Fagoterapia: una alternativa emergente en la era de la multirresistencia antibiótica / Phagotherapy: an emerging alternative in the antibiotic multidrug resistance era

La creciente resistencia antimicrobiana asociada a la crisis en la producción de nuevos antibióticos y las consecuen-cias humanas y económicas de este fenómeno constituyen un complejo escenario que requiere el urgente desarrollo de estrategias antimicrobianas alternativas. Los bacterió-fagos son virus que infectan y lisan bacterias. Si bien se conocen desde hace más de un siglo, en las últimas dos décadas la administración de bacteriófagos ha ganado popularidad en todo el mundo. Existe un extenso cuerpo de evidencia preclínica y clínica que posiciona a la fago-terapia como una de las principales herramientas para el tratamiento de infecciones difíciles de tratar. Aunque esto es conceptualmente promisorio, su implementación está limitada por la escasez de datos clínicos de seguridad y efi-cacia, obtenidos acorde a los estándares científicos actua-les. Esta revisión describe los datos más relevantes acerca de la biología de los fagos, los aspectos farmacocinéticos y farmacodinámicos conocidos hasta la actualidad, los te-mas regulatorios y los resultados clínicos más relevantes

The rising antimicrobial resistance associated with the crisis in new antibiotics production and the human and economic consequences of this phenomenon constitute a complex scenario that requires the urgent development of alternative antimicrobial strategies. Bacteriophages are viruses that infect and lyse bacteria. They have been known for over a century but in the last two decades, phage administration has gained popularity worldwide. There is an extensive body of preclinical and clinical evidence that positions phage therapy as one of the main tools for the treatment of difficult-to-treat infections. Although this is conceptually promising, its implementation is limited by the paucity of clinical data on safety and efficacy, obtained according to current scientific standards. This review describes the most relevant data on phage biology, pharmacokinetic and pharmacodynamic aspects known to date, regulatory issues, and the most relevant clinical results

Establishment and evaluation of a method for phages enrichment by ferric trichloride-polyvinylidene fluoride membrane filter / 中华预防医学杂志

Objective: To establish and evaluate a method of enriching bacteriophages in natural water based on ferric trichloride-polyvinylidene fluoride (FeCl3-PVDF)membrane filter. Methods: Based on the principle of flocculation concentration, the method of recovering bacteriophage from water sample was established by using iron ion flocculation combined with membrane filter. The titer of phage was determined by Agar double layer method. The recovery efficiency of phage was detected by phage fluorescence staining and real-time fluorescence PCR reaction. Water samples from different sources were collected for simulation experiment to evaluate the enrichment effect. At the same time, the sewage discharged from hospitals was taken as the actual water sample, and the common clinical drug-resistant bacteria were used as the host indicator bacteria to further analyze the enrichment effect of FeCl3-PVDF membrane filter rapid enrichment method on the bacteriophage in natural water samples. Results: The method of enrichment of bacteriophages in natural water by iron ion concentration 50 mg/L and PVDF membrane filter was established. The recovery rate of this method for bacteriophage was 93%-100%. Under the multi-functional microscope, it was found that the bacteriophage of the enriched water sample increased significantly and the fluorescence value of the enriched water sample determined by the enzyme labeling instrument was about 13 times as high as that before enrichment. After concentration of the actual water samples from the hospital drainage, the positive rate of bacteriophage isolation in the concentrated group and the non-concentrated group was 23% and 4%, and the fluorescence value in the concentrated group was 2-24 times as high as that of the non-concentrated group. Conclusion: The method of FeCl3-PVDF membrane filter is a simple, efficient and rapid method for enriching bacteriophages in different water samples.

Analysis of genomic information and biological characteristics of a bacteriophage against methicillin-resistant Staphylococcus aureus in patients with median sternal incision infection / 中华烧伤杂志

Objective: To isolate and purify a bacteriophage against methicillin-resistant Staphylococcus aureus (MRSA), and to analyze its genomic information and biological characteristics. Methods: The experimental research methods were adopted. MRSA (hereinafter referred to as host bacteria) solution was collected from the wound of a 63-year-old female patient with the median sternum incision infection admitted to the Second Affiliated Hospital of Army Medical University (the Third Military Medical University). The bacteriophage, named bacteriophage SAP23 was isolated and purified from the sewage of the Hospital by sewage co-culture method and double-layer agar plate method, and the plaque morphology was observed. The morphology of bacteriophage SAP23 was observed by transmission electron microscope after phosphotungstic acid negative staining. The whole genome of bacteriophage SAP23 was sequenced with NovaSeq PE15 platform after its DNA was prepared by sodium dodecyl sulfonate/protease cleavage scheme, and genomic analysis including sequence assembly, annotation, and phylogenetic tree were completed. The bacteriophage SAP23 solution was co-incubated with the host bacterial solution for 4 h at the multiplicity of infection (MOI) of 10.000 0, 1.000 0, 0.100 0, 0.010 0, 0.001 0, and 0.000 1, respectively, and then the bacteriophage titer was measured by the drip plate method to select the optimal MOI, with here and the following sample numbers of 3. The bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for 5, 10, and 15 min, respectively, and the bacteriophage titer was measured by the same method as mentioned above to select the optimal adsorption time. After the bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for the optimal adsorption time, the bacteriophage titers were measured by the same method as mentioned above at 0 (immediately), 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 120 min after culture, respectively, and a one-step growth curve was drawn. The bacteriophage SAP23 solution was incubated at 4, 37, 50, 60, 70, and 80 ℃ and pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 for 1 h, respectively, to determine its stability. A total of 41 MRSA strains stored in the Department of Microbiology of Army Medical University (the Third Military Medical University) were used to determine the host spectrum of bacteriophage SAP23. Results: The bacteriophage SAP23 could form a transparent plaque on the host bacteria double-layer agar plate. The bacteriophage SAP23 has a polyhedral head with (88±4) nm in diameter and a tail with (279±21) nm in length and (22.6±2.6) nm in width. The bacteriophage SAP23 has a linear, double-stranded DNA with a full length of 151 618 bp and 11 681 bp long terminal repeats sequence in the sequence ends. There were 220 open reading frames predicted and the bacteriophage could encode 4 transfer RNAs, while no resistance genes or virulence factors were found. The annotation function of bacteriophage SAP23 genes could be divided into 5 groups. The GenBank accession number was MZ427930. According to the genomic collinearity analysis, there were 5 local collinear blocks in the whole genome between the bacteriophage SAP23 and the chosen 6 Staphylococcus bacteriophages, while within or outside the local collinear region, there were still some differences. The bacteriophage SAP23 belonged to the Herelleviridae family, Twortvirinae subfamily, and Kayvirus genus. The optimal MOI of bacteriophage SAP23 was 0.010 0, and the optimal adsorption time was 10 min. The bacteriophage SAP23 had a latent period of 20 min, and a growth phase of 80 min. The bacteriophage SAP23 was able to remain stable at the temperature between 4 and 37 ℃ and at the pH values between 4 and 9. The bacteriophage SAP23 could lyse 3 of the 41 tested MRSA strains. Conclusions: The bacteriophage SAP23 is a member of the Herelleviridae family, Twortvirinae subfamily, and Kayvirus genus. The bacteriophage SAP23 has a good tolerance for temperature and acid-base and a short latent period, and can lyse MRSA effectively. The bacteriophage SAP23 is a new type of potent narrow-spectrum bacteriophage without virulence factors and resistance genes.

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 to11.0 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.

Prevention and control of antimicrobial resistance using CRISPR-Cas system: a review / 生物工程学报

Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.

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.

Screening and formulation of novel carriers for Xanthomonas bacteriophage to control bacterial leaf blight disease

Aims@#This study was aimed to evaluate the potential of several carriers to formulate the phages and retain their activity under various pH and temperature conditions.@*Methodology and results@#The skim milk, rice flour, corn flour and CalnuXan (calcium and magnesium) as carriers to formulate the isolated phage to maintain its activity under extreme pH and temperature conditions. Two phages formulated with carriers retained their viability at pH 5, pH 7 and pH 9 compared to that of the unformulated phages. Besides, the formulated phages also retained a high titre compared to the unformulated phages when they were exposed to 37 °C and 45 °C. Based on the in vitro study of the formulation, it was applied in the glass house. The plant height, leaf chlorophyll and disease scoring were recorded and analyzed. In the glass house, the rice plant treated with formulated phages showed higher plant height and chlorophyll content than those treated with unformulated or untreated phages. Nonetheless, both formulated and unformulated protected the rice plant, which showed lower disease severity than the untreated group.@*Conclusion, significance and impact of study@#Phage therapy has been used for treating plant diseases caused by pathogenic bacteria. Despite their effectiveness in killing the pathogen in vitro, the results were not reproducible in the field. Bacteriophages (phages) are sensitive to environmental factors and infection efficiency was dropped when exposed to harmful environments. However, this study successfully formulated two novels Xanthomonas phages, as biocontrol agents against bacterial leaf blight (BLB) disease in rice.

Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library / 中国实验血液学杂志

OBJECTIVE@#To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library.@*METHODS@#We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA.@*RESULTS@#A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies.@*CONCLUSION@#A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

Stratifin em Tumores de Cabeça e Pescoço: Um alvo molecular identificado por Phage Display / Stratifin in head and neck tumors: a molecular target identified by Phage Display

Nos últimos anos, houve um aumento na frequência dos casos de tumores de cabeça e pescoço apesar da diminuição do consumo do tabaco e álcool, e isso tem sido atribuído, em parte, à infecção pelo Papilomavírus Humano HPV. Por apresentar baixa sobrevida em 5 anos e ter alta morbidade, tem se buscado novos alvos moleculares para terapias combinadas. Nesse contexto nosso grupo identificou, através da tecnologia de Phage Display, uma sequência peptídica com interação preferencial por células tumorais com relação à células não transformadas, e ensaios adicionais identificaram seu alvo como sendo a proteína Stratifin. Stratifin tem sido reportado como um oncogene em diversos modelos tumorais, entretanto seu papel em carcinoma de células escamosas de cabeça e pescoço (CCECP) permanece desconhecido e poucos trabalhos na literatura reportam sua atividade em CCECP e/ou outro tumores relacionados ao HPV. Dessa forma, o objetivo desse trabalho foi explorar o potencial valor clínico e o papel biológico da Stratifin em CCECP. Dados do perfil de expressão e de metilação assim como dados clínicos foram extraídos em base de dados do The Cancer Genoma Atlas TCGA. Paralelamente, o perfil de expressão de Stratifin foi verificado através de ensaios de RT/qPCR e Western Blot em um painel de linhagens celulares de CCECP que contempla as principais características moleculares para esses tipos tumorais. A partir da observação de que todas as linhagens expressam Stratifin, utilizou-se a tecnologia de CRISPR/Cas9 para modular sua expressão (nocauteando ou superexpressando o gene) de modo a se observar parâmetros relacionados ao processo tumorigênico. Dessa forma, foi possivel verificar os efeitos da Stratifin em ensaios de proliferação, viabilidade após tratamentos com quimioterápicos, irradiação, crescimento livre de ancoragem e clonogenicidade. Como resultados, observamos que expressão aumentada de Stratifin no tecido tumoral quando comparado ao tecido normal, foi positivamente relacionada com o grau histológico, negatividade para HPV, mutação em TP53 e CDKN2A. Biologicamente, o nocaute de Stratifin foi relacionado com maior sensibilidade à quimioterápicos, menor capacidade de formação de colônias, e reduzida capacidade de crescimento livre de ancoragem. Esses resultados sugerem que Stratifin atue como um oncogene em CCECP, entretanto ensaios adicionais devem ser realizados para corroborar esse achados

Over recent years, there has been an increase of head and neck tumors frequency despite the decrease in tobacco and alcohol consumption, and this has been attributed, in part, to Human Papillomavirus infection. Due to its low 5-year survival and high morbidity, new molecular targets for combined therapies have been sought. In this context, our group identified, through Phage Display technology, a peptide sequence with preferential interaction by tumor cells in relation to non-transformed cells, and further assays identified its target as the Stratifin protein. Stratifin has been reported as an oncogene in several tumor models, however its role in head and neck squamous cell carcinoma (HNSCC) remains unknown and few works in the literature report its activity in HNSCC and/or other HPV-related tumors. Therefore, the aim of this study was to explore the potential clinical value and biological role of Stratifin in HNSCC. Expression profile data as well as clinical data were extracted from The Cancer Genome Atlas - TCGA database. In parallel, the expression profile of Stratifin was verified through RT/qPCR and Western Blot assays in a panel of HNSCC cell lines that address the main molecular characteristics for these tumor types. Since all cell lines express Stratifin, CRISPR/Cas9 technology was used to modulate its expression (gene knocking out or overexpressing) in order to check parameters related to the tumorigenic process. Thus, it was possible to verify the Stratifin effects in proliferation assays, viability after chemotherapy treatments, irradiation, anchorage-free growth and clonogenicity. As a result, we observed an increased expression of Stratifin in tumor tissue when compared to normal tissue, which was positively related to histological grade, HPV negativity, mutation in TP53 and CDKN2A. Biologically, knockout of Stratifin was associated with greater sensitivity to chemotherapy, less colony-forming capacity, and reduced anchorage-free growth capacity. These results suggest that Stratifin acts as an oncogene in HNSCC, however additional assays should be performed to corroborate these findings

Development of a luminescence real-time method for monitoring live bacteria during phage lysis / 生物工程学报

The toxin-producing bacterium Vibrio cholerae can cause severe diarrhea and has caused seven global pandemics. Traditional viable cell counts and phage plaques are commonly used to evaluate the efficacy of virulent phage clearance of V. cholerae, but these operations are time-consuming and labor-intensive, and difficult to provide real-time changes. It is desirable to develop a simple and real-time method to monitor V. cholerae during phage lysis. In this study, a luminescence-generating plasmid pBBR-pmdh-luxCDABE was transformed into three O1 serogroup drug-resistant strains of V. cholerae. The results showed that the luminescence value as a monitoring index correlates well with the traditional viable cell count method. Monitoring the number of live cells of V. cholerae by measuring the luminescence allowed real-time analysis of the number of bacteria remaining during phage lysis. This method enables repeated, interference-free, continuous multiple-time-point detection of the same sample without the time delay of re-culture or plaque formation, facilitating real-time monitoring and analysis of the interaction between the phage and the host bacteria.

Advances of phage receptor binding proteins / 生物工程学报

Bacteriophages bind to the bacteria receptor through the receptor binding proteins (RBPs), a process that requires the involvement of complex atomic structures and conformational changes. In response to bacteriophage infection, bacteria have developed a variety of resistance mechanisms, while bacteriophages have also evolved multiple antagonistic mechanisms to escape host resistance. The exploration of the "adsorption-anti adsorption-escape process" between bacteriophages and bacteria helps us better understand the co-evolution process of bacteriophages and bacteria, which is important for the development of phage therapeutic technologies and phage-based biotechnologies. This review summarizes the bacteriophage adsorption related proteins, how bacteriophages escape host resistance based on the RBP alternations, and the recent progress of RBP-related biotechnologies.

Application of CRISPR in evolution analysis, detecting and typing, virulence and antibiotic resistance regulation in food-borne pathogens / 生物工程学报

Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein gene system can limit the horizontal gene transfer, thereby effectively preventing the invasion of foreign gene elements such as bacteriophages. CRISPR arrays of different bacteria are diverse. Based on the differences in the CRISPR system, this review summarizes the application of CRISPR in food-borne pathogen evolution analysis, detection and typing, virulence and antibiotic resistance in recent years. We also address bacterial detection typing method developed based on the characteristics of CRISPR arrays and the association of CRISPR with virulence and drug resistance of food-borne pathogens. The shortcomings of CRISPR in evolution, detection and typing, virulence and resistance applications are analyzed. In addition, we suggest standardizing CRISPR typing methods, improving and expanding the CRISPR database of pathogenic bacteria, and further exploring the co-evolution relationship between phages and bacteria, to provide references for further exploration of CRISPR functions.

Bacteriófagos com potencial aplicabilidade em tubos endotraqueaisavaliação das atividades antibacteriana e antibiofilme / Potential applicability of bacteriophages on endotracheal tubes: evaluation of antibacterial and antibiofilm activities

O objetivo deste estudo foi isolar bacteriófagos com potencial aplicabilidade no controle de biofilme de Pseudomonas aeruginosa em tubos endotraqueais. Os bacteriófagos isolados foram expandidos, titulados e caracterizados quanto ao perfil genômico, morfologia, tipo de material genético, especificidade de hospedeiros, eficiência de plaqueamento, atividade lítica, curva de crescimento e estabilidade às variações de pH e temperatura. A inibição do crescimento planctônico e a atividade antibiofilme, in vitro, foram avaliadas contra 15 cepas de P. aeruginosa. A atividade antibiofilme de tubos endotraqueais revestidos com os bacteriófagos foi avaliada em um modelo de biofilme em fluxo contínuo. A influência dos bacteriófagos sobre os fatores de virulência de P. aeruginosa foi avaliada pela inibição da formação de biofilme, produção de piocianina e proteases extracelulares e expressão dos genes pslA, lasl, lasB e phzH. Os dados referentes a área recoberta por biofilme após o tratamento com os bacteriófagos e a atividade antibiofilme de tubos endotraqueais revestidos apresentaram distribuição não normal e foram analisados em um Modelo Linear Generalizado (α=5%). A influência dos bacteriófagos sobre os fatores de virulência de P. aeruginosa também apresentou distribuição não normal e foi analisada pelo teste de Kruskal-Wallis (α=5%). Todas as demais variáveis apresentaram distribuição normal e variância homogênea e foram analisadas por ANOVA (α=5%). Vinte e cinco bacteriófagos foram isolados a partir de amostras do esgoto doméstico. Do total, 5 bacteriófagos foram selecionados para caracterização integral e avaliação das atividades antibacteriana e antibiofilme. Eles foram designados como vB_PaeM_USP_1, vB_PaeM_USP_2, vB_PaeM_USP_3, vB_PaeM_USP_18 e vB_PaeM_USP_25. Os bacteriófagos pertencem à ordem Caudovirales, família Myoviridae, com genoma constituído por DNA dupla fita (dsDNA), variando de ~62 a ~65 kb e codificam de 65 a 89 proteínas. Os bacteriófagos produziram de 27 a 46 partículas virais após 30 minutos de incubação e foram estáveis às variações de pH e temperatura. Os bacteriófagos exibiram um amplo espectro lítico e foram capazes de infectar P. aeruginosa, incluindo cepas multirresistentes. Eles também reduziram o crescimento de P. aeruginosa na forma planctônica, e a carga microbiana e atividade metabólica quando aplicados a biofilmes associados aos tubos endotraqueais. A área recoberta por biofilme foi significativamente reduzida após a exposição de biofilmes maduros aos bacteriófagos. A aplicação in situ dos bacteriófagos no revestimento de tubos endotraqueais mostrou que o coquetel composto por vB_PaeM_USP_2 e vB_PaeM_USP_18 alterou a colonização bacteriana e o desenvolvimento do biofilme de P. aeruginosa, sem afetar substancialmente a atividade metabólica. Avaliando os fatores de virulência de P. aeruginosa foi observado que os vírus promoveram mudanças no crescimento do biofilme apenas até 8 horas de cocultivo. Também, após 8 horas de cocultivo foi observado que vB_PaeM_USP_1, vB_PaeM_USP_2 e vB_PaeM_USP_3 promoveram filamentação da morfologia bacteriana. A presença de bacteriófagos não alterou a produção de piocianina e proteases extracelulares por P. aeruginosa. No entanto, alterações no nível de expressão de genes relacionados a fatores de virulência foram detectadas, principalmente, após 2 e 48 h de cocultivo. A atividade lítica no biofilme de P. aeruginosa formado por cepas multirresistentes indica que os bacteriófagos isolados neste estudo podem ser considerados bons candidatos para estudos terapêuticos.

The objective of this study was to isolate bacteriophages and potentially apply it against Pseudomonas aeruginosa biofilms on endotracheal tube surfaces. The isolated bacteriophages were propagated, titrated, and characterized in terms of their genomic profile, viral morphology, type of genetic material, host range investigation, efficiency of platting, lytic activity, growth curve, and pH and thermal stability. The inhibition of planktonic growth and antibiofilm activity, in vitro, were evaluated against 15 P. aeruginosa strains. The antibiofilm activity of endotracheal tubes coated with bacteriophages was evaluated in a continuous flow biofilm model. The bacteriophages influence on development of virulence mechanisms on P. aeruginosa was evaluated by the inhibition of biofilm growth, production of pyocyanin and extracellular proteases, and expression of pslA, lasl, lasB and phzH genes. Data referring to the residual aggregated biofilm after treatment with bacteriophages and the antibiofilm activity of endotracheal tubes coated with bacteriophages showed non-normal distribution and were analyzed in a Generalized Linear Model (α=5%). The bacteriophage's influence on development of virulence mechanisms on P. aeruginosa also showed non-normal distribution and was analyzed by Kruskal-Wallis test (α=5%). All other data had normal distribution and homogeneous variance and were analyzed using ANOVA (α=5%). Twenty-five bacteriophages were isolated from domestic sewage samples. Of these, 5 bacteriophages were selected for complete characterization and evaluation of antibacterial and antibiofilm activities. They were designated as vB_PaeM_USP_1, vB_PaeM_USP_2, vB_PaeM_USP_3, vB_PaeM_USP_18 and vB_PaeM_USP_25. All of them belong to the order Caudovirales, Myoviridae family, and they have a double-stranded DNA (dsDNA) genome ranging from ~62 kb to ~65 kb that codes from 65 to 89 proteins. The bacteriophages produced from 27 to 46 particles after 30 minutes of incubation and were pH and heat stable. Bacteriophages exhibited a broad lytic spectrum and were able to infect P. aeruginosa, including multidrug-resistant strains. They also reduced the growth of P. aeruginosa strains in planktonic form, and microbial load and metabolic activity when applied to biofilms associated with endotracheal tubes. Biofilm-coated area were significantly reduced after treatment of mature biofilms with bacteriophages. The in situ application of bacteriophages in endotracheal tubes revealed that the cocktail composed by vB_PaeM_USP_2 and vB_PaeM_USP_18 promoted changes in colonization and biofilm growth processes without, substantially, altering the metabolic activity. Assessing the virulence mechanisms of P. aeruginosa it was observed that the virus promoted changes in P. aeruginosa biofilm growth only up to 8 h of co-incubation. In addition, after 8 h of co-incubation, it was observed that vB_PaeM_USP_1, vB_PaeM_USP_2 and vB_PaeM_USP_3 promoted filamentation of bacterial morphology. Bacteriophage presence did not alter both pyocyanin and protease production by P. aeruginosa. However, changes in the expression level of genes related to virulence factors were detected mainly after 2 and 48 h of co-culture. The lytic activity on multidrug-resistant P. aeruginosa biofilm indicates that isolated bacteriophages in this study may be considered as good candidates for therapeutic studies

Anti-Helicobacter pylori activity of Egyptian medicinal plants and bacteriophages

Background: Helicobacter pylori (H. pylori) is the most common cause of gastric infections worldwide. Due to antibiotic resistance and adverse effects, phytotherapy and phage therapy have been a research focus as an alternative therapy for H. pylori infection. Objectives: To assess the medicinal plant extracts and bacteriophages as a treatment of H. pylori infection. Methodology: Thirty five gastric biopsies were cultured for H. pylori isolation. Screening of medicinal plants extract efficiency was done by Disc diffusion method. Minimum inhibitory concentrations of extracts were assessed. In vivo effect of Punica granatum peel extract was tested by bacterial density and histopathology in rats. Sewage water samples were screened for H. pylori specific bacteriophages. Single plaque isolation technique was used for phage purification. Results: Ten out of 35 (28.57%) patients had positive gastric biopsy for H. pylori by culture. Four out of 10 (40%) isolates were resistant to all antibiotics. Inhibitory effect of Rosemarinus officinalis, Syzygium aromaticum, Rhus coriaria and Ammi visagna on H. pylori was detected. Punica granatum extract was the most efficient in vitro. In vivo, Punica granatum peel extract caused significant reduction of bacterial density (Pty (P<0.05) and enhanced ulcer healing. Sewage water filtrates contained 3 types ofH. pylorispecific bacteriophages. During phagepurification,phage infectivity waslost.Conclusions:Punicagranatumpeel extract revealed better in vivo activity againstH. pylorithanv standard regimen antimicrobials. Other effective plants can be beneficial inH. Pylori infection management .Loss of bacteriophage infectivity may be an obstacle to phage therapy of H. pylori

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

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)

Identification of High-affinity Novel Targeted Peptides for the Treatment of Human Ovarian Cancer

Background: To investigate the potential of the phage display-identified tumor cellbinding peptide as a biomarker of epithelial ovarian cancer using phage display technology. Method: The Ph.D.-7 Phage Display Peptide Library was used to identify the specific conjugated phages with SKOV3 epithelial ovarian cancer cells, while Chinese hamster ovary cells formed the basis. After employing the rapid differential screening method invitro, the enzyme-linked immunosorbent assay (ELISA), DNA sequencing, immunohistochemistry, immunofluorescence, and the competitive inhibition test of synthetic peptides were used to determine the affinity and specificity of the phages with SKOV3 cells. Results: Using bio panning, we screened the phages, showing a 3590-fold increase after the third round. A total of 61 titers of the phage were randomly selected for ELISA and 10 kinds of the phages with an optical density >0.5 were used for DNA sequencing. Clones of the phage TRRNIPN were derived from DNA sequencing based on ELISA, exhibiting both the brown granular phenomenon and green fluorescence. The specific targeted peptide TRRNIPN was incorporated in tumor cells through the competitive inhibition test. Conclusion: The results of our study indicate that the phage display identified polypeptide TRRNIPN may be an effective biomarker for the early diagnosis and targeted therapy of ovarian cancer

Hidrogeis de polivinil-álcool (PVA) contendo o bacteriófago UFV-AREG1 para aplicação como curativo adesivo para a indústria de alimentos / Polyvinyl-alcohol (PVA) hydrogel containing the UFV-AREG1 bacteriophagus for application as a curative adhesive for the food industry

Infecções cutâneas obtidas por cortes nas mãos de manipuladores podem gerar contaminação nos alimentos. O objetivo deste trabalho foi incorporar o bacteriófago UFV-AREG1 em hidrogel de álcool poli(vinílico) – PVA, aplicando-o futuramente como curativo adesivo. Foi adicionado 1,0 mL de solução do fago no PVA e seco em estufa por 48 h. Após, os hidrogeis foram submetidos a testes de Intumescimento, Espectroscopia no infravermelho (FTIR) e efeito antimicrobiano sobre E. coli O157:H7. O intumescimento do PVA-fago foi maior que o PVA-controle (p<0,05). A área de inibição do PVA-fago foi de 3,715 cm2 contra 2,916 cm2 do controle. As análises do FTIR mostraram um pico para o PVA-fago não encontrado no PVA-controle. Foi possível incorporar o bacteriófago em hidrogel de PVA e avaliar sua liberação para posterior aplicação como curativo adesivo.

Interactions Between Atopic Dermatitis and Staphylococcus aureus Infection: Clinical Implications

Staphylococcus aureus commonly colonizes the skin of atopic dermatitis (AD) patients and contributes to the development and exacerbation of AD. Multiple factors are associated with colonization of AD skin by S. aureus, including the strength of S. aureus-corneocyte adhesion, deficiency of antimicrobial peptides, decreased levels of filaggrin and filaggrin degradation products, overexpressed Th2/Th17 cytokines, microbial dysbiosis and altered lipid profiles. S. aureus colonization on AD skin causes skin barrier dysfunction through virulence factors such as superantigens (toxins), enzymes and other proteins. Furthermore, colonization of AD skin by S. aureus exacerbates AD and may contribute to microbial dysbiosis, allergen sensitization, Th2/Th17 polarization, development of atopic march and food allergy in AD patients. Skin colonization of S. aureus, particularly methicillin-resistant S. aureus (MRSA), is one of the major challenges commonly encountered in the management of AD. Bleach bath, and topical or systemic antibiotics could be used to control S. aureus infection on AD skin. However, careful use of antibiotics is required to control the occurence of MRSA. Recently, various strategies, including microbiome transplant, monoclonal antibodies against virulent toxins, vaccines and recombinant phage endolysin, have been studied to control S. aureus infection on AD skin. Further advances in our understanding of S. aureus could provide us with ways to manage S. aureus colonization more effectively in AD patients.