Early detection of a possible multidrug-resistant Acinetobacter baumannii outbreak in the local hospital setting by using random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), oxacillinase gene profiles, and antibiograms.

Background and Objectives: Detecting the source of a potential outbreak of multidrug resistant (MDR) Acinetobacter baumannii is necessary to be investigated. This study aimed to detect the possibility of A. baumannii outbreak in a hospital setting using a combination of random amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR), antibiograms, and the presence of oxacillinase genes. Materials and Methods: The antibiogram of 31 clinical isolates and six environmental isolates of A. baumannii were determined by Vitek® 2 Compact. Oxacillinase genes (OXA-23, -24, -51, and -58) were detected by PCR, and RAPD-PCR was conducted using DAF-4 and ERIC-2 primers. The Similarity Index and dendrogram were generated using GelJ v2.3 software. Results: The antibiograms showed that all MDR A. baumannii isolates has very limited susceptibility to cephalosporins, but mostly susceptible to tigecycline. All isolates were positive for bla OXA-51-like gene, thirty-two of 37 total isolates (86.5%) were positive for bla OXA-23-like gene, and none were positive for bla OXA-24-like and bla OXA-58-like genes. RAPD-PCR showed that the DAF-4 primer on average had more band visualization and lower Similarity Index's variation compared to the ERIC-2. The discriminatory power of DAF-4 was 0.906. There was a significant correlation between the DAF-4 dendrogram pattern with the antibiogram (r=0.494, p<0.001) and the presence of bla OXA-23-like gene (r=0.634, p<0.001) from all ICU A isolates. Six out of fourteen ICU A isolates belonged to the same cluster with >95% Similarity Index, while one clinical isolate having an identical dendrogram and antibiogram pattern with an environmental isolate within this cluster. Conclusion: There is a high probability of MDR A. baumannii outbreak within ICU A detected by multiple analysis of RAPD-PCR, antibiogram and the bla OXA-23-like gene profiles. This combinatorial approach is conceivable to mitigate possible outbreak situations of A. baumannii in the local hospital without sophisticated microbiology laboratory.

Genotypic and antimicrobial susceptibility of Streptococcus agalactiae causing bovine mastitis in the central region of Thailand.

Introduction: Streptococcus agalactiae is a highly contagious pathogen that causes bovine mastitis, leading to significant economic losses. This study aimed to (1) identify and characterize S. agalactiae strains responsible for bovine mastitis by examining their phenotypic and genotypic characteristics in Thai dairy-intensive farming areas and (2) determine their susceptibility profiles to antimicrobial agents. Material and methods: In total, 100 S. agalactiae isolates obtained from clinical and subclinical mastitis cases from 13 dairy herds located in the central region of Thailand were examined. To confirm the identity of the bacterial pathogens, conventional microbiological procedures recommended by the National Mastitis Council (NMC) and the VITEK® 2 system were employed. Results: All 100 isolates were successfully identified as S. agalactiae using the NMC procedure, whereas 94 isolates were identified as S. agalactiae using the VITEK® 2 system. Finally, the S. agalactiae-specific gene dlt S was identified in all the examined isolates using polymerase chain reaction. Capsular polysaccharide (CPS) typing revealed that all strains belonged to CPS type Ia. Multilocus sequence typing identified 33 selected isolates as sequence type 103. Random amplified polymorphic DNA (RAPD) typing yielded 43 RAPD types, with 6 RAPD clusters identified. These results demonstrated a high level of genetic diversity among S. agalactiae within the studied herds. RAPD analysis suggested that specific S. agalactiae strains could persist in dairy farms for 2-12 months. Furthermore, antimicrobial susceptibility testing was performed using the broth microdilution method. Most strains demonstrated susceptibility to ampicillin, penicillin, penicillin/novobiocin, cephalothin, oxacillin, ceftiofur, and erythromycin. Discussion: This study revealed the phenotypic and genotypic characteristics of S. agalactiae isolates responsible for bovine mastitis in the central region of Thailand. The rapid identification of S. agalactiae and application of molecular typing methods can provide valuable epidemiological information regarding S. agalactiae causing mastitis in dairy farms. The antimicrobial susceptibility of S. agalactiae indicates that antimicrobial treatment for control and eradication could be a successful protocol. Our findings revealed that a single clonal strain of S. agalactiae affected the 13 studied farms. Further research is needed to explore the feasibility of vaccine development and application.

Involvement of Dynamic Adjustment of ABA, Proline and Sugar Levels in Rhizomes in Effective Acclimation of Solidago gigantea to Contrasting Weather and Soil Conditions in the Country of Invasion.

Giant goldenrod (Solidago gigantea Aiton) is one of the most invasive plant species occurring in Europe. Since little is known about the molecular mechanisms contributing to its invasiveness, we examined the natural dynamics of the content of rhizome compounds, which can be crucial for plant resistance and adaptation to environmental stress. We focused on rhizomes because they are the main vector of giant goldenrod dispersion in invaded lands. Water-soluble sugars, proline, and abscisic acid (ABA) were quantified in rhizomes, as well as ABA in the rhizosphere from three different but geographically close natural locations in Poland (50°04'11.3″ N, 19°50'40.2″ E) under extreme light, thermal, and soil conditions, in early spring, late summer, and late autumn. The genetic diversity of plants between locations was checked using the random amplified polymorphic DNA (RAPD) markers. Sugar and proline content was assayed spectrophotometrically, and abscisic acid (ABA) with the ELISA immunomethod. It can be assumed that the accumulation of sugars in giant goldenrod rhizomes facilitated the process of plant adaptation to adverse environmental conditions (high temperature and/or water scarcity) caused by extreme weather in summer and autumn. The same was true for high levels of proline and ABA in summer. On the other hand, the lowering of proline and ABA in autumn did not confirm the previous assumptions about their synthesis in rhizomes during the acquisition of frost resistance by giant goldenrod. However, in the location with intensive sunlight and most extreme soil conditions, a constant amount of ABA in rhizomes was noticed as well as its exudation into the rhizosphere. This research indicates that soluble sugars, proline, and ABA alterations in rhizomes can participate in the mechanism of acclimation of S. gigantea to specific soil and meteorological conditions in the country of invasion irrespective of plant genetic variation.

Staphylococcus borealis - A newly identified pathogen of bovine mammary glands.

Twelve Staphylococcus borealis strains, isolated in Canada and Poland from milk of cows with intramammary infections, were characterized phenotypically (biochemical reactions on ID 32 STAPH and Biolog Phenotype MicroArrays™ PM1 and PM2A, ability of biofilm production) and genotypically (random amplified polymorphic DNA). In addition, a genomic comparison was done with S. borealis strains of human and porcine origin using the multilocus sequence typing (MLST) technique. The bovine isolates showed a high degree of phenotypic and genotypic diversity, however, they could be differentiated from human strains by the negative test for urease (found in all but one bovine isolate examined with ID 32 STAPH) and positive reaction for D-galactose (on Biolog phenotype microarray PM1) and D-lactose (on both commercial systems). The MLST method, utilizing six concatenated genes of the total length of ∼2930 bp, revealed that bovine strains (irrespective of the country of origin) show a distinctly greater degree of mutual relationship than to the strains of human and porcine origin, suggesting that S. borealis has evolved independently in these hosts. In conclusion, bovine-specific S. borealis can be involved in intramammary infections in cattle.

Intracellular presence and genetic relationship of Helicobacter pylori within neonates' fecal yeasts and their mothers' vaginal yeasts.

Helicobacter pylori are transmissible from person to person and among family members. Mother-to-child transmission is the main intrafamilial route of H. pylori transmission. However, how it transmits from mother to child is still being determined. Vaginal yeast often transmits to neonates during delivery. Therefore, H. pylori hosted in yeast might follow the same transmission route. This study aimed to detect intracellular H. pylori in vaginal and fecal yeasts isolates and explore the role of yeast in H. pylori transmission. Yeast was isolated from the mothers' vaginal discharge and neonates' feces and identified by internal transcribed spacer (ITS) sequencing. H. pylori 16S rRNA and antigen were detected in yeast isolates by polymerase chain reaction and direct immunofluorescence assay. Genetic relationships of Candida strains isolated from seven mothers and their corresponding neonates were determined by random amplified polymorphic DNA (RAPD) fingerprinting and ITS alignment. The Candida isolates from four mother-neonate pairs had identical RAPD patterns and highly homologous ITS sequences. The current study showed H. pylori could be sheltered within yeast colonizing the vagina, and fecal yeast from neonates is genetically related to the vaginal yeast from their mothers. Thus, vaginal yeast presents a potential reservoir of H. pylori and plays a vital role in the transmission from mother to neonate.

High Frequency of blaOXA-48like Producing Klebsiella pneumoniae Isolated from Nosocomial Infection in Azerbaijan, Iran.

BACKGROUND: Klebsiella pneumoniae is one of the significant agents of hospital-acquired infections. In recent years, carbapenem-resistant K. pneumoniae (CRKP) isolates have been found in numerous epidemics of nosocomial infections. This study aimed to determine carbapenem resistance mechanisms and molecular epidemiological of CRKP infections in Azerbaijan, Iran. MATERIALS AND METHODS: A total of 50 non-duplicated CRKP from January 2020 to December 2020 were isolated form Sina and Imam Reza Hospitals in Tabriz, Iran. Antimicrobial susceptibility testing was performed by the disk-diffusion method. The carbapenem resistance mechanisms were determined by the phenotypic and PCR procedures. CRKP isolates were typed by the Random Amplified Polymorphic DNA PCR (RAPD-PCR) technique. RESULTS: Amikacin was the most effective antibiotics against CRKP isolates. AmpC overproduction was observed in five CRKP isolates. Efflux pump activity was found in one isolate by the phenotypic method. Carba NP test could find carbapenemases genes in 96% of isolates. The most common carbapenemases gene in CRKP isolates were blaOXA-48-like (76%) followed by blaNDM (50%), blaIMP (22%), blaVIM (10%), and blaKPC (10%). The outer membrane protein genes (OmpK36 and OmpK35) were identified in 76% and 82% of CRKP isolates, respectively. RAPD-PCR analysis yielded 37 distinct RAPD-types. Most blaOXA-48-like positive CRKP isolates were obtained from patients hospitalized in intensive care unit (ICU) wards with urinary tract infections. CONCLUSION: The blaOXA-48-like is the main carbapenemase among CRKP isolates in this area. Most blaOXA-48-like producer CRKP strains were collected from the ICU ward and urine samples. To control infections due to CRKP, a strict control program in hospital settings is required.

Evaluating genetic diversity of geographically diverse populations of Embelia ribes Burm f., a highly medicinal woody liana from the Western Ghats of India, using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats (ISSR) markers.

BACKGROUND: Embelia ribes Burm f. (Primulaceae) is a medicinal and vulnerable woody liana distributed throughout India. Embelin, a well-recognized active phytoconstituents in berries, is commonly used in ayurvedic formulations. Due to over-exploitation, the status of the plant is vulnerable. Previous studies on this species mainly focused on its phytochemical analysis, which led to overexploitation and loss of the germplasm. METHODS AND RESULTS: In the present study, 20 RAPD and 18 ISSR markers were employed to assess genetic divergence in 40 genotypes of E. ribes collected from different parts of the Western Ghats of India. In RAPD analysis, all 40 accessions with 20 RAPD primers amplified 282 fragments, with 83.91% average polymorphism and with an average of 14.10 bands per primer. The size of amplicons varied from 200 to 2500 bp. While, ISSR primers produced 203 fragments of which 161 were polymorphic with an average of 11.28 bands per primer with 73.25% average polymorphism. The size of amplicons ranges from 200 to 2500 bp. RAPD and ISSR markers were also assessed by calculating polymorphic information content (PIC) to discriminate the genotypes; the average PIC value for RAPD, ISSR, and combined RAPD + ISSR markers obtained was more than 0.50 suggesting the informativeness of markers. UPGMA analysis based on Jaccard's similarity coefficient for RAPD, ISSR, and RAPD + ISSR data reveals that 40 accessions of E. ribes were depicted in four clusters. The clustering pattern of all individuals in PCoA analysis agreed with the UPGMA dendrograms, which further confirms the genetic relationships explained by cluster analysis. AMOVA analysis of RAPD, ISSR, and combined marker system revealed variation within the population, ranging from 41 to 44%, and among the population, it ranged from 56 to 59%. CONCLUSION: The present study provides an optimized method for evaluating the genetic diversity of Embelia ribes using RAPD and ISSR markers which are useful for further sustainable utilization and conservation of natural populations in the Western Ghats of India.

Bacterial community analysis of infant foods obtained from Chinese markets by combining culture-dependent and high-throughput sequence methods.

In this study, twenty-two baby foods including cereal-based products and powdered infant formula (PIF) obtained from local markets were comprehensively investigated for their bacterial contamination using culture-dependent and high-throughput sequence (HTS) methods. In addition, the genetic diversity and biofilm-forming capacity of the most abundant species were analyzed using random amplified polymorphic DNA (RAPD) and crystal violet staining assay, respectively. Results showed that 170 mesophilic isolates collected from 22 samples were clustered into 15 genera and 41 species. Bacillus (77.65%) was the most prevalent genus, followed by Paenibacillus (7.06%), Alkalibacillus (3.53%), and Lysinibacillus (2.35%). Bacillus licheniformis (49.41%) proved to be the most dominant species in infant foods, and a high genetic diversity with six different RAPD profiles was observed. A total of 87.5% of B. licheniformis isolates were identified as strong biofilm formers, and heterogeneous biofilm-forming ability was observed among the isolates sharing the same RAPD pattern. HTS analysis revealed an 18-fold higher biodiversity at the genus level, and a significantly different bacterial community of infant foods was dominated by Lactococcus, Streptococcus, and Bifidobacterium. Foodborne pathogens including Bacillus cereus, and potentially pathogenic microorganisms such as Acinetobacter baumannii, were identified in infant foods by HTS. The current results could expand the crucial information about bacterial contamination of baby foods.

Gamma Radiation Induced In-Vitro Mutagenesis and Isolation of Mutants for Early Flowering and Phytomorphological Variations in Dendrobium 'Emma White'.

In vitro mutagenesis offers a feasible approach for developing new orchid cultivars through genetic manipulation. In the present study, protocorm-like bodies (PLBs) were exposed to gamma rays (10, 20, 40, 60, 80 Gy) to study in vitro growth responses and induction of mutants in Dendrobium 'Emma White'. Both proliferation and regeneration of PLBs decreased progressively with increasing doses, except for a significantly enhanced growth response at 10 Gy. The optimal dose of gamma radiation for mutagenesis was found in the range 10 to 25 Gy based on the growth reduction curve. Analysis using a high-throughput cell analyzer revealed a significant reduction in nuclear DNA content at > 40 Gy doses. At 10 Gy treatment, the growth attributes, such as root length, plant height and leaf number, were significantly increased by 36%, 26% and 20%, respectively, compared to the control. This increase was significant over other tested doses as well. Testing of random amplified polymorphic DNA markers revealed the presence of detectable polymorphism among gamma mutant plantlets with a polymorphism information content value at 0.41. The gamma-ray-induced earliness in flower development was observed within 294 days post ex vitro growth of 10 Gy mutant compared to the control plants flowered after 959 days. Our results highlight the significance of gamma radiation in inducing enhanced growth, morphological variations and early floral initiation in Dendrobium, providing a basic framework for mutation breeding and improvement of orchids.

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0 mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5 mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5 mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60 days after being transferred to auxins and cytokinin (1.0 and 1.5 mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5 mg Kinetin L-1) along with 200 mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30 days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Genetic Diversity Analysis based on the Virulence, Physiology and Regional Variability in Different Isolates of Powdery Mildew in Pea.

Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops (Pisum sativum L.). To examine the powdery mildew pathogen's morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in the Nilgiris district of Tamil Nadu, India. The pathogen Erysiphe pisi was identified morphologically based on anamorphic characters. Molecular identification of E. pisi isolates was befitted by targeting the internal transcribed spacer (ITS) region of rDNA and specific primers of powdery mildew fungi. The genetic variation between ten different E. pisi isolates collected from topographically distinct mountainous areas was studied using random amplified polymorphic (RAPD). Based on its morphological characteristics, the powdery mildew fungus presented high similarities to E. pisi. Molecular characterization of the ITS rDNA of E. pisi produced 650 bp nucleotides, PMITS (powdery mildew-internal transcribed region) primers produced 700 bp nucleotides, and an Erysiphe specific ITS primer pair amplified and synthesized 560 bp nucleotides. According to the findings, the collected E. pisi strains exhibited a low level of genetic diversity and only a slight differential in virulence on the host. In the study, E. pisi isolates from Anumapuram, Emerald Valley, Indira Nagar, and Thuneri showed a greater disease incidence in the natural field conditions and shared the same genetic lineage with other isolates in UPGMA hierarchical cluster analysis based on RAPD markers. There was no evidence of a link between the occurrence of the disease and these grouped populations.

Effect of using mycotoxin-detoxifying agents in dairy cattle feed on natural whey starter biodiversity.

Natural whey cultures (NWC) are undefined multiple-strain bacterial starter communities that can be affected by even small changes along the entire dairy chain. We applied a multidisciplinary approach to investigate how the addition of 2 mycotoxin-detoxifying agents [sodium smectite and lignocellulose-based material (B1); leonardite and betaine (B2)] to cow diets modified the microbiota of the NWC in manufacture of a Grana-like cheese. Microbiological and flow cytometry analyses showed that the content and viability of lactic acid bacteria (LAB) and the total whey microbiota were not affected by the detoxifying agents, and Streptococcus thermophilus, Lactobacillus helveticus, and Limosilactobacillus fermentum were the dominant taxa. Random amplified polymorphic DNA-PCR fingerprinting and metagenomic analysis highlighted differences in the bacterial community of the NWC and in the relative abundance of Bacteroidetes that increased when B1 and B2 were included in the diet. Two of 6 St. thermophilus biotypes were detected only in control samples; conversely, none of the Lb. helveticus biotypes found in control samples were isolated from B1 and B2. In vitro tests showed that the 2 binders did not significantly affect the development of St. thermophilus, but they stimulated the growth of Lb. helveticus strains recovered only from B1 and B2 NWC. The addition of binders in cow feed can affect the LAB biotypes present in NWC.

Diversidad genética de aedes aegypti en el eje transfronterizo Central-Alto Paraná en Paraguay / Genetic diversity of Aedes aegypti in the central-upper Paraná Cross-Border axis in Paraguay

RESUMEN Objetivos: Conocer la diversidad genética de Aedes aegypti en el corredor vial transfronterizo Central-Alto Paraná de Paraguay, con registros de casos de dengue. Materiales y métodos: Se seleccionaron veinte hembras adultas de la eclosión de huevos de Ae. aegypti procedentes de casas geolocalizadas en los departamentos de Alto Paraná, Caaguazú, Cordillera y Central, entre el 2018 y 2019. Se extrajo ADN del tejido de las hembras para amplificación aleatoria de sus patrones polimórficos mediante amplificación aleatoria del ADN polimórfico por PCR (RAPD-PCR), usando cebadores H3 y B03 a fin de conocer parámetros genéticos de diversidad poblacional. Las relaciones entre las poblaciones de mosquitos según la localidad fueron visualizadas mediante análisis no apareado de la media aritmética. Las áreas idóneas de distribución geográfica real y potencial de estas poblaciones de Ae. aegypti fueron analizadas mediante DIVA-GIS 7.3.0 y MAXENT. Resultados: Se identificaron 40 loci mediante perfiles RAPD-PCR, con diferenciación génica moderada (Gst = 0,12). El corredor transfronterizo presentó condiciones bioclimáticas para la presencia de poblaciones variantes de Ae. aegypti, siendo determinantes en la distribución la precipitación del trimestre más cálido y la temperatura media del trimestre más seco. Conclusiones: Se evidencia que existe diversidad genética moderada en las poblaciones de Ae. aegypti procedentes de zonas con registros de casos de dengue ubicadas en el corredor vial transfronterizo que une los departamentos Central y Alto Paraná de Paraguay. El estudio de variabilidad genética de Ae. aegypti es de gran utilidad para la vigilancia entomoepidemiológica y evaluación de posibles eventos de resistencia al control químico.

ABSTRACT Objective: To determine the genetic diversity of Aedes aegypti in the Central-Alto Paraná cross-border road corridor of Paraguay, an area that has reports of dengue cases. Materials and methods: Twenty adult females were selected from hatching Ae. aegypti eggs from households geolocated in the departments of Alto Paraná, Caaguazú, Cordillera and Central, between 2018 and 2019. DNA was extracted from the tissue of females for amplifying their polymorphic patterns by random amplification of polymorphic DNA by PCR (RAPD-PCR), using primers H3 and B03 in order to identify genetic parameters of population diversity. The relationships between mosquito populations according to locality were observed by unpaired arithmetic mean analysis. We used DIVA-GIS 7.3.0 and MAXENT to analyze the suitable areas of actual and potential geographic distribution of these Ae. aegypti populations. Results: Forty loci were identified by RAPD-PCR profiling, with moderate gene differentiation (Gst = 0.12). The cross-border corridor presented bioclimatic conditions for the presence of variant populations of Ae. aegypti, with precipitation in the warmest quarter and mean temperature in the driest quarter being determinant in the distribution. Conclusions: There is evidence of moderate genetic diversity in Ae. aegypti populations from areas that have reported dengue cases in the cross-border road corridor linking the Central and Alto Paraná departments of Paraguay. The study of genetic variability of Ae. aegypti is very useful for entomo-epidemiological surveillance and evaluation of possible resistance to chemical control.

Molecular study and antifungal susceptibility profile of Trichophyton rubrum and Trichophyton mentagrophytes strains isolated from lesions of humans and cattle.

Background and Objectives: Monitoring of contagious diseases is important to advance our knowledge of their epidemiology and to enable more impressive investigation and prevention efforts. This study aimed to examine antifungal drug susceptibility and molecular analysis of clinical isolates of Trichophyton rubrum and Trichophyton mentagrophytes in humans and cattle. Materials and Methods: A total of 400 patients and 500 cattle were evaluated in this study. Dermatophytosis was confirmed in cases by direct microscopy and culture methods. Antifungal drug susceptibility profiles, MIC50, and MIC90 of isolates were determined using the broth microdilution method. Multiplex-PCR, RAPD PCR, and sequencing methods were used for the genetic analysis of virulence genes and the ITS1 and ITS2 regions, respectively. Results: A total of 175 patients and 120 cattle were diagnosed with dermatophytosis. Dermatophytes showed a remarkable rate (30%) of terbinafine resistance. T. mentagrophytes showed lower susceptibility than T. rubrum (MIC50 =16 µg/mL). Strains harboring Mep1, Mep2, and Mep4 genes had the highest frequency among all genotypes. A RAPD-PCR dendrogram divided T. mentagrophytes and T. rubrum strains into three and six groups, respectively. Conclusion: A notable rate of resistance to terbinafine in isolated dermatophytes was reported in this study. Examination of RAPD-PCR results showed that T. rubrum strains had higher genetic diversity than T. mentagrophytes. Genetic monitoring of dermatophytes must be considered an important factor in providing fungal infection prevention and treatment approaches.

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan.

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.

Domestication affects the composition, diversity, and co-occurrence of the cereal seed microbiota.

Introduction: The seed-associated microbiome has a strong influence on plant ecology, fitness, and productivity. Plant microbiota could be exploited for a more responsible crop management in sustainable agriculture. However, the relationships between seed microbiota and hosts related to the changes from ancestor species to breeded crops still remain poor understood. Objectives: Our aims were i) to understand the effect of cereal domestication on seed endophytes in terms of diversity, structure and co-occurrence, by comparing four cereal crops and the respective ancestor species; ii) to test the phylogenetic coherence between cereals and their seed microbiota (clue of co-evolution). Methods: We investigated the seed microbiota of four cereal crops (Triticum aestivum, Triticum monococcum, Triticum durum, and Hordeum vulgare), along with their respective ancestors (Aegilops tauschii, Triticum baeoticum, Triticum dicoccoides, and Hordeum spontaneum, respectively) using 16S rRNA gene metabarcoding, Randomly Amplified Polymorphic DNA (RAPD) profiling of host plants and co-evolution analysis. Results: The diversity of seed microbiota was generally higher in cultivated cereals than in wild ancestors, suggesting that domestication lead to a bacterial diversification. On the other hand, more microbe-microbe interactions were detected in wild species, indicating a better-structured, mature community. Typical human-associated taxa, such as Cutibacterium, dominated in cultivated cereals, suggesting an interkingdom transfers of microbes from human to plants during domestication. Co-evolution analysis revealed a significant phylogenetic congruence between seed endophytes and host plants, indicating clues of co-evolution between hosts and seed-associated microbes during domestication. Conclusion: This study demonstrates a diversification of the seed microbiome as a consequence of domestication, and provides clues of co-evolution between cereals and their seed microbiota. This knowledge is useful to develop effective strategies of microbiome exploitation for sustainable agriculture.

Random amplified polymorphic DNA and inter simple sequence repeat markers reveals genetic diversity between micro propagated, wild and field cultivated genotypes of Gloriosa superba: an endangered medicinal plant.

Gloriosa superba L., an endangered medicinal plant with global interest due to presence of colchicine, an important alkaloid used in formulations of Indian and Traditional medicine. The plant has become endangered due to its unscientifically exploitation and high medicinal values. In the Present study 10 randomly amplified polymorphic DNA (RAPD) and 6 ISSR markers were employed to assess genetic divergence among micro propagated, wild and field cultivated plants of Gloriosa superba collected from different parts of India. In RAPD analysis, all the 10 accession with 10 RAPD primers amplified 466 fragments, with 96.43 % polymorphism and with an average of 46.6 bands per primer. The size of amplicons varied from 1656 to 100 bp. While, ISSR primers produced 328 fragments of which 298 were polymorphic with an average of 49.7 bands per primer with 91.83% polymorphism. The size of amplicons ranges from 2395 to 181 bp. RAPD, ISSR markers were also assessed by calculating polymorphic information content (PIC) to discriminate the genotypes, Average PIC value for RAPD, ISSR and combined RAPD + ISSR markers obtained was ≤ 0.50 suggesting the informativeness of markers. Jaccard's coefficient ranges from 0.18 to 0.75 (RAPD) and 0.17 to 0.61 (ISSR) and 0.21-0.52 for pooled ISSR and RAPD markers. The clustering pattern based on UPGMA analysis of the genotypes in the combined analysis revealed that the majority of the genotypes remained similar to the ISSR dendrogram, while the RAPD-based dendrogram showed some variation in the clustering of genotypes. The result of PCA scattered plot obtained were in agreement with the UPGMA dendrogram, which further confirms the genetic relationships explain by cluster analysis. Results confirmed that the genotype studied had good genetic diversity and can be used for identification, conservation, and future breeding program of Gloriosa species and consequently for the benefit of the pharmaceutical industries.

Characterization of Streptococcus lutetiensis isolated from clinical mastitis of dairy cows.

Streptococcus lutetiensis, previously termed Streptococcus bovis type II/1, has rarely been associated with bovine mastitis. The objectives of this work were to characterize the molecular diversity, antimicrobial resistance profiles, virulence genes of Strep. lutetiensis (n = 37) isolated from bovine clinical mastitis, as well as its pathogenic effects in a murine mastitis model. Genetic relationships of isolates were determined by random amplified polymorphic DNA (RAPD)-PCR, virulence genes were detected by PCR. Antimicrobial susceptibility testing was carried out by broth microdilution technique. The pathogenic effects of Strep. lutetiensis were studied with 2 infection models: bovine mammary epithelial cells cultured in vitro and murine mammary infection in vivo. Streptococcus lutetiensis isolates were clustered into 5 RAPD-types (A-E), with a dominant type A representing 84% of isolates. Eighteen (49%), 16 (43%), and 9 (24%) isolates were resistant to ceftiofur, tetracycline, and erythromycin, respectively. Prevalence of multidrug resistance (resistant to ≥3 classes of antimicrobials) was 24% (9/37). The most prevalent virulence genes were bca (100%), speG (100%), hly (97%), scpB (95%), and ssa (95%). There was no difference between isolates from mild and moderate cases of bovine mastitis in prevalence of virulence genes. Streptococcus lutetiensis rapidly adhered to and subsequently invaded (1 and 3 h after infection, respectively) bovine mammary epithelial cells, resulting in elevated lactate dehydrogenase release (4 h after infection). Edema and hyperemia were observed in challenged mammary glands and bacteria were consistently isolated at 12, 24, and 48 h after infection. In addition, numerous neutrophils migrated into gland alveoli and interstitium of infected mammary tissue. We concluded that Strep. lutetiensis had potential to spread within a dairy herd and good adaptive ability in bovine mammary cells or tissue, which are generally characteristics of a contagious mastitis pathogen.

Characterisation of Thai strawberry (Fragaria × ananassa Duch.) cultivars with RAPD markers and metabolite profiling techniques.

Strawberries (Fragaria × ananassa Duch.) are one of the most economically important fruit crops worldwide, several commercially viable cultivars are cultivated in the northern region of Thailand. The morphological characters at the young vegetative seedling stage can be very similar, which has hindered breeding efforts. The present study assesses the ability of random amplification of polymorphic DNA (RAPD) markers and metabolomics techniques to distinguish six strawberry cultivars. Both techniques showed congruent results for the leaf tissue and classified the cultivars into three major clusters. For the most different cultivars, Akihime and Praratchatan No.80, fruits were analysed at eight fruit ripening stages. The data highlighted a broad biological variation at the early ripening stages and less biological variation at the mature stages. Key metabolic differences included the polyphenol profile in Praratchatan No.80 and fatty acid synthesis/oxidation in Akihime. In summary, the RAPD and metabolite data can be used to distinguish strawberry cultivars and elucidate the metabolite composition of each phenotype. This approach to the characterisation of genotypes will benefit future breeding programmes.

SCAR marker for identification and discrimination of specific medicinal Lycium chinense Miller from Lycium species from ramp-PCR RAPD fragments.

In the current study, ramp-PCR fragments from improved RAPD (random amplified polymorphic DNA) amplification of Lycium (Goji) species or cultivars were cut and cloned into the vector of pGEM-T. A positive clone 10-5 was screened by PCR amplification, enzymatic digestion, and Sanger sequencing. A SCAR (sequence-characterized amplified region) marker, named Goji 10-5, with 949 nucleotides in length, was identified. Goji 10-5 is specific to Goji species Lycium chinense Miller from Jiangxi in China and Texas in the USA. A BLAST search of this nucleotide sequence in the GenBank database indicated that it shows no identity with any other species, including no any other Lycium species. As a new sequence, we have deposited it in the GenBank database with accession No. MN862323. PCR assays were developed and converted the nucleotide sequence to become a novel molecular marker for Lycium chinense Miller, named Goji 10-5. This marker may be used for the genetic identification of other samples. This study has successfully developed Goji 10-5, a specific SCAR marker to identify L. chinense and distinguish it from other species, including other Lycium species from different locations.