High-throughput screening of Saccharomyces cerevisiae efficiently producing tyrosine / 生物工程学报

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.

Neumonía bullosa secundaria a Elizabethkingia meningoseptica, su evolución clínica y radiológica / Bullous pneumonia secondary to Elizabethkingia meningoseptica, its clinical and radiological evolution

Elizabethkingia meningoseptica es un patógeno oportunista, con una elevada mortalidad y una incidencia en aumento en las terapias intensivas. Se presenta a una paciente de 4 años con antecedente de atresia de vías biliares y trasplante hepático a los 11 meses de vida, que se internó por infección respiratoria aguda baja con hipoxemia. Durante la internación, sufrió un empeoramiento clínico con requerimiento de asistencia respiratoria mecánica. Por fiebre e hipoxemia persistente, se realizó un minilavado broncoalveolar, con cultivo positivo para Elizabethkingia meningoseptica. Recibió vancomicina, trimetoprima/sulfametoxazol y ciprofloxacina durante 14 días, con buena respuesta. Una tomografía de tórax evidenció la presencia de hipoperfusión en mosaico, imágenes quísticas y bronquiectasias bilaterales. Durante los siguientes 2 años, presentó una buena evolución clínica, con escasas intercurrencias respiratorias, síntomas intercrisis aislados y buena tolerancia al ejercicio. En las imágenes de control, se evidenció la resolución de la mayoría de las lesiones iniciales a los 20 meses de su seguimiento.

Elizabethkingia meningoseptica is an opportunistic pathogen with a high mortality and an increasing incidence in the intensive care units. We present a 4-year-old patient with a history of atresia of the biliary tract and a liver transplant at 11 months of age, who was admitted for acute respiratory infection with hypoxemia. During the hospitalization, she required mechanical ventilation. Due to persistent fever and hypoxemia, mini bronchoalveolar lavage was performed with a positive culture for Elizabethkingia meningoseptica. She received vancomycin, trimethoprim/sulfamethoxazole and ciprofloxacin for 14 days with a good response. A chest tomography showed the presence of mosaic hypoperfusion, cystic images, and bilateral bronchiectasis. During the following 2 years, she presented good clinical progress, with scarce respiratory infections, isolated symptoms and good tolerance to exercise. The resolution of the majority of the initial lesions was evidenced at 20 months of follow-up.

Neonato con Ventriculitis causada por Empedobacter Brevis: Reporte de un caso en Perú / Neonate with Ventriculitis caused by Empedobacter Brevis: Report of a case in Peru

RESUMEN Las infecciones causadas por microorganismos poco comunes son objeto de investigación, ya que animar a los investigadores a encontrar las medidas sanitarias necesarias para prevenir y tratar la enfermedad, así como la búsqueda de nuevas luces sobre las interacciones humano-microbios. En este informe se describe el caso de un recién nacido varón diagnosticado de hidrocefalia y mielomeningocele, que desarrolló ventriculitis y sepsis por Empedobacter brevis resistente. Este caso pone de manifiesto la inesperada identificación de esta bacteria en el líquido cefalorraquídeo y su patrón multirresistente, que fue crucial para dar un manejo terapéutico adecuado. Esta bacteria evidencia una mezcla de diferentes etiologías en el análisis del líquido cefalorraquídeo.

ABSTRACT Infections caused by rare micro-organisms are the subject of research, as researchers are encouraged to find the necessary health measures to prevent and treat the disease, as well as the search for new insights into human-microbial interactions. This report describes the case of a newborn boy diagnosed with hydrocephalus and myelomeningocele who developed ventriculitis and sepsis from resistant Empedobacter brevis. This case highlights the unexpected identification of this bacterium in the cerebrospinal fluid and its multi-resistant pattern, which was crucial for proper therapeutic management. This bacterium shows a mixture of different etiologies in the analysis of cerebrospinal fluid.

Biological control of Flavobacterium psychrophilum infection in ayu (Plecoglossus altivelis altivelis) using a bacteriophage PFpW-3 / 대한수의학회지

The efficacy of using a bacteriophage (phage) to control Flavobacterium psychrophilum (F. psychrophilum) infection of ayu (Plecoglossus altivelis altivelis) was evaluated in this study. Intramuscular challenge failed to induce sufficient infection levels; therefore, a newly designed net-scratch challenge method was also used to induce bacterial infection. Administration of phage PFpW-3 in F. psychrophilum-infected ayu showed notable protective effects, increased survival rates and mean times to death. Additionally, the fate of inoculated bacteria and phage in ayu were investigated. Our results suggest that the phage PFpW-3 could be considered an alternative biocontrol agent against F. psychrophilum infections in ayu culture.

Effects of supplementation with antifreeze proteins on the follicular integrity of vitrified-warmed mouse ovaries: Comparison of two types of antifreeze proteins alone and in combination / 대한생식의학회지

OBJECTIVE: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. METHODS: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. RESULTS: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. CONCLUSION: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.

A Case of Flavobacterium ceti Meningitis

No abstract available.

New host record of five Flavobacterium species associated with tropical fresh water farmed fishes from North India

Abstract Yellow pigmented, filamentous, Gram-negative bacteria belonging to genus Flavobacterium are commonly associated with infections in stressed fish. In this study, inter-species diversity of Flavobacterium was studied in apparently healthy freshwater farmed fishes. For this, ninety one yellow pigmented bacteria were isolated from skin and gill samples (n = 38) of three farmed fish species i.e. Labeo rohita, Catla catla and Cyprinus carpio. Among them, only twelve bacterial isolates (13.18%) were identified as Flavobacterium spp. on the basis of morphological, biochemical tests, partial 16S rDNA gene sequencing and phylogenetic analysis. On the basis of 16S rDNA gene sequencing, all the 12 isolates were 97.6-100% similar to six different formally described species of genus Flavobacterium. The 16S rDNA based phylogenetic analysis grouped these strains into six different clades. Of the 12 isolates, six strains (Fl9S1-6) grouped with F. suncheonense, two strains (Fl6I2, Fl6I3) with F. indicum and the rest four strains (Fl1A1, Fl2G1, Fl3H1 and Fl10T1) clustered with F. aquaticum, F. granuli, F. hercynium and F. terrae, respectively. None of these species except, F. hercynium were previously reported from fish. All the isolated Flavobacterium species possessed the ability of adhesion and biofilm formation to colonize the external surface of healthy fish. The present study is the first record of tropical freshwater farmed fishes as hosts to five environmentally associated species of the Flavobacterium.

Flavobacterium ceti From Blood Samples of a Korean Patient With Alcoholic Liver Cirrhosis

No abstract available.

Culture conditions optimization and high cell density fermentation of recombinant bacteria producing heparinase II from Flavobacterium heparinum / 生物工程学报

Heparinase II (Hep II) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase II (His-Hep II) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 degrees C, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 degrees C for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep II increased to 9 436 U/L.

Composition of Extracellular Polymeric Substances (EPS) produced by Flavobacterium columnare isolated from tropical fish in Brazil

Thirty nine isolates of Flavobacterium columnare from Brazilian fish farms had their carbohydrate composition of EPS evaluated by high efficiency liquid chromatography, using the phenol-sulfuric acid method of EPS. The occurrence of capsules on F. columnare cells was not directly related to biofilm formation, and the predominant monosaccharide is glucose.

Phylogeny of Flavobacteria Group Isolated from Freshwater Using Multilocus Sequencing Analysis

Sequence analysis of the 16S rRNA gene has been widely used for the classification of microorganisms. However, we have been unable to clearly identify five Flavobacterium species isolated from a freshwater by using the gene as a single marker, because the evolutionary history is incomplete and the pace of DNA substitutions is relatively rapid in the bacteria. In this study, we tried to classify Flavobacterium species through multilocus sequence analysis (MLSA), which is a practical and reliable technique for the identification or classification of bacteria. The five Flavobacterium species isolated from freshwater and 37 other strains were classified based on six housekeeping genes: gyrB, dnaK, tuf, murG, atpA, and glyA. The genes were amplified by PCR and subjected to DNA sequencing. Based on the combined DNA sequence (4,412 bp) of the six housekeeping genes, we analyzed the phylogenetic relationship among the Flavobacterium species. The results indicated that MLSA, based on the six housekeeping genes, is a trustworthy method for the identification of closely related Flavobacterium species.

Hematology and productive performance of nile tilapia (Oreochromis niloticus) naturally infected with Flavobacterium columnare

Columnaris disease is one of the main causes of mortality in tilapia rearing and is responsible for large economic losses worldwide. Hematology is a tool that makes it possible to study organisms' physiological responses to pathogens. It may assist in making diagnoses and prognoses on diseases in fish populations. The hematological variables of nile tilapia were studied in specimens with a clinical diagnosis of columnaris disease and in specimens that were disease-free. The total erythrocyte count, hemoglobin rate, hematocrit percentage, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), organic defense blood cell percentages (leukocytes and thrombocytes) and hepatosomatic and splenosomatic index were determined. The results showed that there were changes in the erythrocytic series and in organic defense blood cells, in the fish infected with the bacterium, with reductions in erythrocytic variables and significant increases in the numbers of circulating lymphocytes and neutrophils.

A Flavobacterium lindanitolerans strain isolated from the ascites sample of a Chinese patient with EV71 virus infection / 生物医学与环境科学（英文）

A strain of Flavobacterium lindanitolerans isolated from a sick child's ascites was described. The 16S rRNA gene of the strain was 100% identical to that of Flavobacterium lindanitolerans which was first identified in India in 2008. It was first described that the isolate required X factor (Hemin) for growth in the optimal conditions of 37 °C with 5% CO(2). The isolate produced indole and H(2)S. It did not present hemolytic feature on blood agar.

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.

Plasmid profiling of Flavobacterium psychrophilum isolates from ayu (Plecoglossus altivelis altivelis) and other fish species in Japan

In order to evaluate the genetic variability of the causative agent of cold water disease (CWD), plasmid profiling was used to characterize Flavobacterium (F.) psychrophilum isolates (n = 169). Size analysis of plasmids in F. psychrophilum isolates (n = 128) from several fish species demonstrated that six kinds of plasmids were harbored, and ayu isolates had different profiles compared to other isolates. Moreover, multiple isolates (n = 41) from CWD outbreaks in 2002 to 2003 at a single ayu farm were examined to determine differences between isolates from successive outbreaks and showed different profiles by the sources of seedlings.

Diversity of marine gliding bacteria in Thailand and their cytotoxicity

Eighty-four marine gliding bacteria were isolated from specimens collected in the Gulf of Thailand and the Andaman Sea. All exhibited gliding motility and swarm colonies on cultivation plates and they were purified by subculturing and micromanipulator techniques. Their 16S rRNA genes were amplified by the polymerase chain reaction (PCR) technique. The phylogenetic analysis indicated that the represented isolates can be separated into six different clads (gr 1 - gr 6) within the Cytophaga-Flavobacterium-Bacteriodes (CFB) group. Group 1 formed a remote linear, with only 90 percent sequence similarity, from Flavobacteriaceae bacterium which indicated a potentially novel taxonomic group. Groups 2 and 3 were identified as the recently proposed Tenacibaculum mesophilum and Fulvivirga kasyanovii respectively. Groups 4, 5 and 6, consisting of the largest number of the members, were identified as Rapidithrix thailandica, Aureispira marina and Aureispira maritima respectively. The isolates were cultivated in four different cultivation media (Vy/2, RL 1, CY and SK) and the crude extracts were submitted to screen cytotoxicity using a sulphorodamine B (SRB) assay. The results from cytotoxic screening showed that groups 2, 4 and 6 were capable of producing the cytotoxic metabolites against selected human cell lines (breast adenocarcinoma (MCF-7), colon cancer (HT-29), cervical cancer (HeLa) and oral cancer (KB)). However, groups 1, 3 and 5 did not produce metabolites with cytotoxicity when cultivated in the same cultivation media as the previous groups. CY medium was the only cultivation medium which could yield the cytotoxic metabolites against MCF-7.

Occurrence of heavy metals and antibiotic resistance in bacteria from internal organs of american bullfrog (Rana catesbeiana) raised in Malaysia

A total of 40 bacteria have been successfully isolated from internal organs of the American bullfrog (Rana catesbeiana) raised in Malaysia, namely, eight isolates of Aeromonas spp., 21 of Edwardsiella spp., six of Flavobacterium spp. and five of Vibrio spp. In terms of antibiotic susceptibility testing, each isolate was tested against 21 antibiotics, resulting in 482 (57.3 percent) cases of sensitivity and 61 (7.3 percent) cases of partial sensitivity. Meanwhile, 297 (35.4 percent) bacterial isolates were registered as resistant. The multiple antibiotic resistance (MAR) index of each bacterial species indicated that bacteria from raised bullfrogs have been exposed to tested antibiotics with results ranging from 0.27 to 0.39. Additionally, high percentages of heavy metal resistance among these isolates were observed, with values ranging from 85.0 to 100.0 percent. The current results provided us information on bacterial levels of locally farmed bullfrogs exposed to copper, cadmium, chromium as well as 21 types of antibiotics.(AU)

Studies on flavobacterium columnare isolated from fresh water fish

Flavobacterium columnre is one of the most important bacterial pathogens of wild, farmed and ornamental fish in warm, and also in fresh and sea water causing columnaris disease. In the present study one hundred and forty Nile tilapia were collected from different localities of farms from El-Behera governorate and Kafr El-sheikh provinces. The clinical signs of the disease were detected and the pathological lesions were recorded. Bacteriological examination of samples of naturally infected fish with Flavobacterium columnare revealed that the characteristics colonies on cytophaga agar media. Sixty seven suspected isolates were isolated and biochemically identified by API-20 E system. The distribution of the gained isolates among organs denoted 33 isolates from gills [49.25%], 20 isolates from kidneys [29-85%], 12 isolates from deep layer of dorsal musculature [17.91%], one isolate from both spleen and intestine [1.49%], but it was not isolated from the liver. The pathogencity experiment indicated wide variation among Flavobacterium columnare isolates. Isolate number 10 was the most virulent isolate as it caused 100% mortality two weeks post injection followed by isolate number 7, 3 caused 45, 25% mortality. The clinical signs and the post mortem examination revealed the same signs and lesions of natural infection with Flavobacterium columnare beside severe oedematous swelling at site of injection. The median lethal dose [LD50] of the highly virulent isolate was 10[5] viable bacterial cells /ml. Stress factors, temperature, PH, unionized ammonia and dissolved oxygen affects on the pathogencity of the organism. In vitro antibiotic sensitivity test of 5 isolates revealed that the most effective drug Cefotaxime [60%]. While the less effective one, were Neomycin, Norfloxacin and Rifampcin [100%] then Doxycyclin, Cephalexin, Florfenicel, Gentamycin and Marbocyl [80%] then Cefalor, Tetracycline and Ciprofloxacin [60%]. On the other hand, Erthromycin, Ampicillin [80%] had no effect

Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions / 生物工程学报

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.

Antibacterial activity of plant extracts from Brazil against fish pathogenic bacteria / Atividade antibacteriana de extratos de plantas do Brasil contra bactérias patogênicas para peixes

The aim of this work was to evaluate the antibacterial activity of Brazilian plants extracts against fish pathogenic bacteria. Forty six methanolic extracts were screened to identify their antibacterial properties against Streptococcus agalactiae, Flavobacterium columnare and Aeromonas hydrophila. Thirty one extracts showed antibacterial activity.

O objetivo deste trabalho foi avaliar a atividade antibacteriana de extratos de plantas brasileiras contra bactérias patogênicas para peixes. A atividade antibacteriana de quarenta e seis extratos metanólicos de plantas foi avaliada contra os agentes Streptococcus agalactiae, Flavobacterium columnare e Aeromonas hydrophila. Trinta e um extratos apresentaram atividade antibacteriana.