Population genetic and evolution analysis of Vibrio isolated from Turkish fish farms

The genus Vibrio includes important pathogenic species for human and aquatic organisms such as Vibrio cholera, V. parahaemolyticus, V. vulnificus, V. anguillarum or V. harveyi. At present, Vibrionaceae family consists of >190 described species, classified into nine genera. Vibrio are widespread in shellfish, finfish and marine ecosystems and show resourceful ecologies, which recognized as one of the most diverse bacterial groups for illuminating the genome evolution. In the present study, to clarify the relationship among aquatic species in the genus, a multilocus sequence analysis (MLSA) and typing (MLST) approach was applied to characterize 51 Vibrio isolates from Turkish fish farms, 146 strains deposited in the PubMLST database and 59 type strains from GenBank. For all studied isolates (n = 256), diversity analysis, population structure, determination of recombination, demographic history and gene flow were performed based on the MLST scheme. Vibrio isolates, subjected to the study, showed a high diversity within the Vibrio population and also genetic interactions into the genus. We found 17 new described sequence types by MLST analysis that were isolated from rainbow trout, sea bream and sea bass in Turkish fish farms, which clearly indicate that the genes underwent recombination frequently. While predominant sequence types were found in the presented study, differences of genotypes need to be evaluated in a disease situation or preventing measurements. The findings about genetic recombination possibly helps to understand differences of Vibrio infections in fish. Furthermore, elucidating of genetic variability within species shed light on providing effective measurements in aquaculture by vaccine production and drug applicationsThis research was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) [No: 118O420]. This research was carried out in accordance with the ethical standards of the 2012-14/04 Local Ethics Commission report. We are pleased to Dollvet Veterinary Vaccine Pharmaceutical Biological Substance Production Industry and Trade Inc. for supporting us with strainsS

Plazmidi s višestrukom antibiotičkom rezistencijom, koji se mogu upotrijebiti za kloniranje bez pojave lažnih transformanata i genetičku modifikaciju pomoću fragmenata DNA umnoženih metodom PCR

We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.Konstruirali smo dva plazmida koji se mogu upotrijebiti za kloniranje, kao kalupi za inaktivaciju gena pomoću fragmenata DNA umnoženih metodom PCR, mutagenezu i konstrukciju kaseta za indukciju kromosomskih translokacija. Prema našim spoznajama ovo su prvi plazmidi koji istodobno omogućavaju selekciju bakterija na ampicilinu, kanamicinu i higromicinu, te selekciju kvasca Saccharomyces cerevisiae na geneticinu (G418) i higromicinu B. Istodobno korištenje do tri antibiotičke rezistencije omogućuje precizni odabir rekombinanata i gotovo potpunu eliminaciju lažno pozitivnih transformanata, dok jedinstvena restrikcijska mjesta olakšavaju kloniranje fragmenata DNA. Osim toga, ove smo plazmide uspješno upotrijebili kao kalupe za sintezu fragmenata DNA umnoženih metodom PCR, pomoću kojih je inducirana kromosomska translokacija u kvascu sustavom BIT (engl. bridge-induced translocation). Stanice u kojima smo potaknuli translokaciju imale su očekivane kromosomske rearanžmane i bile su rezistentne na G418 i higromicin B. Plazmidi konstruirani u ovom radu mogu se jednostavno primijeniti u mnogim molekularno-biološkim istraživanjima

Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications

Improved Gustatory Sensitivity in Morbidly Obese Patients After Laparoscopic Sleeve Gastrectomy

Introduction: The reduction in the preferences for sweet and fat containing tastes in obese patients who underwent bariatric surgery was relatively well shown; however, there are only limited data on the changes in the sensitivity of other tastes like sour, salty, and bitter

Expression of pro-apoptotic and anti-apoptotic proteins in granulosa cells of women with diminished ovarian reserve

Purpose To evaluate the expressions of caspase-3 and cytochrome c and heat shock protein 70 (Hsp70) in granulosa cells (GCs) from women with normal ovarian reserve (NOR) and diminished ovarian reserve (DOR) undergoing intracytoplasmic sperm injection (ICSI). Methods GCs were collected from 117 infertile women during oocyte retrieval. Patients were classified into four groups as follows: DOR-COC score of 0, DOR-COC score of I, NOR-COC score of 0, and NOR-COC score of I. The caspase-3, cytochrome c, and Hsp70 analyses were performed immunohistochemically in GCs. The ICSI outcomes were evaluated prospectively. Results The clinical pregnancy and live birth rates were higher in DOR-COC score of I (15, 30.6%; 14, 38.9%) and NOR-COC score of I (19, 38.77%; 19, 52.7%) groups, compared with DOR-COC score of 0 (12, 24.4%; 3, 6.1%) and NOR-COC score of 0 (3, 6.1%; 0%) groups (p = 0.0001; 0.00002), respectively. Caspase-3 and cytochrome c expression levels were higher in DOR-COC score of 0 (23, 65.7%; 25, 71.4%) and NOR-COC score of 0 groups (19, 61.3%; 20, 64.5%), compared with DOR-COC score of I (8, 32%; 9, 36%) and NOR-COC score of I groups (7, 26.9%; 8, 30.8%) (p = 0.00297; p = 0.002), respectively. Lower expression levels of Hsp70 were found in DOR-COC score of 0 (11, 31.4%) and NOR-COC score of 0 groups (10, 32.3%), compared with DOR-COC score of I (16, 64%) and NOR-COC score of I groups (20, 76.9%) (p = 0.001), respectively. Hsp70 expression levels were positively correlated with the number of day 3 good-quality embryo and negatively correlated with estradiol levels in the DOR group. Conclusion Our data suggest that COC score of 0 is associated with increased expression levels of apoptotic proteins, decreased expression levels of anti-apoptotic protein, and poor ICSI clinical outcomes in women with and without DOR

Postpartum Bilateral Sacroiliitis caused by Brucella Infection

Early diagnosis of this septic sacroiliitis is difficult because symptoms are nonspecific during the postpartum period. In this case we dicscuss about a patient with bilateral buttock pain unresolved with painkillers and rest, after an induction delivery. A 31-year-old woman was presented to our clinic on the second week of postpartum period with bilateral buttock pain. She was subfebrile and had no apparent abnormality on her pelvic X-ray. The pain was so severe that she was unable to walk properly. Sacroiliac MRI during the acute episode of pain showed bone marrow oedema and fluid within the bilateral sacroiliac joint. She was found seropositive for brucellosis and the patient completely recovered with antibiotherapy treatment. We stopped our patient from breastfeeding when the Rose Bengal test turned out positive. Brucella sacroiliitis should be considered in puerperium period women when buttock pain and difficulty in walking are present and pain is unresponsive to analgesics

Description of a Novel Fish Pathogen, Plesiomonas shigelloides subsp. oncorhynchi, Isolated from Rainbow Trout (Oncorhynchus mykiss): First Genome Analysis and Comparative Genomics

Plesiomonas shigelloides is the only species in its genus, and has zoonotic importance due to the serious implications resulting from the consumption of contaminated seafood. This is the first report on the genomic features of the whole-genome sequence (WGS) of P. shigelloides strain V-78, recovered from diseased rainbow trout, Oncorhynchus mykiss. The genome of P. shigelloides V-78 consists of 4,478,098 base pairs (bp), which encode 3730 proteins, and has a G + C content of 51.1%. The bioinformatics analysis of WGS of V-78 confirmed the presence of 121 tRNA genes and 42 rRNA genes (15 genes for 5S rRNA, 13 genes for 16S rRNA, and 14 genes for 23S rRNA). Comprehensive genome analyses revealed that the strain encodes for secondary metabolites, antimicrobial resistance, and virulence genes. The strain V-78 has 31 known antibiotic resistance models, which encode many antimicrobial resistances. In addition, strain V-78 has 42 different virulence genes, such as adhesion, secretion system, and motility. The digital DNA–DNA hybridization value against P. shigelloides NCTC 10360 was 74.2%, while the average nucleotide identity value was 97.1%. Based on the scrutinized analysis of genomic data, strain V-78 should be considered a novel subspecies of P. shigelloides, for which Plesiomonas shigelloides subsp. oncorhynchi is proposed.This research was funded by Bursa Uludag University grant number TGA-2022-1052.Peer reviewe

Diversidad del género Pseudomonas en piscifactorías de Turquía

Trabajo presentado en la XVIII Reunión del Grupo de Taxonomía, Filogenia y Biodiversidad de la Sociedad Española de Microbiología (XVIII TAXON), celebrada online del 21 al 23 de octubre de 2020.Pseudomonas es el género que presenta el mayor número de especies dentro de las bacterias Gram negativas. Se distinguen varios grupos filogenéticos, siendo el de Pseudomonas fluorescens el mayoritario. Se ha realizado un estudio de la presencia de especies del género Pseudomonas en centros de acuicultura de Turquía entre los años 2013 a 2018. Se han aislado 90 cepas de Pseudomonas en agar TSA y agar sangre cultivadas a 28 ºC durante 24-48 horas. Los análisis por secuencia del ARN ribosómico 16S indicaron que 51 de los aislados correspondían a especies conocidas del género Pseudomonas. El análisis del gen de la subunidad D del factor sigma 70, rpoD, puso de manifiesto la presencia de posibles nuevas especies. El análisis multigénico de secuencias (MLSA) de los genes ARN ribosómico 16S, gyrB, rpoB y rpoD permitió identificar 51 aislados mayoritariamente dentro del grupo fluorescens: 18 P. haemolytica, 12 P. meridiana, 8 P. lactis, 3 P. migulae, 3 P. weihenstephanensis, 2 P. mandelii, 1 P. fluorescens, 1 P. simiae, 1 P. lurida, 1 P. lundensis y 1 P. proteolytica. Otros 29 aislados presentaron porcentajes entre 90-96 % en el análisis de semejanza de sus secuencias nucleotídicas del gen rpoD, valores por debajo de los valores establecidos para cepas de una misma especie en el género Pseudomonas y que supondrían unas seis posibles nuevas especies. Se han caracterizado taxonómicamente 13 aislados de una de estas posibles nuevas especies. La especie más próxima en el análisis MLSA fue P. baetica (P. koreensis SG) con una similitud del 95.5-95.0%. Los análisis de ácidos grasos son los característicos del género. Los perfiles proteicos por WC-MALDI-TOF MS ubican a los aislados en el subgrupo de P. koreensis. Los análisis genómicos separan estas cepas en dos posibles nuevas especies con valores de ANIb del 94.0-93.7% y GGDC del 58.6 -58.2% entre ambas. Los valores de ANIb y GGDC son de 88.2-88.0 y 36.4-36.7% respectivamente con P. baetica. Se proponen dos nuevas especies: P. anatoliensis, con P9T como representante y P. iridis, con P42T como representante. Estos resultados indican que más de la tercera parte de los aislados corresponden a posibles nuevas especies, indicando que la diversidad en el grupo de P. fluorescens sigue aumentando, pese al gran número de especies que lo componen.Peer reviewe

Diversidad del género Pseudomonas en piscifactorías de Turquía

Trabajo presentado en la XVIII Reunión del Grupo de Taxonomía, Filogenia y Biodiversidad de la Sociedad Española de Microbiología (XVIII TAXON), celebrada online del 21 al 23 de octubre de 2020.Pseudomonas es el género que presenta el mayor número de especies dentro de las bacterias Gram negativas. Se distinguen varios grupos filogenéticos, siendo el de Pseudomonas fluorescens el mayoritario. Se ha realizado un estudio de la presencia de especies del género Pseudomonas en centros de acuicultura de Turquía entre los años 2013 a 2018. Se han aislado 90 cepas de Pseudomonas en agar TSA y agar sangre cultivadas a 28 ºC durante 24-48 horas. Los análisis por secuencia del ARN ribosómico 16S indicaron que 51 de los aislados correspondían a especies conocidas del género Pseudomonas. El análisis del gen de la subunidad D del factor sigma 70, rpoD, puso de manifiesto la presencia de posibles nuevas especies. El análisis multigénico de secuencias (MLSA) de los genes ARN ribosómico 16S, gyrB, rpoB y rpoD permitió identificar 51 aislados mayoritariamente dentro del grupo fluorescens: 18 P. haemolytica, 12 P. meridiana, 8 P. lactis, 3 P. migulae, 3 P. weihenstephanensis, 2 P. mandelii, 1 P. fluorescens, 1 P. simiae, 1 P. lurida, 1 P. lundensis y 1 P. proteolytica. Otros 29 aislados presentaron porcentajes entre 90-96 % en el análisis de semejanza de sus secuencias nucleotídicas del gen rpoD, valores por debajo de los valores establecidos para cepas de una misma especie en el género Pseudomonas y que supondrían unas seis posibles nuevas especies. Se han caracterizado taxonómicamente 13 aislados de una de estas posibles nuevas especies. La especie más próxima en el análisis MLSA fue P. baetica (P. koreensis SG) con una similitud del 95.5-95.0%. Los análisis de ácidos grasos son los característicos del género. Los perfiles proteicos por WC-MALDI-TOF MS ubican a los aislados en el subgrupo de P. koreensis. Los análisis genómicos separan estas cepas en dos posibles nuevas especies con valores de ANIb del 94.0-93.7% y GGDC del 58.6 -58.2% entre ambas. Los valores de ANIb y GGDC son de 88.2-88.0 y 36.4-36.7% respectivamente con P. baetica. Se proponen dos nuevas especies: P. anatoliensis, con P9T como representante y P. iridis, con P42T como representante. Estos resultados indican que más de la tercera parte de los aislados corresponden a posibles nuevas especies, indicando que la diversidad en el grupo de P. fluorescens sigue aumentando, pese al gran número de especies que lo componen.Peer reviewe