Genetic diversity and virulence properties of caprine Trueperella pyogenes isolates.

BACKGROUND: Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in various animal species, including goats. So far, only limited knowledge of phenotypic and genotypic properties of T. pyogenes isolates from goats has been gathered. In our study, we characterized the phenotypic and genotypic properties of caprine T. pyogenes isolates and established their relationship by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). RESULTS: From 2015 to 2023, 104 T. pyogenes isolates were obtained from 1146 clinical materials. In addition, two T. pyogenes isolates were obtained from 306 swabs collected from healthy goats. A total of 51 T. pyogenes isolates were subjected to detailed phenotypic and genotypic characterization. The virulence genotype plo/nanH/nanP/fimA/fimC/luxS was predominant. All of the tested isolates showed the ability to form a biofilm but with different intensities, whereby most of them were classified as strong biofilm formers (72.5%). The high level of genetic diversity among tested caprine T. pyogenes isolates (19 different RAPD profiles) was observed. The same RAPD profiles were found for isolates obtained from one individual, as well as from other animals in the same herd, but also in various herds. CONCLUSIONS: This study provided important data on the occurrence of T. pyogenes infections in goats. The assessment of virulence properties and genetic relationships of caprine T. pyogenes isolates contributed to the knowledge of the epidemiology of infections caused by this pathogen in small ruminants. Nevertheless, further investigations are warranted to clarify the routes of transmission and dissemination of the pathogen.

Identification and Characterization of Human Breast Milk and Infant Fecal Cultivable Lactobacilli Isolated in Bulgaria: A Pilot Study.

During the last few decades, the main focus of numerous studies has been on the human breast milk microbiota and its influence on the infant intestinal microbiota and overall health. The presence of lactic acid bacteria in breast milk affects both the quantitative and qualitative composition of the infant gut microbiota. The aim of this study was to assess the most frequently detected cultivable rod-shaped lactobacilli, specific for breast milk of healthy Bulgarian women and fecal samples of their infants over the first month of life, in 14 mother-infant tandem pairs. Additionally, we evaluated the strain diversity among the most common isolated species. A total of 68 Gram-positive and catalase-negative strains were subjected to identification using the MALDI-TOF technique. Predominant cultivable populations belonging to the rod-shaped lactic acid bacteria have been identified as Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, and Limosilactobacillus reuteri. Also, we confirmed the presence of Lactiplantibacillus plantarum and Lactobacillus gasseri. Up to 26 isolates were selected as representatives and analyzed by 16S rRNA sequencing for strain identity confirmation and a phylogenetic tree based on 16S rRNA gene sequence was constructed. Comparative analysis by four RAPD primers revealed genetic differences between newly isolated predominant L. rhamnosus strains. This pilot study provides data for the current first report concerning the investigation of the characteristic cultivable lactobacilli isolated from human breast milk and infant feces in Bulgaria.

Analysis of antimicrobial resistance and genetic diversity of Acinetobacter baumannii in a tertiary care hospital in Haikou City.

This study addresses the distribution and antimicrobial resistance of Acinetobacter baumannii (A. baumannii) in a medical facility in Haikou City, aiming to provide essential insights for enhancing in-hospital treatment and prevention strategies. We conducted a retrospective analysis of 513 A. baumannii isolates collected from a tertiary care hospital in Haikou between January 2018 and December 2020, focusing on their antimicrobial resistance patterns. Random Amplified Polymorphic DNA (RAPD) analysis was performed on 48 randomly selected A. baumannii strains. Using Gel-pro4.0 and NTSYSspc2.10 software, we constructed dendrograms to assess the genetic diversity of these strains. Our results indicate that males between 60 and 70 years old are particularly vulnerable to A. baumannii infections, which are most frequently detected in sputum samples, with a detection rate exceeding 70%. Alarmingly, over 50% of the isolates were identified as multi-drug resistant. The RAPD-PCR fingerprinting cluster analysis demonstrated substantial genetic diversity among the strains. Using primer OPA-02 at a 45% similarity coefficient, the strains were categorized into four groups (A-D), with group A being predominant (39 strains). high-prevalence areas like the Neurosurgery and Intensive Care Medicine Wards require enhanced surveillance and targeted interventions to manage Group C infections effectively. Additionally, the varied presence of other groups necessitates customized strategies to address the specific risks in each ward. Similarly, primer 270 at a 52% similarity coefficient classified the strains into five groups (E-I), with group E being most common (36 strains). The study highlights a concerning prevalence of antimicrobial resistance, particularly multi-drug resistance, among A. baumannii strains in the Haikou hospital. The significant genetic diversity, especially within groups A and E, underscores the need for tailored hospital treatment protocols and prevention measures. These findings contribute to the growing body of research on antimicrobial resistance, emphasizing the urgent need for effective management strategies in healthcare settings.

Trueperella Pyogenes-Strain Diversity and Occurrence in Dairy Herds.

Trueperella (T.) pyogenes is a mastitis-causing pathogen formerly known to cause severe clinical mastitis (CM), especially during the summer, leading to milk losses and low recovery rates. Unfortunately, its transmission behavior within herds is unclear. The diversity and occurrence of T. pyogenes were monitored to gain an initial insight into the infection transmission behavior of T. pyogenes in dairy herds and to lay a foundation for following targeted investigations. CM milk samples were collected from German herds, and one Swedish farm was sampled for isolates from subclinical mastitis. All in all, 151 T. pyogenes isolates from 16 herds were isolated, identified by MALDI TOF analysis and typed with RAPD PCR. Of these, 17 isolates originated from subclinical mastitis cases. We found that T. pyogenes mastitis occurred year-round, and clinical mastitis cases were caused by multiple strains (31 affected animals/28 strains). Instances of multiple cows being infected with the same T. pyogenes strain were rare and typically only involved a small number of animals at a time. However, if several quarters of a cow were affected, it was likely the same strain. Unlike clinical infections, subclinical T. pyogenes infections, in one investigated farm, harbored a dominant strain. Additionally, we found that T. pyogenes infections tended to persist and stay within a herd for a minimum of 7 months in the same or different cows.

Evaluation of Primers OPF-01, P54, and 1253 to Identify A. fumigatus, A. flavus, and A. niger from Polymorphic Patterns Obtained by RAPD-PCR.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys.

BACKGROUND: Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. OBJECTIVES: The present study was conducted in isolation and identification of ORT from slaughtered turkeys. METHODS: Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence-associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. CONCLUSIONS: This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region-specific vaccines for future use.

Development of SCAR Markers for Genetic Authentication of Metarhizium acridum.

In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

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.

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.

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.

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.

Cloning, sequencing, and characterizing of soil antibiotic active-producing Streptomyces species-specific DNA markers.

Association of the antibiotic activity of the soil Streptomyces isolates to their genetic profiles analyzed through RAPD-PCR fingerprints prompted us here in this study to use the most common bands as specific markers to identify homologous proteins within these isolates by cloning, sequencing, and characterizing these markers. Six out of twelve DNA bands ranged between 600 and 1350 bp previously obtained by RAPD-PCR analysis were purified out of the RAPD gels, and then cloned into pGEM-T Easy vector system. Success of the cloning process was confirmed by digesting purified plasmids with EcoRI. The clones namely No. 54, 55, 20, 56, 57, and 58 were sequenced using the DNA BigDye Terminator Sequencing System utilizing the M13 primer. Results indicated that the size of the inserted sequences is 599, 566, 522, 870, 857, and 254 bp, in clones No. 54. 55, 20, 56, 57, and 58, respectively. Homologous proteins of the six cloned sequences generated by DNA blast software indicated that the highest score of protein homology was scored for clone No. 54 with 87 % homology to putative secreted pectate lyase [Streptomyces coelicolor A3(2)]. The other clones showed less homology with 77 % homology for the clones No. 55 and 56, 73 % homology for the clone No. 20, and 55 % homology for the clones No. 57 and 58. The association of homologous proteins to the reported RAPD pattern is confirmed here for the first time, and the resulting DNA cloned fragments deserve further molecular analysis.

Application of multi-functional lactic acid bacteria strains in a pilot scale feta cheese production.

Feta cheese is the most recognized Greek Protected Designation of Origin (PDO) product in the world. The addition of selected autochthonous lactic acid bacteria (LAB) strains to cheese milk as adjunct cultures is gaining more attention, since they can impact the nutritional, technological and sensory properties of cheeses, as well as improve the safety of the product. The aim of this study was to produce Feta cheese with enhanced quality and safety, and distinctive organoleptic characteristics by applying autochthonous lactic acid bacteria (LAB) with multi-functional properties as adjunct cultures. Feta cheeses were produced with the commercial lactococcal starter culture and the addition of 9 LAB strains (Lactococcus lactis SMX2 and SMX16, Levilactobacillus brevis SRX20, Lacticaseibacillus paracasei SRX10, Lactiplantibacillus plantarum FRX20 and FB1, Leuconostoc mesenteroides FMX3, FMX11, and FRX4, isolated from artisanal Greek cheeses) in different combinations to produce 13 cheese trials (12 Feta trials with the adjunct LAB isolates and the control trial). In addition, Feta cheese manufactured with FMX3 and SMX2 and control Feta cheese were artificially inoculated (4 log CFU/g) with Listeria monocytogenes (a cocktail of 4 acid or non-acid adapted strains). Cheese samples were monitored by microbiological and physicochemical analyses during ripening, and microbiological, physicochemical, molecular and sensory analyses during storage at 4°C. The results showed that after manufacture, the LAB population was ca. 9.0 log CFU/g at all samples, whereas during storage, their population declined to 6.5-7.0 log CFU/g. In the Listeria inoculated samples, Listeria was absent after 60 days (end of ripening) and after 90 days in the adjunct culture, and in the control trials, respectively. Moreover, the addition of selected strains, especially Lcb. paracasei SRX10, led to cheeses with desirable and distinctive organoleptic characteristics. Furthermore, randomly amplified polymorphic PCR (RAPD-PCR) molecular analysis confirmed that the multi-functional LAB strains were viable by the end of storage. Overall, the results of this study are promising for the use of autochthonous strains in various combinations with the commercial starter culture to satisfy industry requirements and consumer demands for traditional and high added value fermented products.

Understanding Age-Related Longitudinal Dynamics in Abundance and Diversity of Dominant Culturable Gut Lactic Acid Bacteria in Pastured Goats.

Understanding gut lactic acid bacteria (LAB) in healthy hosts is an important first step in selecting potential probiotic species. To understand the dynamics of LAB in healthy goats, a cohort of thirty-seven healthy new-born goat kids was studied over a ten-month period. Total LAB was quantified using SYBR green qPCR. Seven hundred LAB isolates were characterized using microscopy, M13 RAPD genotyping and 16S rDNA sequencing. The highest and lowest LAB counts were detected at one week and ten months of age, respectively. Diverse LAB species were detected, whose identity and prevalence varied with age. The main isolates belonged to Limosilactobacillus reuteri, Limosilactibacillus fermentum, Lactobacillus johnsonni, Ligilactobacillus murinus, Ligilactobacillus salivarius, Limosilactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus agilis, Lactobacillus acidophilus/amyolovolus, Pediococcus spp. and Enterococcus spp. Uniquely, L. reuteri and Pediococcus spp. were most common in pre- and peri-weaned goats, while Lactobacillus mucosae and Enterococcus spp. were predominant in goats one month and older. Based on RAPD genotyping, L. reuteri had the highest genotypic diversity, with age being a factor on the genotypes detected. This data may be relevant in the selection of age-specific probiotics for goats. The findings may also have broader implications by highlighting age as a factor for consideration in probiotic bacteria selection in other animal hosts.

Molecular Epidemiology of Bulgarian Clinically Significant Staphylococcus aureus Isolates.

Severe infections due to highly virulent and resistant Staphylococcus aureus pose a serious health threat in Bulgaria and worldwide. The purpose of this study was to explore the clonal spread of recent clinically significant methicillin-susceptible S. aureus (MSSA) isolates from inpatients and outpatients treated in three university hospitals in Sofia, Bulgaria, during the period 2016-2020 and evaluate the relationship between their molecular epidemiology, virulence profiling, and antimicrobial resistance. A total of 85 isolates (invasive and noninvasive) were studied using RAPD analysis. Ten major clusters (A-K) were identified. The first major cluster A (31.8%) was found to be predominant during 2016 and 2017 and was widespread in two hospitals, unlike its case in the following years, when it was found to be replaced by newer cluster groups. All MSSA members of the second most common cluster F (11.8%) were recovered from the Military Medical Academy, mainly during 2018-2020, and were determined to be susceptible to all other groups of antimicrobials, except for penicillins without inhibitors because they harboured the blaZ gene. The newer cluster I, with 9.4% of the isolates absent in 2016-2017, showed significantly higher virulence and macrolide resistance (42.9%) due to ermB and ermC. All the isolated MSSA in groups F and I were nosocomial and mostly invasive. In conclusion, this 5-year study demonstrates the molecular epidemiology of MSSA infections in three Bulgarian hospitals. Findings can be helpful for the understanding of staphylococcal infection distribution in hospital settings and their prevention.

Improved RAPD Method for Candida parapsilosis Fingerprinting.

Recently, methods based on the analysis of arbitrarily amplified target sites of genome microorganisms have been extensively applied in microbiological studies, and especially in epidemiological studies. The range of their application is limited by problems with discrimination and reproducibility resulting from a lack of standardized and reliable methods of optimization. The aim of this study was to obtain optimal parameters of the Random Amplified Polymorphic DNA (RAPD) reaction by using an orthogonal array as per the Taguchi and Wu protocol, modified by Cobb and Clark for Candida parapsilosis isolates. High Simpson's index values and low Dice coefficients obtained in this study indicated a high level of interspecies DNA polymorphism between C. parapsilosis strains, and the optimized RAPD method proved useful in the microbiological and epidemiological study.

Morphological and molecular comparison as a useful tool for identification of the three centric marine diatoms (Bacillariophyceae: Chaetoceros).

The objective of this study was to identify morphological and molecular comparison of three marine Chaetoceros species using microscopic observations, sequence analysis of 18S rDNA, random amplified polymorphic DNA (RAPD-PCR) barcoding and nuclear magnetic resonance (NMR) spectroscopy. Chaetoceros were obtained from three different algae laboratories: Center of Excellence for Marine Biotechnology (CEMB), Chanthaburi Coastal Fisheries Research and Development (CHAN) and Institute of Marine Science, Burapha University (BIM). Genomic DNA for the RAPD-PCR analysis was extracted using the phenol-chloroform method, followed by 18S rDNA amplification. The blast results of 18S rDNA sequence confirmed the significantly matched to C. gracilis for Chaetoceros BIM and CHAN and C. muelleri for Chaetoceros CEMB(e-value = 0.0, identity = 99%). The RAPD-PCR results revealed differences in the three Chaetoceros isolates with polymorphisms between 30.43% and 60.00%, and Chaetoceros CEMB showed high polymorphic bands. Scanning electron microscopy revealed that Chaetoceros CEMB were larger and had larger setae compared to the other isolates (P < 0.05). The results of the NMR characterization of metabolites were consistent with the results of the sequence and morphological analyses. The concentrations of several metabolites, including chlorophyll c1, chlorophyll a, Myo-inositol, fucoxanthin, astaxanthin, lutein and zeaxanthin, were lower in Chaetoceros CEMB than in Chaetoceros BIM and CHAN. However, high concentrations of fatty acids, such as oleic acid, linoleic acid, α-linolenic acid and arachidic acid, were observed in all isolates. Generally, the results of this study will aid future studies examining the diversity of Chaetoceros in various cultural environments.

Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.

Evaluation of Apical and Molecular Effects of Algae Pseudokirchneriella subcapitata to Cerium Oxide Nanoparticles.

Cerium oxide engineered nanoparticles (nCeO2) are widely used in various applications and are, also, increasingly being detected in different environmental matrixes. However, their impacts on the aquatic environment remain poorly quantified. Hence, there is a need to investigate their effects on non-target aquatic organisms. Here, we evaluated the cytotoxic and genotoxic effects of <25 nm uncoated-nCeO2 on algae Pseudokirchneriella subcapitata. Apical (growth and chlorophyll a (Chl a) content) and genotoxic effects were investigated at 62.5-1000 µg/L after 72 and 168 h. Results demonstrated that nCeO2 induced significant growth inhibition after 72 h and promotion post 96-168 h. Conversely, nCeO2 induced enhanced Chl a content post 72 h, but no significant changes were observed between nCeO2-exposed and control samples after 168 h. Hence, the results indicate P. subcapitata photosynthetic system recovery ability to nCeO2 effects under chronic-exposure conditions. RAPD-PCR profiles showed the appearance and/or disappearance of normal bands relative to controls; indicative of DNA damage and/or DNA mutation. Unlike cell recovery observed post 96 h, DNA damage persisted over 168 h. Thus, sub-lethal nCeO2-induced toxicological effects may pose a more serious threat to algae than at present anticipated.

Agarose Gel Electrophoresis-Based RAPD-PCR-An Optimization of the Conditions to Rapidly Detect Similarity of the Alert Pathogens for the Purpose of Epidemiological Studies.

Agarose gel electrophoresis is a well-known tool to detect DNA fragments amplified in polymerase chain reaction (PCR). Its usefulness has also been confirmed for epidemiological studies based on restriction fragments length polymorphism (RFLP), usually performed using pulsed-field gel electrophoresis (PFGE). Little is known on the effectiveness for alert-pathogen epidemiological studies of another less time-consuming and costly technique called randomly amplified polymorphic DNA-PCR (RAPD-PCR). Meanwhile, its usefulness is believed to be comparable to RFLP-PFGE. Therefore, the aim of the study was to establish and optimize the conditions of agarose gel electrophoresis following RAPD-PCR for 19 Enterococcus faecium strains derived from epidemic outbreaks at intensive care units. An application of different PCR primers, primer combinations, and, in particular, agarose gel concentrations and electrophoresis conditions revealed the usefulness of this relatively fast and inexpensive method based on RAPD-PCR for epidemiological studies without a compulsion to use the specialized equipment necessary for RFLP-PFGE.