Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to 11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Recombinant antibodies against Iranian cobra venom as a new emerging therapy by phage display technology

The production of antivenom from immunized animals is an established treatment for snakebites; however, antibody phage display technology may have the capacity to delivery results more quickly and with a better match to local need. Naja oxiana, the Iranian cobra, is a medically important species, responsible for a significant number of deaths annually. This study was designed as proof of principle to determine whether recombinant antibodies with the capacity to neutralize cobra venom could be isolated by phage display. Methods: Toxic fractions from cobra venom were prepared by chromatography and used as targets in phage display to isolate recombinant antibodies from a human scFv library. Candidate antibodies were expressed in E. coli HB2151 and purified by IMAC chromatography. The selected clones were analyzed in in vivo and in vitro experiments. Results: Venom toxicity was contained in two fractions. Around a hundred phage clones were isolated against each fraction, those showing the best promise were G12F3 and G1F4. While all chosen clones showed low but detectable neutralizing effect against Naja oxiana venom, clone G12F3 could inhibit PLA2 activity. Conclusion: Therefore, phage display is believed to have a good potential as an approach to the development of snake antivenom.(AU)

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 10−6 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

Isolamento e caracterização genômica de bacteriófagos quanto ao seu potencial de uso terapêutico em infecções causadas por enterobactérias

O crescente surgimento de resistência bacteriana aos antibióticos convencionais é um grave problema que precisa ser enfrentado, seja pela descoberta de novas substâncias antimicrobianas, naturais ou sintéticas, ou através da pesquisa de terapias alternativas que sejam economicamente acessíveis. A terapia de fagos é uma dessas alternativas. Trata-se de uma forma de controle biológico, baseado em vírus específicos que infectam e destroem células bacterianas: os bacteriófagos. No entanto, esta é uma fonte terapêutica ainda pouco explorada. Esse trabalho utilizou o cultivo, isolamento e sequenciamento do genoma, além de técnicas de genômica de alto desempenho para isolar e caracterizar o genoma de bacteriófagos específicos para a linhagem enteroinvasiva de Escherichia coliATCC 43893, visando o entendimento e a definição do ciclo de infecção desses vírus (líticos ou lisogênicos).

A metodologia utilizada nessa pesquisa possibilitou o isolamento de 12 vírus. 8 diferentes linhagens virais tiveram seu material genético extraído e purificado, apresentando bom rendimento e quantidade reduzida de DNA bacteriano contaminante. O sequenciamento do genoma desses 8 vírus foi realizado usando a plataforma de nova geração MiSeq. Foi analisada a diversidade genética desses bacteriófagos e verificou-se que são vírus da ordem Caudovirales, sendo 2 da família Siphoviridae e 6 da família Myoviridae. Apenas um deles mostrou potencial de ter ciclo lisogênico, os outros sete vírus não continham nenhum gene que sugerisse isso. Entretanto, apesar dos bacteriófagos isolados não terem apresentado genes relacionados ao ciclo lisogênico, análises mais aprofundadas devem ser realizadas para comprovar que são realmente exclusivamente líticos, já que muitos não apresentam seu genoma completo e mais de 50% dos genes anotados não têm função definida

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Characterization of bacteriophages infecting clinical isolates of Pseudomonas aeruginosa stored in a culture collection

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.

Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO) were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8) pfu/mL) without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL), after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

Nitrogen-deficient microalgae are rich in cell-surface mannose: potential implications for prey biorecognition by phagotrophic protozoa / Microalgas com deficiência de nitrogênio são enriquecidas com manose na superfície celular: potenciais implicações para reconhecimento de presas por protozoários fagotróficos

Flow cytometry was used to quantify the abundance of mannose-linked glycoconjugates on microalgae precultured using low- or high-nitrate media. Nitrogen-deficient microalgae were richer in cell-surface mannose than nitrogen-sufficient. Findings are discussed in view of recent research which reveals mannose-specific 'feeding receptors' assist prey biorecognition by phagotrophic protozoa that ingest microalgae.

Citometria de fluxo foi usada para quantificar a abundância de glicoconjugados com manose em precultivos de microalgas usando meios com baixo e alto teor de nitrato. Microalgas com deficiências de nitrogênio tinham mais manose na superfície celular do que as com nitrogênio suficiente. Resultados são discutidos com base nas pesquisas recentes que revelam receptores específicos para manose que auxiliam no reconhecimento da presa por protozoários fagotróficos que ingerem microalgas.

Isolation of Vibrio cholerae 0139 phages to develop a phage typing scheme.

Five V. cholerae 0139 phages isolated from different parts of India have been used for phage typing study. A strain isolated from Nagpur city (NPR-4) was used as the host for phage propagation. All but one of the 260 strains of V. cholerae 0139 were found to be typeable and could be clustered into 8 distinct phage types as revealed by lytic patterns. Phage type 1 was the predominant type (61.15%) followed by type 2 (18.46%). The strains isolated from Madras exhibited 7 out of 8 phage types. These newly isolated phages could be adopted for phage typing of V. cholerae 0139 strains as an epidemiological tool.

Partial characterization of bacteriophages active against rhizobium leguminosarum Isolated from some Egyptian soils

Seventeen rhizobiophages infective against Rhizobium leguminosarwn were isolated from field grown Vicia faba in AI-Ibrahimia [Sharkia Governorate-Egypt]. Morphology, host range and inactivation pattern of the phage to heat and UV-light were studied. Isolated phages had isometric heads and tails which were contractile [2 phages], long and non contractile [7 phages], or very short tails [8 phages]. The phages were members of Bradley's basic morphological groups A, B and C. Most of these phages showed, specificity for their host [R. leguminosarune local strain] and distinct rates of sensitivity against heat and UV

Isolation, purification and ultrastructure of bacteriophages from natural mosquito breeding places in Egypt

Two bacteriophages were isolated from field collected samples representing two different mosquito breeding places. The phage AB-1 [isolated from Abheit Village, Fayoum Governorate "seepage water"] and the phage GA-2 [isolated from El-Gabal El-Asfer, Qalyoubia Governorate "sewage drain water"] were purified. Both bacteriophages were ultrastructurally described with respect to their morphology, dimensions, phases of bacterial attack and lysogeny. No major differences were observed between both isolated phages in relation to specificity, however, they were isolated from two different types of breeding places and two different geographic areas as well. This study may assume a wide host range of the isolated phages and reflect how bacterial insecticides used for mosquito larval control could be inhibited by such bacteriophage

Recombinantes estables entre el bacteriofago M13 y el plasmido pHR322 / Stable recombinants between the bacteriophage M13 and the plasmid PHR322

Se han aislado dos recombinantes entre el fago M13 y el plásmido pHR322, analizando el contenido plasmídico de una centena de clones obtenidos por transducción. El estudio de la estructura de los dos recombinates muestra que un fragmento del genoma de M13 ha sido integrado a pHR322. En los dos casos, ese fragmento contiene una parte de la región replicativa del fago y está insertado en el replicón de pHR322 o cerca del mismo. El hecho de que los replicones del fago y del plásmido parezcan estar involucrados en el evento de recombinación sugiere que el mismo es facilitado cuando la replicación comienza. En ningún caso ha sido posible aislar un recombinante llevando los genomas enteros de pHR322 y M13. Esto es debido, sin duda, a la inestabilidad de la molécula recombinante

Características de los bacteriófagos virulentos CAI y A25 de estreptococos del grupo A / Characteristic of CAI and A25 bacteriophages from group A streptococci

Los estreptococos del grupo A son capaces de provocar enfermedades en el hombre. El estudio genético de los factores de patogenicidad de estas bacterias ha estado limitado a la transducción (por bacteriófagos), sin embargo, las complejas características de los correspondientes bacteriófagos no se han estudiado suficientemente. En el presente trabajo se investigaron las características fisicoquímicas de las partículas, proteínas y ADN de los bacteriófagos CA1 y A25 de estreptococos del grupo A. Los principales resultados mostraron que el fago CA1 es idéntico morfológicamente al A25, presentando una cabeza hexagonal (60 nm) con una larga cola no contráctil (180 nm), según los datos de la microscopía electrónica. La estructura de la partícula de ambos fagos está constituida por 10 polipéptidos electroforéticamente diferentes. Los ADNs de ambos fagos presentan características similares: densidad de flotación 1,709 g/cm a la tres CsCI; dos zonas de temperatura de fusión a 80- y 88-C; ambos fagos presentan moléculas de ADN lineales y de doble hebra de una longitud de 38,3ñ 1,1 kb. Los datos de los análisis de homología mostraron una complementariedad total entre los ADNs de estos fagos. La secuencia de los ADNs de los bateriófagos A25 y CA1 contiene sitios de reconocimiento para seis endonucleasas (Hae II, Msp I, Ben I, Hind III y Bsu RI); basado en los análisis de restricción se construyó el mapa físico preliminar para ambos ADNs

Caracterización de una enzima asociada a un bacteriófago que despolimeriza el sacárido capsular de Klebsiella pneumoniae K-8 / Characterization of a bacteriophage associated enzyme which de polimerises the capsular polysaccharide of Klebsisella pneumoniae

Se aisló un bacteriólogo de K. pneumoniae K-8 el cual induce la síntesis de una enzima que despolimeriza el polisacárido capsular de la cepa huésped. la despolimerasa fue purificada y se determinaron algunas condiciones que influyen en su actividad, como son el tiempo de incubación, las concentraciones de enzima y substrato, el pH, la temperatura de incubación y la constante de Michaelis (Km). Se encontró que la enzima hidroliza parcialmente el polisacárideo. No se detectaron azúcares reductores como productos de la hidrólisis enzimática

Adhesion of cholera phage to glass surfaces at high inactivation temperatures.

Decimal dilutions of cholera phage heated in test tubes at the temperature range of 65 degrees to 70 degrees showed an erratic behaviour in that the residual counts had no relationship to the quantity of phage originally present in the tubes. If the contents of the heated tubes were decanted off and the empty tubes washed repeatedly with broth, the recovery of phage from successive washings of the tubes was much higher than what would be expected on the basis of the simple dilution effect of washings. The data presented indicate that the heating causes loose adhesion of phage to the wall of the glass tubes from where they can be detached by washing or shaking. The facts that E. coli phage T1 and also cholera phages tested with two different broths have given similar results, suggest that some general property of the phage itself is responsible for the phenomenon observed. The phenomenon appears to be different from the adsorption of phage to glass filters at lower temperature range described by earlier workers.

Thermal inactivation of cholera phages.

Thermal inactivation of seven cholera phages have been tested over the temperature range between 50 degrees to 70 degrees C. It was found that the phages vary widely in their heat sensitivity, Mukerjee's phages III being the most sensitive of the whole group. With all the phages over the temperature range studies, the inactivation curve seem to follow the pattern of virus thermal inactivation in general, the inactivation proceeding initially at a rapid rate, which in about 15 minutes time, gradually changes to a slower rate, each component tending to follow kinetics of the first order. The difficulty of explaining this phenomenon on the basis of population heterogeneity has been discussed.