-19. Fifteen major groups at 75% similarity coefficient were obtained and high similarities were found among genetic profile of Delhi and Uttar Pradesh isolated strains, whereas the strains of Karnataka state separated in other groups. Out of 28 isolates of P. agglomerans, 12 showed antagonistic property against R. solanacearum in vitro conditions. Isolate DRP-7 showed highest inhibition zone (2.8 cm2) against pathogenic bacterium (R. solanacearum) followed by 2.5 cm2 in DRP-8 and UPP-20. Plant growth promoting activities of P. agglomerans isolates were tested in vitro situation. Majority of the isolates of P. agglomerans had good potentiality to solubilize the phosphorus, produce siderophores, indole acetic acid and ammonia. The result showed that the isolates DRP-7 and UPP-20 have both antagonistic and growth promoting ability, which is a good candidate to manage wilt disease and promote growth of tomato plants.

Metabolic and Genetic Diversities of Rhizobacteria Isolated from Degraded Soil of Ethiopia

Genetic and metabolic diversities are sources of biodiversity that provides adaptation and could help to cope with environmental stresses. Thus, the study was initiated to assess the metabolic and genetic diversities of rhizobacteria from degraded soil through BOX-PCR and 16S rRNA sequencing. A total of eighty isolates were subjected to carbohydrate and amino acid utilization tests, BOX PCR and 16S rRNA profiling. The phenotypic profiling comprised utilization of C and N sources revealed metabolic versatility of the strains (14.29 to 100%). Accordingly, Ochrobactrum, Pseudomonas and Klebsiella spp expressed notable metabolic versatilities. BOX-PCR showed greater diversity with high degree of polymorphism. Simpson’s Index of Diversity (0.883) took the leading position with Bacillus species. The 16S rRNA showed that the isolates assigned to phyla Firmicutes (21.92%) and Proteobacteria (78.08%). The most dominant species were Pseudomonas (23%) and Ochrobactrum (21%). Metabolic and genetic diversities of the rhizobacteria reveal their potential for plant growth improvement under water deficient soil.

Strain variability and pathogenic potential of tdhcarrying Vibrio parahaemolyticus isolated from diseased Penaeus monodon

A disease outbreak in 42-d-old black tiger shrimp Penaeus monodon juveniles from a commercial aquaculture farm in Kerala, India, was investigated. The cause of the disease outbreak was confirmed as Vibrio parahaemolyticus by biochemical tests, PCR targeting the toxR gene and pathogenicity testing of the isolates. All of the isolates tested negative by PCR specific for V. parahaemolyticus associated with acute hepatopancreatic necrosis disease (AHPND), implicating vibriosis unrelated to AHPND as the cause of mortality. Among the 19 isolates obtained, 2 possessed the tdh gene (coding for thermo-stable hemolysin), whereas none of the isolates possessed trh. The LD50 value of 8 isolates of V. parahaemolyticus from diseased and apparently healthy shrimp ranged from 2.7 × 104 to 4.9 × 105 CFU ml-1 by immersion challenge of P. monodon postlarvae. BOX-PCR and dendrogram analysis of the bacterial isolates revealed that the isolates from moribund and apparently healthy shrimp formed separate clusters, indicating that these isolates originate from separate clones. The isolates from moribund shrimp including tdh-positive V. parahaemolyticus clustered together. The present study represents the first report of tdh-positive V. parahaemolyticus causing disease in a shrimp farm.

First Report of Pseudomonas syringae pv. aptata Causing Bacterial Leaf Spot on Common Beet (Beta vulgaris) in Ohio

In July 2017, common beet leaves (cv. Red Ace) from an open field in Erie County, Ohio, showing foliar blight symptoms were received by the OSU Vegetable Pathology Laboratory. Another beet sample (cv. Boro) from Wayne County, Ohio, displaying the same symptoms was received in June 2018. Symptoms consisted of pale brown circular water-soaked necrotic lesions, 2 to 3 mm in diameter, surrounded by a red halo. Bacterial streaming was observed from the lesions when viewed microscopically. Pieces of symptomatic tissue ∼0.25 cm² were surface sterilized in 70% ethanol for 30 s, rinsed in sterile water, placed in 100 µl of sterile water, and minced with a sterile blade. After 30 s, 10 µl of the resulting suspension was streaked onto Pseudomonas F agar medium (PF) and incubated in the dark for 48 h at 28°C. White creamy colonies fluorescent on PF when exposed to UV light were selected. Isolates from both samples were gram negative, oxidase negative, arginine dihydrolase negative, levan positive, and caused a hypersensitive reaction 24 h after infiltration into tobacco (Nicotiana tabacum) leaves. None of the isolates caused soft rot when inoculated at 10⁷ CFU/ml onto sterile potato slices. Box PCR (Louws et al. 1994) profiles were similar for strain SM200-2017 (Erie County) and SM61-2018 (Wayne County), with an additional band in SM200-2017. Both strains were subjected to whole-cell DNA extraction and multilocus sequence analysis of the housekeeping genes sigma factor 70, rpoD; glyceraldehyde-3-phosphate dehydrogenase, gapA; DNA gyrase B, gyrB; and citrate synthase, gltA (Hwang et al. 2005). Sequences were compared with those of the reference pathotype strain Pseudomonas syringae pv. aptata (Psa) CFBP 1617 deposited in the PAMDB database (Almeida et al. 2010). Both SM200-2017 (MK085510) and SM61-2018 (MK085511) showed 100% identity to the rpoD gene. SM200-2017 gapA sequence (MK085516) shared 99% homology with the gapA sequence of Psa CFBP 1617, and SM61-2018 gapA sequence (MK085517) was 100% identical. Similarly, the gyrB sequences from SM200-2017 (MK085515) and SM61-2018 (MK085514) were 99 and 100% similar to Psa CFBP 1617, respectively. No match was found for SM200-2017 and SM61-2018 gltA sequences (MK085512 and MK085513, respectively) when compared with those of Psa CFBP 1617. However, the sequences were 100% identical to Psa sequence KX226440 (Arabiat et al. 2016) deposited in the NCBI database. Koch’s postulates were confirmed in a greenhouse by spray inoculating 4-week-old beet seedlings (cv. Red Ace) with 10⁶ CFU/ml water suspensions of SM200-2017 or SM61-2018, or with sterile water. Plants were maintained at 27°C with a 14-h photoperiod, in plastic bags for the first 48 h. Symptom development was monitored daily. Water-soaked necrotic lesions similar to those observed on the original samples appeared 10 days after inoculation, whereas mock-inoculated plants did not show any symptoms. Symptomatic leaves were collected and processed as described above. Colonies with the same morphology on PF and with the same biochemical characteristics previously described were isolated. Box PCR profiles matched those of the original strains. To our knowledge, this is the first report of bacterial leaf spot of beet caused by Psa in Ohio. Common beets are cultivated throughout Ohio and can be severely affected under disease-conducive environmental conditions.

Genetic Diversity of Proteus Mirabilis Isolated from Urine Specimens by Box PCR

Genetic Diversity of Proteus Mirabilis Isolated from Urine Specimens by Box PCR

Phosphate solubilizers as antagonists for bacterial leaf blight with improved rice growth in phosphorus deficit soil

Multifaceted phosphate solubilizing bacteria (PSB) promote growth of plants by acting both as biofertilizer and biopesticide. The present study aimed to use native beneficial rhizobacteria to suppress rice Bacterial Leaf Blight (BLB) pathogen as well as to combat the plant in P deficiency without reduction in crop yield. In present investigation, of 50 PSB isolated from rice rhizosphere, three bacterial strains inhibited the BLB pathogen (18–25 mm), solubilized phosphorus (up to197 µg mL−1) produced IAA (1.7–14 µg mL−1). Gluconic acid produced by phosphate solubilizing antagonistic bacteria and molecular confirmation through amplification of glucose dehydrogenase genes (gcd) showed that gluconic acid may have dual role in P solubilization as well as BLB suppression. Plate germination assay showed that increase in vigor index (27%), root diameter (29%), root length volume-1 (57%) and root projection area (43%) were observed in inoculated seedlings as compared to uninoculated control. Plant inoculation studies under net house conditions showed BLB disease suppression (up to 56%) along with increase in plant P contents (up to 0.47% in straw and 1.26% in seed) in treatments inoculated with Pseudomonas spp. MR11, MR34 and Bacillus sp. MR42. Increase in the activity of defense related enzymes Phenylalanine ammonia lyase (PAL: 5.165 activity as µmole cinnamic acid g−1. f. wt.), Catalase (CAT: 2.88 activity g−1. f. wt.), Peroxidase (POD: 2.4 activity g−1. f. wt.), β, 1–3 glucanase (283 activity g−1. f. wt.) and Polyphenol oxidase (PPO: 65 activity g−1. f. wt.) were observed in response to inoculation of P-solubilizing antagonistic bacteria. BOX PCR finger printing and viable cell count coupled with Fluorescent in situ hybridization showed the competence and survival of inoculated bacteria in rhizosphere and phyllosphere. The findings of the current research work strongly encouraged the use of PSB strains for BLB suppression in phosphorus deficient soils.

The unravelled Enterococcus faecalis zoonotic superbugs: Emerging multiple resistant and virulent lineages isolated from poultry environment.

This study aimed to investigate the zoonotic potential by virtue of phylogenetic analysis, virulence and resistance gene profiles of Enterococcus faecalis originating from poultry environment. The ERIC, BOX and RAPD PCR analysis showed the clustering of E.faecalis strains (n=74) into five groups (G1-G5) and fifteen sub-clusters (B1-B15), which share 50%-80% similarities with ATCC E.faecalis and clinical strains of human infection. E.faecalis strains harboured seven enterocins genes including ent1097 (85%), entB (84%), enterolysinA (51%), entSEK4 (51%), entL50 (31%), entA (25.7%) and ent1071 (14.9%). The highest prevalence of gelE-sprE (90%), lip-fl (90%) followed by cylL (62%), hyl (60%), katA (16%) and cylA (5.4%) was observed in poultry isolates. The fsr operon and gelE-sprE was co-associated in 66.2% strains. E.faecalis also harboured biofilm and endocarditis-associated genes, including efaAfs (97%), ebp-pilli (ebpABC and srtC 69.9%-80%), asa1 (71%), agg (55%), ace (54%) and esp-Tim (3%). Despite all found sensitive to vancomycin, 98.6% strains were multi-drug resistant to five to twelve tested antimicrobials. An increased-level of resistance (≥32μg/ml) was observed to ampicillin (8.1%), meropenem (21.6%), chloramphenicol (73.4%), erythromycin (90.5%), tetracycline (100%) and high-level resistance to kanamycin (79.7%) and gentamicin (52.7%). The multi-drug resistant E.faecalis (MDRe.f) were carried pbp4 (90%), tetL (90%), tetM (70%), ermB (81%), cat (52.7%), acc6-aph2 (58.1%), aaph(3)-III (49.9%), gyrA (97%) and parC (98%) genes. Moreover, these MDRe.f were also harboured, hospital-associated marker IS16 (58%) and pheromone responsive genes, that is ccf (88%), cpd (74%), cob (62%) and eep (66%). Thus, regardless of the distinct phylogenetic background of E.faecalis of poultry origin, ATCC E.faecalis and clinical strains of human origin, we found major similarities in virulence, resistance gene profiles and mobile genetic elements (IS16 and pheromone responsive plasmids), supporting the zoonotic/reverse zoonotic risk associated with this organism.

Rep-PCR Genomic Fingerprinting Revealed Genetic Diversity and Population Structure among Ethiopian Isolates of Pseudocercospora griseola Pathogen of the Common Bean (Phaseolus vulgaris L.)

Angular leaf spot (Pseudocercospora griseola) is one of the most devastating diseases affecting common bean production in most parts of Ethiopia. Thus, use of common bean varieties with durable resistance is the most effective and economical control measure. Knowledge about the genetic variability and population structure of the pathogen populations is important for a successful common bean improvement program. The objective of this study was to determine the genomic diversity existing among and between P. griseola isolates which were obtained from the field survey collection of diverse common-bean growing areas of Ethiopia. The study used the repetitive extragenic palindromic elements-polymerase chain reaction protocol to fingerprinting DNA sequence diversity. To study the genetic diversity, we analysed molecular data from 79 single-spore isolates of the P. griseola pathogen. Hence, Molecular Analysis of Variance (AMOVA) and cluster analysis revealed the existence of high genetic diversity within and among P. griseola isolates. ERIC PCR produced 21 different patterns of clusters, whereas, REP-PCR and BOX PCR produced 11 and 5 different patterns of clusters respectively. This is because of some isolates that shared the same BOX patterns could be distinguished by the ERIC and REP finger printing patterns. The ERIC-, BOX- and REP-PCR combined fingerprinting patterns discriminated 25 different patterns among the 79 monosporic P. griseola isolates were produced at cut-off 77% genetic similarity matrix. These discriminated clusters revealed the existence of genetic diversity within and among the isolates of P. griseola collected from the diverse common bean growing regions of Ethiopia.

Bifidobacterium callitrichidarum sp. nov. from the faeces of the emperor tamarin (Saguinus imperator).

Three Gram-stain-positive, non-spore-forming, microaerophilic and fructose-6-phosphate phosphoketolase positive strains were isolated from a faecal sample of an adult subject of the emperor tamarin (Saguinus imperator). Given that the isolates revealed identical BOX PCR profiles, strain TRI 5T was selected as a representative and characterized further. Comparative analysis of 16S rRNA gene sequence similarity revealed that strain TRI 5T was closely related to Bifidobacterium saguini DSM 23967T (96.4 %) and to Bifidobacterium longum subsp. longum ATCC 15708 (96.2 %). Multilocus sequence analyses of five housekeeping genes showed the close phylogenetic relatedness of this strain to Bifidobacterium breve DSM 20213T (hsp60 94.1 %), Bifidobacterium saguini DSM 23967T (clpC 91 %), Bifidobacterium avesanii DSM 100685T (dnaG 80.3 %), Bifidobacterium longumsubsp. infantis ATCC 15697T (dnaJ 85.3 %) and Bifidobacterium longumsubsp. longum ATCC 15708 (rpoB 93 %), respectively. The peptidoglycan type was A3β, with an interpeptide bridge comprising l-Orn (Lys) - l-Ser - l-Ala - l-Thr - l-Ala. The DNA G+C content of strain TRI 5T was 60.9 mol%. Based on the data provided, strain TRI 5T represents a novel species of the genus Bifidobacterium for which the name Bifidobacteriumcallitrichidarum sp. nov. is proposed. The type strain is TRI 5T (=DSM 103152T=JCM 31790T).

Rapid RHD Zygosity Determination Using Digital PCR.

Paternal zygosity testing is used for determining homo- or hemizygosity of RHD in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the Rhesus box PCR, which can be difficult to interpret and unreliable, particularly for black African populations. DNA samples extracted from 53 blood donors were analyzed using 2 multiplex reactions for RHD-specific targets against a reference (AGO1)2 to determine gene dosage by digital PCR. Results were compared with serological data, and the correct genotype for 2 discordant results was determined by long-range PCR (LR-PCR), next-generation sequencing, and conventional Sanger sequencing. The results showed clear and reliable determination of RHD zygosity using digital PCR and revealed that 4 samples did not match the serologically predicted genotype. Sanger sequencing and long-range PCR followed by next-generation sequencing revealed that the correct genotypes for samples 729M and 351D, which were serologically typed as R1R2 (DCe/DcE), were R2r' (DcE/dCe) for 729M and R1r″ (DCe/dcE), R0ry (Dce/dCE), or RZr (DCE/dce) for 351D, in concordance with the digital PCR data. Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal RH zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous RHD-positive individuals.

Molecular assessment of virulence determinants, hospital associated marker (IS16gene) and prevalence of antibiotic resistance in soil borne Enterococcus species.

Enterococci, no more regarded as GRAS (Generally Recognized As Safe) organism, are emerging as an important source of nosocomial infections worldwide. The main contributors in pathogenesis of enterococci are the presence of various virulent factors and antibiotic resistance genes. We aimed to examine the prevalence, dissemination, antibiotic resistance and virulent factors associated with enterococci from bulk soil (BS). A total of 372 enterococci were isolated from 500 soil samples. PCR was used to identify the isolates up to species level and for carriage of 16 virulence genes including hospital associated marker (i.e. IS16). E.faecium (77%), E.faecalis (10%), E.hirae (4%) and E.casseliflavus (1%) were the major species isolated. The efaAfs was the most dominant gene (100%), followed by gelE (78.9%), sprE (76.3%) and esp (13%) in E.faecalis isolates. The E.faecium carried largely efaAfm (86.8%) and acm (50.3%) genes. Presence of entP (10%), entA (8.3%) and entB (6.9%) genes was detected mostly in E.faecium, while enlA (18%) and ef1097 (2.6%) was only detected in E.faecalis isolates. 50% E.faecalis and 2% E.faecium isolates harbored IS16, while five E.faecalis harbored both IS16 and espTIM genes providing strong evidence about the presence of espTIM gene on 64Kb pathogenicity island. BOX and RAPD PCR analysis revealed high degree of genetic variation within the species. Degree of resistance against 12 major antibiotics showed chloramphenicol as the most effective and meropenom as the least effective antibiotic. Presence of multiple antibiotic resistant, virulent and hospital associated enterococci in bulk soil represents a potential source for further dissemination to humans and animals and poses potential impact on public health.

Pseudomonas cerasi sp. nov. (non Griffin, 1911) isolated from diseased tissue of cherry

Eight isolates of Gram-negative fluorescent bacteria (58T, 122, 374, 791, 963, 966, 970a and 1021) were obtained from diseased tissue of cherry trees from different regions of Poland. The symptoms resembled those of bacterial canker. Based on an analysis of 16S rDNA sequences the isolates shared the highest over 99.9% similarity with Pseudomonas ficuserectae JCM 2400T and P. congelans DSM 14939T. Phylogenetic analysis using housekeeping genes gyrB, rpoD and rpoB revealed that they form a separate cluster and confirmed their closest relation to P. syringae NCPPB 281T and P. congelans LMG 21466T. DNA–DNA hybridization between the cherry isolate 58T and the type strains of these two closely related species revealed relatedness values of 58.2% and 41.9%, respectively. This was further supported by Average Nucleotide Identity (ANIb) and Genome-to-Genome Distance (GGDC) between the whole genome sequences of strain LMG 28609T and closely related Pseudomonas species. The major cellular fatty acids are 16:0 and summed feature 3 (16:1 ω7c/15:0 iso 2OH). Phenotypic characteristics differentiated the novel isolates from other closely related species. The G+C content of the genomic DNA of strain 58T was 59%. The diversity was proved by PCR MP and BOX PCR, eliminating the possibility that they constitute a clonal population. Based on the evidence of this polyphasic taxonomic study the eight strains are considered to represent a novel species of the genus Pseudomonas for which the name P. cerasi sp. nov. (non Griffin, 1911) is proposed. The type strain of this species is 58T (=LMG 28609T=CFBP 8305T).

Evaluation of genetic and phenotypic consistency of Bacillus coagulans MTCC 5856: a commercial probiotic strain.

Commercial probiotics preparation containing Bacillus coagulans have been sold in the market for several decades. Due to its high intra-species genomic diversity, it is very likely that B. coagulans strain may alter in different ways over multiple years of production. Therefore, the present study focuses to evaluate the genetic consistency and probiotic potential of B. coagulans MTCC 5856. Phenotypic and genotypic techniques including biochemical profiling, 16S rRNA sequencing, GTG 5″, BOX PCR fingerprinting, and Multi-Locus-Sequence typing (MLST) were carried out to evaluate the identity and consistency of the B. coagulans MTCC 5856. Further, in vitro probiotic potential, safety and stability at ambient temperature conditions of B. coagulans MTCC 5856 were evaluated. All the samples were identified as B. coagulans by biochemical profiling and 16S rRNA sequencing. GTG 5″, BOX PCR fingerprints and MLST studies revealed that the same strain was present over 3 years of commercial production. B. coagulans MTCC 5856 showed resistance to gastric acid, bile salt and exhibited antimicrobial activity in in-vitro studies. Additionally, B. coagulans MTCC 5856 was found to be non-mutagenic, non-cytotoxic, negative for enterotoxin genes and stable at ambient temperature (25 ± 2 °C) for 36 months. The data of the study verified that the same strain of B. coagulans MTCC 5856 was present in commercial preparation over multiple years of production.Electronic supplementary materialThe online version of this article (doi:10.1007/s11274-016-2027-2) contains supplementary material, which is available to authorized users.