Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

Molecular typing and virulence characteristics of Escherichia coli strains isolated from hospital and community acquired urinary tract infections.

BACKGROUND: The main causes of hospital- and community-acquired urinary tract infections (UTIs) are a group of Escherichia coli (E. coli) strains with multiple virulence factors known as uropathogenic E. coli. METHODS AND RESULTS: One hundred E. coli isolates from the urine specimens of hospital- and community-acquired UTI patients were characterized based on their virulence factors and genetic relatedness using PCR and RAPD‒PCR, respectively. Among all, the traT (71%), sitA (64%), ompT (54%), malX (49%), ibeA (44%), tsh (39%), hlyD (18%) and cnf1 (12%) genes had the highest to lowest frequencies, respectively. There was no significant difference between the frequency of tested virulence genes in E. coli isolates from inpatients and outpatients. The frequency of the hlyD gene was significantly greater in E. coli isolates from patients hospitalized in gynecology, dermatology and intensive care unit (ICU) wards than in those from other wards. Eight virulence gene patterns were common among the isolates of inpatients in different wards of the same hospital, of which five patterns belonged to the isolates of inpatients in the same ward. More E. coli isolates with similar virulence gene patterns and greater genetic similarity were found in female patients than in male patients. The analysis of the RAPD‒PCR dendrograms revealed more genetic similarities among the E. coli isolates from inpatients than among those from outpatients. CONCLUSION: Our findings indicate the presence of a wide variety of virulence factors in E. coli isolates and the possibility of spreading the same clones in different wards of the hospital.

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.

Biodiversity and antibiotic resistance profile provide new evidence for a different origin of enterococci in bovine raw milk and feces.

Enterococci are widely distributed in dairy sector. They are commensals of the gastrointestinal tract of animals, thus, via fecal contamination, could reach raw milk and dairy products. The aims of this study were: 1) to investigate the enterococcal diversity in cow feces and milk samples and 2) to evaluate the antibiotic resistance (AR) of dairy-related enterococci and their ability to transfer resistance genes. E. faecalis (59.9%), E. faecium (18.6%) and E. lactis (12.4%) were prevalent in milk, while E. faecium (84.2%) and E. hirae (15.0%) were dominant in bovine feces. RAPD-PCR highlighted a high number of Enterococcus biotypes (45 from milk and 37 from feces) and none of the milk strains exhibited genetic profiles similar to those of feces biotypes. A high percentage of enterococci isolated from milk (71%) were identified as multidrug resistant and resistance against streptomycin and tetracycline were widespread among milk strains while enterococci from feces were commonly resistant to linezolid and quinupristin/dalfopristin. Only E. faecalis strains were able to transfer horizontally the tetM gene to Lb. delbrueckii subsp. lactis. Our results indicated that Enterococcus biotypes from milk and bovine feces belong to different community and the ability of these microorganisms to transfer AR genes is strain-dependent.

Enhanced ε­poly­L­lysine production in Streptomyces species by combining interspecific hybridization with multiple antibiotic resistance.

To improve the ε-PL production in wild-type strains of Streptomyces. albulus, Streptomyces. noursei, Streptomyces. rochei and Streptomyces. yunnanensis, the interspecific hybridization based on protoplast fusion was first performed. Two-species hybridizations failed to obtain hybrids with significant increase in ε-PL production, but four-species hybridizations succeed in acquiring many high-yield hybrids. 16S rDNA homology alignment and RAPD confirmed that the hybrid HX17 was restructured by integrating gene fragments from S. albulus and S. rochei with S. noursei as the carrier. S. noursei HX17 was subsequently suffered from mutagenesis and genome shuffling combining with multiple antibiotic resistance, and a mutant S. noursei GX6 was obtained with ε-PL yield of 2.23 g/L in shake-flask fermentation. In fed-batch fermentation, the ε-PL production of GX6 reached 47.2 g/L, which was increased by 95.6% to 136.8% over the wild parents. Ribosomal genes associated with antibiotics were sequenced and majority of mutant strains had mutations at different sites, indicating that the increase of antibiotic resistance was strongly associated with them. This research proved that combining interspecific hybridization with multiple antibiotic resistance was as an effective approach to rapidly improve the ε-PL production in Streptomyces species.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Gardnerella vaginalis ventilatory acquired pneumonia among patients with trauma.

Gardnerella vaginalis (G. vaginalis) is a bacterium rarely responsible for systemic infections and is exceptionally isolated from bronchopulmonary samples. Here, we report here two patients with trauma who were diagnosed with a G. vaginalis ventilatory acquired pneumonia (VAP) via mini bronchoalveolar lavage (mini-BAL). According to our observations, G. vaginalis was the only microorganism with a significant threshold and the identification was obtained by a reliable mean. There is no recommendation for antibiotic treatment for invasive G. vaginalis infection. We treated these infections with Cefotaxim and Metronidazole which clinically improved the infection. To determine whether the two patients were infected by the same strain, we used a random amplified polymorphic DNA (RAPD) technique. The two G. vaginalis organisms had distinct RAPD profiles, suggesting the absence of cross-transmission. These two cases of trauma and G. vaginalis VAP suggest that this infection cannot be ruled out and should alert the clinician to treat it.

Determination of antibiotic resistance patterns and genotypes of Escherichia coli isolated from wild birds.

BACKGROUND: Curbing the potential negative impact of antibiotic resistance, one of our era's growing global public health crises, requires regular monitoring of the resistance situations, including the reservoir of resistance genes. Wild birds, a possible bioindicator of antibiotic resistance, have been suggested to play a role in the dissemination of antibiotic-resistant bacteria. Therefore, this study was conducted with the objective of determining the phenotypic and genotypic antibiotic resistance profiles of 100 Escherichia coli isolates of gull and pigeon origin by using the Kirby-Bauer disk diffusion method and PCR. Furthermore, the genetic relationships of the isolates were determined by RAPD-PCR. RESULTS: Phenotypic antibiotic susceptibility testing revealed that 63% (63/100) and 29% (29/100) of E. coli isolates were resistant to at least one antibiotic and multidrug-resistant (MDR), respectively. With the exception of cephalothin, to which the E. coli isolates were 100% susceptible, tetracycline (52%), kanamycin (38%), streptomycin (37%), ampicillin (28%), chloramphenicol (21%), trimethoprim/sulfamethoxazole (19%), gentamicin (13%), enrofloxacin (12%) and ciprofloxacin (12%) resistances were detected at varying degrees. Among the investigated resistance genes, tet(B) (66%), tet(A) (63%), aphA1 (48%), sul3 (34%), sul2 (26%), strA/strB (24%) and sul1 (16%) were detected. Regarding the genetic diversity of the isolates, the RAPD-PCR-based dendrograms divided both pigeon and gull isolates into five different clusters based on a 70% similarity threshold. Dendrogram analysis revealed 47-100% similarities among pigeon-origin strains and 40-100% similarities among gull-origin E.coli strains. CONCLUSIONS: This study revealed that gulls and pigeons carry MDR E. coli isolates, which may pose a risk to animal and human health by contaminating the environment with their feces. However, a large-scale epidemiological study investigating the genetic relationship of the strains from a "one health" point of view is warranted to determine the possible transmission patterns of antibiotic-resistant bacteria between wild birds, the environment, humans, and other hosts. Video Abstract.

Phytotoxicity and genotoxicity study of reactive red 141 dye on mung bean (Vigna radiata (L.) Wilczek) seedlings.

BACKGROUND: Reactive Red (RR) 141 dye is widely used in various industrial applications, but its environmental impact remains a growing concern. In this study, the phytotoxic and genotoxic effects of RR 141 dye on mung bean seedlings (Vigna radiata (L.) Wilczek) were investigated, serving as a model for potential harm to plant systems. METHODS AND RESULTS: Short-term (14 days) and long-term (60 days) experiments in paddy soil pot culture exposed mung bean seedlings to RR 141 dye. The dye delayed germination and hindered growth, significantly reducing germination percentage and seedling vigor index (SVI) at concentrations of 50 and 100 ml/L. In short-term exposure, plumule and radical lengths dose-dependently decreased, while long-term exposure affected plant length and grain weight, leaving pod-related parameters unaffected. To evaluate genotoxicity, high annealing temperature-random amplified polymorphic DNA (HAT-RAPD) analysis was employed with five RAPD primers having 58-75% GC content. It detected polymorphic band patterns, generating 116 bands (433 to 2857 bp) in plant leaves exposed to the dye. Polymorphisms indicated the appearance/disappearance of DNA bands in both concentrations, with decreased genomic template stability (GTS) values suggesting DNA damage and mutation. CONCLUSION: These findings demonstrate that RR 141 dye has a significant impact on genomic template stability (GTS) and exhibits phytotoxic and genotoxic responses in mung bean seedlings. This research underscores the potential of RR 141 dye to act as a harmful agent within plant model systems, highlighting the need for further assessment of its environmental implications.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

BACKGROUND: It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. METHODS: Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. RESULTS: The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41 µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100 mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1 pg/µl while root gDNA remained amplifiable at 10 pg/µl. Genomic DNA obtained was also suitable for sequencing. CONCLUSION: This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Fine mapping of a new common bean anthracnose resistance gene (Co-18) to the proximal end of Pv10 in Indian landrace KRC-5.

KEY MESSAGE: Mapping and fine mapping of bean anthracnose resistance genes is a continuous process. We report fine mapping of anthracnose resistance gene Co-18 which is the first anthracnose gene mapped to Pv10. The discovery of resistance gene is a major gain in the bean anthracnose pathosystem research. Among the Indian common bean landraces, KRC-5 exhibit high levels of resistance to the bean anthracnose pathogen Colletotrichum lindemuthianum. To precisely map the anthracnose resistance gene, we used a Recombinant Inbred Line (F2:9 RIL) population (KRC-5 × Jawala). The inheritance test revealed that KRC-5 carries a dominant resistance gene temporarily designated as Co-18. We discovered two RAPD markers linked to Co-18 among 287 RAPD markers. These RAPD markers were eventually developed into SCARs (Sc-OPR15 and Sc-OPF6) and flank Co-18 on chromosome Pv10 at a distance of 5.3 and 4.2 cM, respectively. At 4.0-4.1 Mb on Pv10, we detected a SNP (single-nucleotide polymorphism) signal. We synthesized 58 SSRs and 83 InDels from a pool of 135 SSRs and 1134 InDels, respectively. Five SSRs, four InDels, and two SCARs were used to generate the high-density linkage map, which led to the identification of two SSRs (SSR24 and SSR36) that are tightly linked to Co-18. These two SSRs flank the Co-18 to 178 kb genomic region with 13 candidate genes including five NLR (nucleotide-binding and leucine-rich repeat) genes. The closely linked markers SSR24 and SSR36 will be used in cloning and pyramiding of the Co-18 gene with other R genes to develop durable resistant bean varieties.

Involvement of UV-C-induced genomic instability in stimulation рlant long-term protective reactions.

The study of the mechanisms affecting single stress factor impact on long-term metabolic rearrangements is necessary for understanding the principles of plant protective reactions. The objective of the study was to assess the involvement of UV-C-induced genomic instability in induction рlant long-term protective reactions. The study was carried out on two genotypes of chamomile, Perlyna Lisostepu (PL) variety and its mutant, using UV-C pre-sowing seed radiation exposure at dose levels 5-15 kJ/m2. Multiple DNA damages under different exposure doses were studied on plant tissues during the flowering stage using - ISSR-RAPD DNA marker PCR. In the cluster analysis of changes within the amplicon spectra as an integral group the Jacquard similarity index was used. The results of the study suggest that genomic instability is a link between the direct effects of UV-C exposure and stimulation of metabolic rearrangements at the final stages of ontogeny. A hypothetical scheme for the transformation of primary UV-C DNA damage into long-term maintenance of genomic instability signs has been proposed.

Mitigation of arsenic poisoning induced oxidative stress and genotoxicity by Ocimum gratissimum L.

Arsenic toxicity is a major problem across the world due to geogenic activity and has been supposed to generate free radicals and genotoxicity among the arsenic-poisoned population. There is a need to find suitable free radical quenching compounds for the arsenic-induced free radical-affected population. In the present study, Na3AsO3- induced oxidative stress and genotoxicity were evaluated in Oryctolagus cuniculus L, and quenching competency of Ocimum species was examined by applying enzymatic and non-enzymatic in vitro tests, comet assay, and Random Amplified Polymorphic Deoxyribonucleic acid - Polymerase Chain Reaction (RAPD-PCR) methods. In the present study, oxidative damage due to Na3AsO3 intoxication in O. cuniculus L has been confirmed followed by substantive genotoxicity, and in a further study, it has also been reported that the extract of O. gratissimum L lowers the oxidative stress in experimental animals confirmed by a decrease in Malondialdehyde (MDA) 4.78 ± 0.05 (nmol/mg protein), and an increase in Glutathione (GSH) 2.87 ± 0.50 (µmoles/mg proteins), Superoxide Dismutase (SOD) 1.78 ± 0.03(Units/mg protein), Catalase (CAT) 2.72 ± 0.02 (µmoles of H2O2 consumed/min/mg proteins) and Glutathione peroxidase (GPX) 7.43 ± 0.01 (µg of glutathione utilized/min/mg protein). A positive impact of extract of O. gratissimum L on protection of genotoxicity has been also confirmed by Random Amplified Polymorphic DNA (RAPD) based reduction in polymorphic bands of Deoxyribonucleic acid (DNA) from 6.5 to 3.16 and comet assay-based increase in head DNA % (87.86 ± 1.58), tail moment (1.07 ± 0.27) and decrease in tail DNA % (12.13 ± 1.58) & tail length (8.2 ± 1.46) at 5% P in lymphocytes. A significant level reduction in free radicals and reduction in DNA polymorphism has proved the competency of test material for the development of suitable antidotes against arsenicosis.

Metal accumulation and genetic adaptation of Oryza sativa to Cadmiun and Chromium heavy metal stress: A hydroponic and RAPD analyses.

This research was performed to assess the influence of Cd and Cr metals on growth, pigments, antioxidant, and genomic stability of Oryza sativa indica and Oryza sativa japonica were investigated under hydroponic conditions. The results revealed that significant metal influence on test crop growth, pigment content, metal stress balancing antioxidant activity in a dose dependent manner. Since, while at elevated (500 ppm) concentration of Cd as well as Cr metals the pigment (total chlorophyll, chlorophyll a, b and carotenoids) level was reduced than control; however antioxidant activity (total antioxidant, H2O2, and NO) was considerably improved as protective mechanisms to combat the metal toxicity and support the plant growth. Furthermore, the test crops under typical hydroponic medium (loaded with Cd and Cr as 200, 300, 400, and 500 ppm) growth conditions, effectively absorb the metals from medium and accumulated in the root and least quantity was translocated to the shoot of this test crops. Furthermore, typical RAPD analysis with 10 universal primers demonstrated that the genomic DNA of the test crops was adaptable to develop metal resistance and ensure crop growth under increased concentrations (500 ppm) of tested heavy metals. These findings suggest that these edible crops have the ability to accumulate Cd along with Cr metals, and additionally that their genetic systems have the ability to adapt to metal-stressed environments.

Characterization of Coniella granati Isolates Causing Pomegranate Decline in Italy.

Coniella granati, the causal agent of pomegranate crown rot, twig blight, and fruit decay, is an emerging worldwide pathogen with a heavy impact on pomegranate cultivation. In this study, we report the rapid spread of the fungus in Italian pomegranate orchards associated with crown rot symptoms and provide new results on fungal development, baseline sensitivity to different fungicides, and intraspecific variability by analyzing 11 isolates, representative of populations of the pathogen from comparable pomegranate orchards in different regions of Italy. In vitro assays showed that 25 to 30°C was the optimal range for both colony growth and conidial germination, corroborating the results previously obtained for Californian and Greek isolates. According to the baseline sensitivity assay on the response of colony growth and conidial germination to 10 fungicides, fludioxonil, thiophanate-methyl, tebuconazole, and cyprodinil were the most effective. Random amplified polymorphic DNA (RAPD) analysis, carried out using fourteen 10-mer primers, showed very low intraspecific variability (similarity coefficient >0.95), probably as a result of the recent spread of the pathogen in Italy and the uncommon occurrence of the sexual process as a source of genetic variability. In summary, this study provides new knowledge on C. granati that will be helpful for improving pomegranate crown rot management.

In vitro Genotoxicity and In silico Docking Analyses of the Essential Oils of Thuja orientalis.

Two main objectives were pursued to assess the reliability of Thuja orientalis essential oils (TOEO). The first objective was to extract TOEO, analyze them by GC-MS, and determine their in vitro genotoxicity against selected plants using the RAPD-PCR method. The second objective was to evaluate the in-silico toxicity of TOEO. The binding sites and energies of each content was calculated against B-DNA. In-silico analyses were performed using a simulation program, AutoDock Vina, and Toxicity Estimation Software Tools. 3-carene, cedrol, and 2-pinene were identified as the predominant components. In vitro studies showed that the TOEO had a more significant impact on reducing genomic stability in wheat compared to the amaranth. The lowest stability was determined as 39.78 % in wheat and 53.58 % in amaranth. Cedrol (-5,7 kcal/mol) and selinene (-5,6 kcal/mol) exhibited the highest binding affinity. The toxicity test indicated that components other than cyclohexene may have toxic effects, none of them were predicted to be mutagenic, and LD50 (mol/kg) values could vary between 1.33 and 1.55.

Antimicrobial resistance and epidemic clustering of late-onset neonatal infections in a Brazilian intensive care unit.

Nosocomial infections in the neonatal intensive care unit (NICU) tend to cluster and multidrug-resistant (MDR) pathogens are rising in developing countries. We did a retrospective cohort study of neonates admitted to a NICU in Brazil with late-onset neonatal sepsis (LOS) confirmed by blood culture from October 2012 to December 2016 and from July 2018 to December 2021. We defined a cluster of infection when at least two cases of LOS occurred within two different time intervals: 15 and 30 days with the same pathogen in different patients. A random amplified polymorphic DNA (RAPD) was performed from samples from one of these clusters. A logistic regression model was applied having death as the outcome and the infection with an MDR pathogen as the exposure of interest. There were 987 blood cultures from 754 neonates, 621 (63%) were gram-positive cocci, 264 (30%) were gram-negative rods and 72 (7%) fungi. A third of Enterobacterales were resistant to cefepime and a third of non-fermenting glucose rods were resistant to carbapenems. There were 100 or 104 clusters of infection in the 15- or 30-day interval, respectively. A RAPD analysis from an outbreak of MDR Acinetobacter baumannii showed that all five samples belonged to a single clone. An infection with an MDR pathogen was associated with death (OR 1.82, 95% CI 1.03-3.21). In conclusion, clusters of infections in a Brazilian NICU are a frequent phenomenon as seen elsewhere. They suggest cross-transmission of pathogens with increasing antimicrobial resistance and should prompt intensified surveillance and infection control measures.

Comparative genomics analysis of Streptococcus iniae isolated from Trachinotus ovatus: novel insight into antimicrobial resistance and virulence differentiation.

BACKGROUND: Streptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24. RESULTS: The results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24. CONCLUSION: Comparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.

Mutagenic effects of a commercial glyphosate-based herbicide formulation on the soil filamentous fungus Aspergillus nidulans depending on the mode of exposure.

Glyphosate-based herbicides (GBH) are the most used pesticides worldwide. This widespread dissemination raises the question of non-target effects on a wide range of organisms, including soil micro-organisms. Despite a large body of scientific studies reporting the harmful effects of GBHs, the health and environmental safety of glyphosate and its commercial formulations remains controversial. In particular, contradictory results have been obtained on the possible genotoxicity of these herbicides depending on the organisms or biological systems tested, the modes and durations of exposure and the sensitivity of the detection technique used. We previously showed that the well-characterized soil filamentous fungus Aspergillus nidulans was highly affected by a commercial GBH formulation containing 450 g/L of glyphosate (R450), even when used at doses far below the agricultural application rate. In the present study, we analysed the possible mutagenicity of R450 in A. nidulans by screening for specific mutants after different modes of exposure to the herbicide. R450 was found to exert a mutagenic effect only after repeated exposure during growth on agar-medium, and depending on the metabolic status of the tested strain. The nature of some mutants and their ability to tolerate the herbicide better than did the wild-type strain suggested that their emergence may reflect an adaptive response of the fungus to offset the herbicide effects. The use of a non-selective molecular approach, the quantitative random amplified polymorphic DNA (RAPD-qPCR), showed that R450 could also exert a mutagenic effect after a one-shot overnight exposure during growth in liquid culture. However, this effect was subtle and no longer detectable when the fungus had previously been repeatedly exposed to the herbicide on a solid medium. This indicated an elevation of the sensitivity threshold of A. nidulans to the R450 mutagenicity, and thus confirmed the adaptive capacity of the fungus to the herbicide.

Molecular genotyping of Salmonella spp. isolated from cheese samples of local stores in Al-Diwaniyah city, Iraq.

Background: Food safety is an important subject that the global cheese industry increases awareness of. This urges these economic sectors to elevate the level of research to minimize cheese contamination with pathogenic bacteria, such as Salmonella. Aim: Based on these merits, this study was conducted to genotype Salmonella spp. isolated from cheese samples of local stores in Al-Diwaniyah City, Iraq. Methods: The study used 41 samples of local fresh unsalted white cheese in a selective-growth-based isolation of Salmonella. These isolates were confirmed utilizing a slide-agglutination (SA) test and VITEK® 2 system (V2S). Then, the isolates were subjected to conventional PCR and sequencing techniques that both targeted the 16S rRNA gene. For subtyping, the Salmonella isolates were subjected to a random amplified polymorphic DNA (RAPD)-PCR method. Results: The results of both SA and V2S revealed the presence of 14 (34.2%) isolates of Salmonella spp. in the cheese samples. The PCR confirmed 6 (42.9%) of these isolates, which further were defined with close nucleotide similarity (98.03%) and (97.88%) to different world isolates, such as Salmonella enterica subsp. Arizonae and Salmonella enterica subsp. enterica serovar Typhi, respectively. The RAPD-PCR findings showed different fragments for all the tested isolates. Conclusion: The present study indicates that the samples of the local fresh unsalted white cheese contain different Salmonella genotypes, which could be originated from different contamination sources.