Sex identification in Nepenthes adrianii from Baturraden Botanical Garden: Genetic analysis using RAPD Markers

Nepenthes adrianii is one of pitcher plant species that grows endemically in Mount Slamet, Central Java. At present, it is one of pitcher plant collections of Baturraden Botanical Garden. Since N. adrianii is dioecious and both sexes are difficult to distinguish morphologically, early sex determination supporting its conservation at Baturraden Botanical Garden is needed. One approach can be performed with the use of RAPD molecular markers. Hence, this study aims to know whether differences in RAPD pattern between male and female N. adrianii exist or not and also to find out what the differences are. Genomic DNAs were extracted from leaves of 4 males, 2 females, and 2 sexually unidentified individuals. The extracted DNAs were then used to analyze DNA variation between male and female N. adrianii employing RAPD technique. As many as five oligonucleotide primers (OPA-15, OPK-16, OPP-15, OPP-08, and OPO-08) were used to amplify N. adrianii DNA. The results showed that one primer, i.e. OPK-16 (5’-GAGCGTCGAA-3’), produces a specific band of approximately 290 bp which is only found in female plants. It is assumed that this band is related to gene(s) controlling sex determination in N. adrianii. The RAPD marker can be used for the sex determination of young N. adrianii seedlings.

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of β-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Diversity of Escherichia coli from the enteric microbiota in patients with Escherichia coli bacteraemia

ABSTRACTObjectiveThe increase in Escherichia coli bloodstream infections mandates better characterisation of the relationship between commensal and invasive isolates. This study established a simple approach to characterize diversity of E. coli in the gut reservoir from patients with either E. coli bacteraemia, other Gram-negative bacteraemia, or patients without bacteraemia not receiving antibiotics. Stool or rectal swabs from patients in the three groups were cultured on selective chromogenic agar. Genetic diversity of E. coli in gut microbiota was estimated by RAPD-PCR.ResultsEnteric samples from E. coli bacteraemia patients yielded a median of one E. coli RAPD pattern (range 1-4) compared with two (range 1-5) from groups without E. coli bacteraemia. Of relevance to large-scale clinical studies, observed diversity of E. coli among E. coli bacteraemia patients was not significantly altered by sample type (rectal swab or stool), nor by increasing the colonies tested beyond ten. Overall, hospitalised patients demonstrated an apparently limited diversity of E. coli in the enteric microbiota and this was further reduced in those with E. coli bacteraemia. The reduced diversity of E. coli within the gut during E. coli bacteraemia raises the possibility that dominant strains may outcompete other lineages in patients with bloodstream infection.

SCAR MARKER DEVELOPMENT FOR THE CORRECT IDENTIFICATION OF IRIS ENSATA

Objective: The objective of this research was to develop the RAPD based SCAR marker for the correct identification of the Iris ensata Thunb. (I. ensata) plant from its adulterants.Methods: Five samples of I. ensata. from the different geographical area were used in this study. The plant genomic DNA was isolated with the CTAB method with some modification (as dried samples were also used). After that, polymorphism was checked with the help of the 10-mer random primers of OPAA and BG series. Then, the bands of interest were eluted and cloned into pGEMT easy vector for the sequencing. Finally, the sequence is used to develop the SCAR primers (Ir-f andIr-R) specific for I. ensata and the developed primers also validated with respect to the market samples.Results: A putative 580 bp sequence specific for Iris ensata was identified from the randomly amplified polymorphic DNA (RAPD) analysis. To overcome the main limitation of RAPD it has been converted into SCAR markers. So that, this specific band was then eluted, cloned and sequenced. After that, SCAR primers (Ir-F and Ir-R) were synthesized by using this sequence. For the validation of the synthesized SCAR primers, they were tested with respect to the market samples. The amplicon of 260 bp was produced by the SCAR primers in the authentic I. ensata but market samples did not produce any bands with the synthesized SCAR primers.Conclusion: The results of this study show a high level of polymorphism in the RAPD pattern of the different accessions of the plant. Furthermore, this study results in the successful development of the RAPD based SCAR marker for the identification of the I. ensata.

Random amplified polymorphic DNA (RAPD) analysis of Ornithobacterium rhinotracheale strains isolated from chickens in Turkey

Six field strains of Ornithobacterium rhinotracheale isolated from chickens in Elazig province located in the East of Turkey were typed by serotyping and random amplified polymorphic DNA assay using a random primer (OPG-11). Using the AGP test used for serotyping, serotype A was found to be the predominant serotype, only one strain was serotyped as serotype B. By RAPD assay, the tested ORT strains were found to have different RAPD profiles. In addition, the RAPD assay showed almost similar DNA profiles among the tested strains of the serotypes A, B, D and E. The strain of serotype C did give a different RAPD profile. Within strains of the same serotype (A), different profiles were found but the strain of serotype (B) had an identical profile as strains of serotype A. This study suggests that more genotypes of ORT strains are present within the same serotype and thus that no relationship exists between the RAPD pattern of ORT and their serotype.

Isolation and characterization Shiga toxin-producing Escherichia coli from sheep and goats inJordanwith evidence of multiresistant serotype O157:H7

Ninety-three rectal swabs of lambs and young goats from two extensively and two intensively managed herds in Jordanwere taken and examined for Shiga toxin-producing Escherichia coli (STEC). The bacteriological examination included the preenrichment of rectal swabs in EC broth with novobiocin, and a subsequent parallel isolation on enterohemolysin agar and immunomagnetic separation with cultivation on CT-SMAC. The STEC O157:H7 strains were demonstrated in 8 of 32 diarrheic lambs 1- to 3-weeks old in one sheep herd with intensive milk production. In the remaining three herds, serogroups O128, O78, O15 and serotype O128:K85 of STEC strains were the most frequent findings. The presence of stx2, ehlyA and eaeA genes in all STEC O157:H7 isolates was confirmed by PCR. In two untypable STEC isolates, stx2 and ehlyA genes were detected. In other STEC non-O157 isolates, only stx1 a ehlyA genes were found. All STEC O157:H7 isolates were resistant against sulphonamides and chloramphenicol, five were also resistant against ampicillin and streptomycin, one against co-trimoxazole. One isolate was resistant against ampicillin, ampicillin-sulbactam, cephalosporins (cefazolin, cefuroxime), monobactams (aztreonam), sulphonamides, co-trimoxazole, aminoglycosides, tetracycline and chloramphenicol. Compared the resistant STEC O157:H7 isolates, the multiresistant isolate had a different RAPD pattern. Of 36 STEC non-O157 isolates, one isolate was resistant against sulphonamides and co-trimoxazole, and another one against ampicillin, streptomycin, sulphonamides and co-trimoxazole. STEC isolates resistant against antimicrobial agents were demonstrated only in herds with intensive management.

The RAPD analysis of several cultivars of grapevine (Vitis viniferaL.) and their clones

Nine identification RAPD markers (Moravcová et al. 2003) were used to distinguish 24 clones and grapevine cultivars. No polymorphism was detected among all the tested clones of Chardonnay, Pinot gris and Zweigeltrebe from Polešovice. Pinot noir, Pinot gris, Pinot blanc and Pinot Meunier were indistinguishable within clones, they also showed the identical RAPD profile within cultivars (except discussed sample No. 26). On the other hand, Auxerrois as a relative to cultivars of Pinot group showed unique patterns and may be classified as a different cultivar. Some irregularities within the cultivars of Pinot family from Oblekovice were also found, several of them gave different results from those expected: Pinot blanc sample 26 has the RAPD profile typical of Chardonnay. A new abnormal RAPD pattern as a marker of typical Chardonnay and Pinot profiles was observed in two cases. While RAPD banding patterns could not distinguish between the known clones, they were useful for distinguishing between phenotypically similar cultivars and for assessing the origins of cultivars thought to have originated as sports.