Polymorphic DNA Polymerase Chain Reaction Research Articles

Limited genetic variability and spatial population structure in grasshoppers between natural and metal-contaminated areas in Egypt.

Pollutants in an environment can have long-term implications for the species living there, resulting in local adaptations with implications for their genetic structure. Heavy metal pollutants infiltrate soils and groundwater, bioaccumulate in food webs, and negatively impact biota. In this study, we investigated the degree to which the genetic structure and variability of the slender green-winged grasshopper (Aiolopus thalassinus (Fabricius) (Orthoptera: Acrididae)) were impacted by heavy metal pollution and distance. We used the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method to examine the genetic variability of populations in 3 heavy metal-polluted and 3 unpolluted locations across varying geographical distances in Egypt. The heavy metal concentrations of cadmium, copper, lead, and zinc were measured from the grasshopper tissue and soils. Sixty-nine unique and polymorphic bands were produced by 4 primers. Cluster and principal component analyses separated the populations inside and outside Cairo into 2 main branches, which were further divided into smaller branches corresponding to their geographical regions. We found no differences in the Shannon genetic diversity index between populations or with increasing heavy metal concentrations in either the soil or the grasshopper tissue. Our results showed a greater genetic variation among populations than between populations within the same location, indicating populations within locations were less differentiated than those between locations. The moderate correlation between genetic similarity and spatial distance suggests geographical isolation influenced grasshopper population differentiation. Based on the RAPD analysis, environmental pollutants and geographical distances impact the A. thalassinus population structure, potentially restricting gene flow between sites even at small spatial scales.

Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio.

Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.

Screening and evaluating microorganisms with broad-spectrum biogenic amine-degrading ability from naturally fermented dry sausage collected from Northeast China

This study aimed to screen autochthonous strains with broad-spectrum biogenic amine (BA) degradation ability from traditional dry sausages and to evaluate their BA-degrading ability in dry sausages. A total of 120 strains were isolated from dry sausages collected from various regions in Northeast China, and 35 of 120 isolates were identified as non-BA producing strains by the in vitro agar method. The random amplified polymorphic DNA polymerase chain reaction technique genotyped these 35 isolates into 18 biotypes. Moreover, high performance liquid chromatography (HPLC) quantification showed that six strains (Latilactobacillus sakei MDJ6; Lactiplantibacillus plantarum SH7; Weissella hellenica DQ9; Staphylococcus saprophyticus JX18 and SYS8; and Macrococcus caseolyticus SYS11) of the 18 biotypes exhibited broad-spectrum BA-degrading ability, all of which had various levels of amine oxidase activity with monoamine oxidase and diamine oxidase activities ranged of 6.60–619.04 and 26.32–352.81 U/mg protein, respectively. These six strains were subsequently inoculated into dry sausages and the results showed that they exhibited varying degrees of BA-degrading ability, of which strain Lat. sakei MDJ6 allowed to have less BA production on dry sausage with a final concentration of 61.33 mg/kg.

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.

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. 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. 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. 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.

Molecular variability of Fusarium oxysporum f. sp. lini isolates by RAPD analysis

Seven isolates of Fusarium oxysporum f. sp. lini collected from different linseed growing areas in India were analyzed by random amplified polymorphic DNA-polymerase chain reaction. Two isolates (FOL 1 and FOL 2) found non-pathogenic to linseed crop. Twenty random decamer primers were used for amplification. All the primers revealed scorable polymorphism among the isolates and a total of 156 bands were recorded. Amplification patterns of non-pathogenic Fusarium isolates were entirely distinct from those of pathogenic isolates. Seventeen primers generated isolate specific bands which would be highly useful in the identification and characterization of Fusarium isolates. RAPD markers showed substantial genetic diversity among the different isolates with maximum of 0.89 between FOL1 and FOL6. UPGMA cluster analysis was used to generate a dendogram showing relationship between them. Isolates were clustered in to three groups corresponding to their genetic similarity and physiological reactions. RAPD markers can be quick and reliable alternative for differentiating isolates of Fusarium oxysporum f. sp. liniin to different groups.

Outbreak of Burkholderia multivorans among patients at two acute-care hospitals in California, August 2021–July 2022

Background:Burkholderia multivorans are gram-negative bacteria typically found in water and soil. B. multivorans outbreaks among patients without cystic fibrosis have been associated with exposure to contaminated medical devices or nonsterile aqueous products. Acquisition can also occur from exposure to environmental reservoirs like sinks or other hospital water sources. We describe an outbreak of B. multivorans among hospitalized patients without cystic fibrosis at 2 hospitals within the same healthcare system in California (hospitals A and B) between August 2021 and July 2022. Methods: We defined confirmed case patients as patients without cystic fibrosis hospitalized at hospital A or hospital B between January 2020 to July 2022 with B. multivorans isolated from any body site matching the outbreak strain. We reviewed medical records to describe case patients and to identify common exposures. We evaluated infection control practices and interviewed staff to detect exposures to nonsterile water. Select samples from water, ice, drains, and sink splash zone surfaces were collected and cultured for B. multivorans in March 2022 and July 2022 from both hospitals. Common aqueous products used among case patients were tested for B. multivorans. Genetic relatedness between clinical and environmental samples was determined using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic polymerase chain reaction (Rep-PCR). Results: We identified 23 confirmed case patients; 20 (87%) of these were identified at an intensive care unit (ICU) in hospital A. B. multivorans was isolated from respiratory sources in 18 cases (78%). We observed medication preparation items, gloves, and patient care items stored within sink splash zones in ICU medication preparation rooms and patient rooms. Nonsterile water and ice were used for bed baths, swallow evaluations, and ice packs. B. multivorans was cultured from ice and water dispensed from an 11-year-old ice machine in the ICU at hospital A in March 2022 but no other water sources. Additional testing in July 2022 yielded B. multivorans from ice and a drain pan from a new ice machine in the same ICU location at hospital A. All products were negative. Clinical and environmental isolates were the same strain by RAPD and Rep-PCR. Conclusions: The use of nonsterile water and ice from a contaminated ice machine contributed to this outbreak. Water-related fixtures can serve as reservoirs for Burkholderia, posing infection risk to hospitalized and immunocompromised patients. During outbreaks of water-related organisms, such as B. multivorans , nonsterile water and ice use should be investigated as potential sources of transmission and other options should be considered, especially for critically ill patients.Disclosures: None

Genotyping-driven diversity assessment of biocontrol potent Bacillus spp. strain collection as a potential method for the development of strain-specific biomarkers.

Bacillus species are among the most researched and frequently applied biocontrol agents. To estimate their potential as environmentally friendly microbial-based products, reliable and rapid plant colonization monitoring methods are essential. We evaluated repetitive element-based (rep) and Random Amplified Polymorphic DNA (RAPD) PCR (Polymerase Chain Reaction) genotyping in a diversity assessment of 251 strains from bulk soil, straw, and manure samples across Serbia, highlighting their discriminative force and the presence of unique bands. RAPD 272, OPG 5, and (GTG)5 primers were most potent in revealing the high diversity of a sizable environmental Bacillus spp. collection. RAPD 272 also amplified a unique band for a proven biocontrol strain, opening the possibility of Sequence Characterized Amplified Region (SCAR) marker design. That will enable colonization studies using the SCAR marker for its specific detection. This study provides a guide for primer selection for diversity and monitoring studies of environmental Bacillus spp. isolates.

Genetic Diversity of Candida spp. Isolates Colonizing Twins and Their Family Members.

A wide range of options for studying Candida species are available through genetic methods. Twins, particularly monozygotic ones and their families may be fitting subjects for studying those microorganisms. The question is: How specific can yeast flora be in an individual? The study aimed to analyze the strain relatedness among commensal yeasts isolated from various parts of the bodies of healthy people and to compare correlations between the genotypes of the isolates. Yeasts were isolated from 63 twins and their family members (n = 25) from the oral cavity, anus, interdigital space and navel. After species identification, Candida albicans (n = 139), C. parapsilosis (n = 39), C. guilliermondii (n = 25), C. dubliniensis (n = 11) and C. krusei (n = 9) isolates were analyzed using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) optimization method. The similarities between the strains were calculated based on the Dice (Sab) coefficient and are displayed graphically as dendrograms. Using cluster analysis, the following relatedness was distinguished: 13 genotypes and three unique (Un) patterns among C. albicans; 10 genotypes and four Un patterns among C. parapsilosis; three genotypes and one Un pattern among C. guilliermondii and C. dubliniensis; and three genotypes among C. krusei isolates. The presence of identical, similar or both genotypes among the strains isolated from family members shows the transmission of yeasts between ontocenoses in the same person and between individuals. The similarity between the genotypes of C. albicans, C. guilliermondii, C. dubliniensis and C. krusei was more remarkable than between the genotypes of C. parapsilosis in the strains isolated from ontocenoses of the same individual and their family members. The degrees of genetic similarity between Candida spp. strains isolated from monozygotic twins and those obtained from their relatives did not differ.

The Human Milk Microbiota Produces Potential Therapeutic Biomolecules and Shapes the Intestinal Microbiota of Infants.

Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four Levilactobacillus brevis strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six Lactiplantibacillus plantarum strains by PCR analysis and in vitro antibacterial studies. 1H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain, Limosilactobacillus fermentum MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel Lactobacillus strains expressing specific functional biomolecules.

Antimicrobial susceptibility and genotyping of microorganisms isolated from sputum culture of children with cystic fibrosis in an Iranian referral children's hospital.

Chronic bacterial infections of the airways are present in most patients with cystic fibrosis (CF). Although most pathogens are acquired from the environment, there is great evidence of patient-to-patient transmission. Therefore, evaluating the genetic variation of strains isolated from CF patients is recommended for the purpose of examining hospital infection. The aim of this study was to determine the antibiotic susceptibility pattern and genotyping of Staphylococcus aureus and Pseudomonas aeruginosa strains isolated from sputum samples of children with CF referred to asingle pediatric CF center in Tehran, Iran. In this cross-sectional study, the antimicrobial susceptibility profiles of strains isolated from patients with CF during 1year were determined. Pseudomonas aeruginosa and S.aureus isolates were genotyped using the random amplified polymorphic DNA polymerase chain reaction method and were analyzed using GelComparII software. Of 534patients with CF, 384 had negative sputum cultures (72%), and 94strains of P.aeruginosa (18%) and 53strains of S.aureus (10%) were isolated. The mean age of the patients was 8.22 ± 5.7years (range, 2months to 18years). The P.aeruginosa strains showed high sensitivity to ceftazidime (96%), piperacillin/tazobactam (96%), and imipenem (94%). All strains of S.aureus were susceptible to vancomycin, and 13% of the strains were methicillin-resistant S.aureus. High resistance to penicillin (92%) and erythromycin (88.5%) were reported. The results of P.aeruginosa genotyping revealed that there were six major clusters in this hospital. Also, based on the analysis of genotyping results, S.aureus strains were obtained from five clusters, most of which were located in cluster B1 (34isolates, 64%). The results of this study show the possibility of strains being transferred from one part of the hospital to another (especially from the respiratory ward to other areas). Hence, high attention should be paid to the basic methods of preventing infection.

Long-Term Lacticaseibacillus rhamnosus GG Storage at Ambient Temperature in Vegetable Oil: Viability and Functional Assessments.

Vegetable oils with varying saturated fat levels were inoculated with Lacticaseibacillus rhamnosus GG (LGG), subjected to different heat treatments in the absence and presence of inulin and stored for 12 months at room temperature. After storage, the heat-treated probiotics actively grew to high concentrations after removal of the oils and reculturing. The bacterial samples, regardless of aerobic or anaerobic conditions and treatment methods, showed no changes in their growth behavior. The random amplified polymorphic DNA-polymerase chain reaction, antimicrobial, morphology, and motility tests also showed no major differences. Samples of LGG treated with a higher antioxidant content (Gal400) showed reduced inflammatory and anti-inflammatory properties. These findings have been confirmed by metabolite and genome sequencing studies, indicating that Gal400 showed lower concentrations and secretion percentages and the highest number of single nucleotide polymorphisms. We have shown proof of concept that LGG can be stored in oil with minimum impact on probiotic in vitro viability.

Microbial Analysis of Obturators During Maxillofacial Prosthodontic Treatment Over an 8-Year Period.

The aim of the study was to investigate the microbial colonization (by Candida species, anaerobic and facultative anaerobic bacteria) of maxillary obturators used for the restoration of maxillary defects, including during radiotherapy. Retrospective cohort study. Fifteen patients requiring a maxillary obturator prosthesis had swabs of their obturators and adjacent tissues taken at different stages of their treatment over a period of 8 years. Identification of microbial species from the swabs was carried out using randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis, checkerboard DNA–DNA hybridization, CHROMagar Candida chromogenic agar, and DNA sequencing. Candida species were detected in all patients and all patients developed mucositis and candidiasis during radiotherapy which was associated with an increase in colonization of surfaces with Candida spp ., particularly C albicans. Microbial colonization increased during radiotherapy and as an obturator aged, and decreased following a reline, delivery of a new prosthesis, or antifungal treatment during radiotherapy. Microbial colonization of maxillary obturators was related to the stage of treatment, age of the obturator material, radiotherapy and antifungal medications, and antifungal treatment may be recommended if C albicans colonization of palatal tissues is greater than 105 colony-forming units per cm2 following the first week of radiotherapy.

RAPD-PCR-Based Fingerprinting Method as a Tool for Epidemiological Analysis of Trueperella pyogenes Infections.

In this study, a Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method for genetic typing of Trueperella pyogenes, an opportunistic bacterial pathogen, was designed. The method optimization was performed for 37 clinical T. pyogenes strains isolated from various infections in different animal species. Optimal conditions for reliable and reproducible DNA fingerprinting were determined according to the modified Taguchi method. The developed method was assessed regarding its typeability, reproducibility, and discriminatory power using the Hunter’s and Gatsons’ index of discrimination. A high degree of genetic diversity was shown between the studied strains, which represented 31 genotypes. The generated RAPD profiles were relatively complex and simultaneously easy to interpret due to the wide size range of amplicons. The discriminatory index of the designed method was sufficiently high; thus, only strains epidemiologically related displayed identical RAPD genotypes. In conclusion, the DNA fingerprinting of T. pyogenes by the developed RAPD-PCR method is a reliable typing tool that may allow a better understanding of the epidemiology as well as pathogenesis of infections caused by this pathogen.

Selection of Polymorphic Patterns Obtained by RAPD-PCR through Qualitative and Quantitative Analyses to Differentiate Aspergillus fumigatus.

The objective of this work was to use the random amplification of the polymorphic DNA–polymerase chain reaction (RAPD-PCR) technique to select polymorphic patterns through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and A. tubingensis. Twenty-seven Aspergillus isolates from different species were typified using phenotypic (macro- and micromorphology) and genotypic (partial BenA gene sequencing) methods. Thirty-four primers were used to obtain polymorphic patterns, and with these a qualitative analysis was performed to select the primers that presented species-specific patterns to distinguish each species. For the quantitative selection, a database was built from the polymorphic patterns and used for the construction of logistic regression models; later, the model that presented the highest value of sensitivity against specificity was evaluated through ROC curves. The qualitative selection showed that the primers OPA-19, P54, 1253 and OPA-02 could differentiate the species. A quantitative analysis was carried out through logistic regression, whereby a species-specific correlation of sensitivity and specificity greater than 90% was obtained for the primers: OPC-06 with a 96.32% match to A. flavus; OPF-01 with a 100% match to A. fumigatus; OPG-13 with a 98.01% match to A. tubingensis; and OPF-07 with a 99.71% match to A. niger. The primer OPF-01 discriminated the four species as well as closely related species. The quantitative methods using the selected primers allowed discrimination between species and showed their usefulness for genotyping some of the species of medical relevance belonging to the genus Aspergillus.

Assessment of larvicidal and genotoxic potentials of extracts of Hyptis suaveolens against Culex quinquefasciatus based on enzyme profile and RAPD-PCR assay

Vector control strategies have focused on the development of effective and ecofriendly alternatives. In the present study, investigation of larvicidal and genotoxic effects of leaves of Hyptis suaveolens from four different extraction solvents (aqueous, hexane, methanol and acetone) on fourth instar larvae of Culex quinquefasciatus was carried out. Extraction was done using soxhlet apparatus and the characteristics functional group of active constituents were identified using Fourier Transform Infrared spectrophotometer. Larvicidal activities were screened using three different concentrations (50, 150 and 250 mg/mL) following WHO standard protocol and mortality was recorded after 24, 48 and 72 hr. Hexane extract showed the highest mortality (27.92, 38.75, 90.42 %; LC50: 272.5, 191.3, 114.8 mg/mL), followed by aqueous extract (20.83, 34.58, 59.58 %; LC50: 496.6, 392.9, 208.1 mg/mL) and acetone extract (20.83, 32.08, 59.58 %; LC50: 1111.2, 393.6, 266.1 mg/mL) and methanol extract (17.92, 29.17, 52.92 %; LC50: 466.0, 400.1, 272.3 mg/mL). Enzyme profile such as superoxide dismutase (SOD), glutathione-S-transferase (GST), catalase (CAT), glutathione (GSH) and alkaline phosphatase (ALP) were significantly altered in the larvae exposed to the four extracts. Phytochemical screening of all solvents extract revealed the presence of saponins, flavonoids, terpenoids and alkaloids as common constituents. Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) profile implied hexane and aqueous extracts altered the DNA of larvae. Furthermore, FTIR Spectroscopic analysis revealed phenols, alcohols, aliphatic primary amines and saponins as the major groups in the extracts. Conclusively, this study established the lethal potential of extracts of H. suaveolens as alternative plant-based and eco-friendly larvicide against Cx. quinquefasciatus.

Isolation and Identification of Lactic Acid Bacteria from Natural Whey Cultures of Buffalo and Cow Milk.

In southern Italy, some artisanal farms produce mozzarella and caciocavallo cheeses by using natural whey starter (NWS), whose microbial diversity is responsible for the characteristic flavor and texture of the final product. We studied the microbial community of NWS cultures of cow’s milk (NWSc) for the production of caciocavallo and buffalo’s milk (NWSb) for the production of mozzarella, both from artisanal farms. Bacterial identification at species and strain level was based on an integrative strategy, combining culture-dependent (sequencing of the 16S rDNA, species/subspecies-specific Polymerase Chain Reaction (PCR) and clustering by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) and culture-independent (next-generation sequencing analysis, NGS) approaches. Results obtained with both approaches showed the occurrence of five species of lactic acid bacteria in NWSb (Lactococcus lactis subsp. lactis, Lactobacillus fermentum, Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactobacillus helveticus) and five species in NWSc (Lc. lactis subsp. lactis, Enterococcus faecium, and S. thermophilus, Lb. helveticus, and Lb. delbrueckii), with the last two found only by the NGS analysis. Moreover, RAPD profiles, performed on Lc. lactis subsp. lactis different isolates from both NWSs, showed nine strains in NWSb and seven strains in NWSc, showing a microbial diversity also at strain level. Characterization of the microbiota of natural whey starters aims to collect new starter bacteria to use for tracing microbial community during the production of artisanal cheeses, in order to preserve their quality and authenticity, and to select new Lactic Acid Bacteria (LAB) strains for the production of functional foods.

Functional Characteristics and Molecular Identification of Interspecific Hybrids from Genus Capsicum

The taxonomy of the genus Capsicum has distinguished more than twenty species of peppers with only C. annuum L. being a vegetable of economic importance. The remaining species can, however, be used in breeding via interspecies hybridization as a source of valuable functional traits. The aim of the study was to obtain interspecific hybrids of peppers with increased agronomic potential and their molecular identification. Based on their agromorphological characteristics, the obtained hybrids (‘Anchi’ × C. frutescens L.)F1 and (‘Anchi’ × C. baccatum var. pendulum)F1 formed fruits that had the combined functional traits typical of the cultivar ‘Anchi’, with a high content of soluble solids and dry matter characteristic of pollinators. To identify hybrids and their parental forms, the random amplified polymorphic DNA polymerase chain reaction (PCR-RAPD) and inter simple sequence repeat polymerase chain reaction (PCR-ISSR) techniques were used. The polymorphic products obtained in both methods totalled 65% and 59%, respectively, and they allowed for the identification of all tested genotypes, excluded the possibility of self-pollination of ‘Anchi’ plants, and confirmed the hybrid characteristics of the crossbreed (‘Anchi’ × C. frutescens L.)F1.

DNA polymorphism and relationships among the three different riverine populations of spotted snakehead (Channa punctata)

The current study was aimed to investigate the DNA polymorphism of C. punctata from three different rivers of northern region of India. For this, C. punctata of river Ganga and its tributaries (river Gomti and river Ken) was carried out using RAPD-PCR (Random amplified polymorphic DNA-polymerase chain reaction). A total of 54 loci were amplified by five selected primers out of 20 having GC content of 60% to 70%. The fish population of river Ganga showed highest genetic polymorphism with 33 (61.11%) polymorphic loci out of 54 loci whereas a total of 55.56% and 46.30% DNA polymorphism were recorded in the fish populations of Ken and Gomti respectively. Maximum genetic identity and minimum genetic distance was recorded between the fish populations of Ganga and Ken showing that these two populations are more similar to each other than to Gomti population. Dendogram based on genetic distance using UPGMA also confirmed that Ganga and Ken population are closer to each other.

RAPD Fingerprinting and Genetic Diversity of Salmonella Spp. Isolated from Broiler and Layer Flocks in Karbala, Iraq.

Salmonellosis in poultry is one of the most significant bacterial infections causing mortality, reduced production, and serious economic losses. This study aimed to study the molecular diversity among Salmonella isolates and investigate the epidemiological spread of these bacteria in broiler and layer chicken flocks in five different farms in Karbala, Iraq, using random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR). In total, 217 cloac a swabs were collected from the farms, out of which 129 and 88 swabs were taken from broiler and layer chickens. The samples were screened by PCR for S. enterica subsp. enterica using primers specific for the invA gene. Afterward, RAPD-PCR with uniplex or multiplex octamer primers was applied to genotype the isolates. The incidence rate of Salmonella infections in broilers and layers was estimated to be 27.9% and 12.5%. The uniplex primers P2 and P3, along with the multiplex primers yielded discriminatory patterns. Moreover, the RAPD typing showed a diverse range of banding patterns of Salmonella spp. Dendrograms created through GelJ software revealed various Salmonella genotypes in broilers and layers. The RAPD-PCR could be used as an accurate and fast tool to identify genetic relatedness among Salmonella spp. The obtained results would assist researchers in epidemiological studies and controlling salmonellosis in poultry fields.

Antibiotic resistance and phylogenetic comparison of human, pet animals and raw milk Staphylococcus aureus isolates

The present study was conducted to compare the S. aureus isolates from different sources in the basis of resistance phenotypic and genotypic features and phylogenetic differences. Total of 70 S. aureus isolates (including 25 human, 25 raw milk and 20 pet animal isolates) were subjected to the antimicrobial susceptibility testing, polymerase chain reaction (PCR) detection of the resistance genes and DNA fingerprinting using random amplification of polymorphic DNA–PCR (RAPD-PCR) to survey the variability of the isolates. Among 70 S. aureus, 55 (78.5%) isolates were MRSA. The isolates showed the highest antibiotic resistance to methicillin, ampicillin and penicillin (78.5%) and showed the lowest resistance to ciprofloxacin (12.8%). ErmB and tetM resistance genes were present in all isolates and the vanA gene was not detected in any of the isolates. Thirteen distinct clusters were identified in RAPD-PCR fingerprinting. Statistical analysis showed that the isolates without resistance to antibiotics were significantly in associated with raw milk origin (P < 0.05). According to the results of the study, S. aureus strains with pets and raw milk origin are significant sources of antibiotic-resistant isolates such as MRSA. They are also carriers of resistance genes that can be transmit to human isolates and cause drug resistance in human infections. Identifying the source of these infections is possible with a reliable genotyping method such as RAPD-PCR.