Los inicios de la fagoterapia en Chile: los doctores Mercedes Perez Matus y Hugo Vaccaro Kosovich

Mercedes Pérez Matus y Hugo Vaccaro Kosovich fueron destacados médicos y microbiólogos de la cátedra ordinaria de Bacteriología de la Facultad de Medicina de la Universidad de Chile. En 1931, ambos médicos fueron contratados por la Facultad de Medicina para reorganizar la convulsionada Cátedra de Bacteriología luego de la crisis política de 1931. En el mismo período el destacado investigador del instituto Pasteur Eugéne Wollman vino a Chile a dirigir el Instituto Sanitas (1929-1931), incorporando en nuestro país el conocimiento sobre los bacteriófagos y las técnicas para su aislamiento. La prolongada labor docente y de investigación de Vaccaro y Pérez se extendió por casi 40 años (1931-1970). Publicaron numerosos artículos científicos, siendo uno de sus temas preferidos, en los primeros años, el estudio de los bacteriófagos que aprendieron junto a Wollman. En la década de los 40, bajo el liderazgo de los Dres. Vaccaro y Pérez, se inició la fagoterapia en Chile.

Mercedes Perez Matus and Hugo Vaccaro Kosovich were distinguished doctors and microbiologists from the ordinary chair of Bacteriology at the Faculty of Medicine of the University of Chile. In 1931, both doctors were hired by the F aculty of Medicine to reorganize the convulsed Chair of Bacteriology after the political crisis of 1931. In the same period, the prominent Pasteur Institute researcher Eugene Wollman came to Chile to direct the Sanitas Institute (1929-1931), incorporating in our country the knowledge about bacteriophages and the techniques for their isolation. The long teaching and research work of Vaccaro and Pérez spanned almost 40 years (1931-1970). They published numerous scientific articles, being one of their favorite topics, in the early years, the study of bacteriophages that they learned together with Wollman. In the 1940s, under the leadership of Drs. Vaccaro and Pérez, phage therapy began in Chile.

Isolation and Phenotypic Characterization of Lytic Bacteriophage with Activity against Pseudomonas aeruginosa

Aims: To perform the isolation and phenotypic characterization of bacteriophage with lytic activity against Pseudomonas aeruginosa. To demonstrate that this type of viral agent can be isolated from the environment and used for the biocontrol of resistant bacterial types, such as Pseudomonas aeruginosa. Study Design: This study was an experimental study. Place and Duration of Study: The study was conducted at, Bacteriology and Mycology Laboratory in the Veterinary Hospital at the School of Agricultural Sciences, Innovation and Business of the University of Passo Fundo (ESAN/UPF) and Center for Diagnosis and Research in Animal Health of the University of Passo Fundo (CDSA/UPF), between April 2022 and June 2022. Methodology: Samples of untreated water were inoculated with the host bacterium strain Pseudomonas aeruginosa ATCC 27853 in an enriched media After the incubation period in, a phage filtrate was obtained by centrifugation followed by filtration. We verified the presence of bacteriophages using spot test and we carried out its purification by the method of sterile toothpick plate transfer on bacterial overlay semi-solid agar. Amplification was performed using an SM buffer elution procedure to produce a stock of viral material. Through assays in Petri dishes with bacterial overlay, we performed titration and phenotypic characterization regarding the lysis spectrum and efficiency of phage infection in the host. Results: We managed to isolate a morphologically characterized lytic bacteriophage with approximately 1 mm of diameter, high clarity in the inhibition area, the presence of halo and well-demarcated edges. The bacteriophage, named as Pseudomonas aeruginosa Phage UPF_PaBP1, demonstrated the infection capacity of the target bacteria in all tested dilutions and a stock preparation with a titre of 6.5 x 10? PFU/ml was obtained for future use. Conclusion: The isolated phage showed strong lytic activity against the bacterial host, a finding that nourishes our expectations regarding the use of this phage as a biocontrol agent and phage therapy.

Role of alternatives to antibiotics in mitigating the antimicrobial resistance crisis

Antimicrobial resistance (AMR) is a burgeoning challenge of global priority, warranting immediate action to prevent the explosion of multidrug-resistant (MDR) pathogens. Indiscriminate antimicrobial use is the most important driver for AMR. AMR has led to depletion of the antibiotic pipeline and developing new antibiotics is extremely challenging due to technical and financial issues and also resistance emerges as soon any new antibiotic is introduced. At present, preserving the power of existing antibiotics by prudent use and curtailing spread of pathogens by infection prevention and control (biosecurity) in both humans and animals are the best available options to defer AMR crisis. Meanwhile, to reduce dependence on antibiotics, other alternatives such as vaccines, antibodies, pattern recognition receptors, probiotics, bacteriophages, peptides, phytochemicals, metals, and antimicrobial enzymes are being explored. This review provides an overview of various promising, potential and under investigative strategies as alternatives to antibiotics.

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

Phage therapy and its application and prospects in skin and genitourinary tract infections / 中华皮肤科杂志

The emergence of a large number of drug-resistant bacteria has brought severe challenges to clinical anti-infection treatment. Phage therapy is considered to be a very promising method to cope with this dilemma, which has been explored and applied in many disciplines. Its efficacy has also been confirmed in clearing skin and mucous membrane infections, and it has become a hot spot in international research. The use of phage cocktails, phage lysozymes and phage proteins has shown good efficacy in the treatment of drug-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella, Bacillus proteus, etc. This review summarizes the background, characteristics, development of phage therapy, and its application and prospects in skin and genitourinary tract infections.

In vitro lytic efficiency of Staphylococcus aureus bacteriophages in bacteria from bovine mastitis: a meta-analysis / Eficiência lítica in vitro de bacteriófagos de Staphylococcus aureus em bactérias de mastites bovina: uma meta-análise

ABSTRACT: Bacteriophages have been investigated as alternative to the treatment of bacterial infections, including bovine mastitis, in production animals. In this meta-analysis, we evaluated in vitro efficiency of phages of Staphylococcus aureus against S. aureus, which is involved in the etiology of bovine mastitis. Seventeen studies were included and the bacterial lytic activity was extracted using proportion analysis. The lytic efficiency of phages was obtained in this meta-analysis using a random-effects model [significant difference (P<0.05)]. Forest plots were used to graphically represent the efficiency of phages on bacterial isolates. Most phages (e.g., CS1, DW2, ΦSA011, ΦSA012, ΦSA022, ΦSA023, ΦSA024, ΦSA025, ΦSA037, ΦSA038, ΦSA039, ΦSA041, ΦSA042, ΦSA043, ΦSA044, MSA6, Ufv-aur2 to Ufv-aur11, SAH-1, SPW, vB_SauM_JS25, SaPh1 to SaPh6, SA, SANF, SA2, ΦSA012, ΦSA039, phi11, phiIPLA88, phiIPLA35, phiIPLA-RODI, phiIPLA-C1C, SAJK-IND, vBSP-A1, vBSP-A2, STA1.ST29, EB1.ST11, EB1.ST27, Remus, and ISP) were efficiently lytics or infected most S. aureus isolates, demonstrating 80% (P<0.05) lytic efficiency. The phages SA, SANF and SA2, also demonstrated lytic activity or infected the non-Staphylococcus aureus and Macrococcus caseolyticus isolates. In this meta-analysis, we compared and demonstrated the in vitro efficiency and host range of S. aureus phages. Additionally, the phages represent an alternative to be researched to treat bovine mastitis in dairy cattle caused by the prevalent microorganism, S. aureus.

RESUMO: Os bacteriófagos têm sido investigados como alternativa ao tratamento de infecções bacterianas em animais de produção, incluindo a mastite bovina. Nesta meta-análise, avaliamos a eficiência in vitro de fagos de Staphylococcus aureus contra S. aureus envolvidas na etiologia da mastite bovina. Dezessete estudos foram incluídos e a atividade lítica bacteriana foi extraída usando análise de proporção. A eficiência lítica dos fagos foi obtida nesta meta-análise, usando um modelo de efeitos aleatórios (diferença significativa (P <0,05)). Os gráficos de Forest plots foram usados para representar graficamente a eficiência dos fagos em isolados bacterianos. Os fagos avaliados, na sua grande maioria, (por exemplo, CS1, DW2, ΦSA011, ΦSA012, ΦSA022, ΦSA023, ΦSA024, ΦSA025, ΦSA037, ΦSA038, ΦSA039, ΦSA041, ΦSA042, ΦSA043, ΦSAf e U0f, ua04 SPW, vB_SauM_JS25, SaPh1 a SaPh6, SA, SANF, SA2, ΦSA012, ΦSA039, phi11, phiIPLA88, phiIPLA35, phiIPLA-RODI, phiIPLA-C1C, SAJK-IND, vBSP-A1, vBSP-A2, STA1.ST29, EB1.ST11, EB1.ST27, Remus, e ISP) foram eficientemente líticos ou infectaram a maioria dos isolados de S. aureus, demonstrando 80% (P < 0,05) de eficiência lítica. Os fagos SA, SANF e SA2 também demonstraram atividade lítica ou infectaram os isolados Staphylococcus não-aureus e Macrococcus caseolyticus. Nesta meta-análise, comparamos e demonstramos a eficiência in vitro e gama de hospedeiros de fagos de S. aureus. Adicionalmente, os fagos representam uma alternativa a ser pesquisada para o tratamento da mastite bovina em gado leiteiro causada pelo microrganismo prevalente, ou seja S. aureus.

Application of bacteriophage technique in the treatment of alcoholic liver disease / 临床肝胆病杂志

Alcoholic hepatitis (AH) is a severe alcohol-associated liver disease with a mortality rate as high as 40%. A recent study reveals that the exotoxin (cytolysin)-secreting gut bacterium Enterococcus faecalis is a critical factor for AH, which can be eliminated by bacteriophages, and therefore, the use of bacteriophages for the treatment of AH provide a new treatment option for AH. This article introduces this pioneering study and the knowledge of bacteriophages and cytolysin, so as to provide a theoretical basis for the clinical research on AH.

Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm / 中华烧伤杂志

Objective@#To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient, and to study its biological characteristics, genomic information, and effects on bacterial biofilm.@*Methods@#(1) In 2018, pan-drug resistant Klebsiella pneumoniae UA168 (hereinafter referred to as the host bacteria) solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Ruijin Hospital) was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method, drip plate method, and double-agar plate method. The bacteriophage was named as phage KP168 and the plaque morphology was observed. (2) The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis, and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining. (3) The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients′ blood in Ruijin Hospital by the drip plate method, and then the lysis rate was calculated. (4) The phage KP168 solution at a initial titer of 9.3×1011 plaque-forming unit (PFU)/mL (400 μL per tube) and the host bacteria solution at a concentration of 1×109 colony-forming unit (CFU)/mL (4 mL per tube) were conventionally shaking cultured together for 4 hours at multiplicity of infection (MOI) of 10.000, 1.000, 0.100, 0.010, or 0.001, respectively (1 tube per MOI). The titer of phage KP168 was measured by the double-agar plate method (the measurement method was the same below) to select the optimal MOI. The experiment was repeated three times. (5) The host bacteria solution at a concentration of 1×109 CFU/mL (4 mL per tube) and the phage KP168 solution at an adjusted titer of 5×107 PFU/mL (400 μL per tube) were mixed at the MOI of 0.005. The plaques were counted 0 (immediately), 1, 2, 3, 4, 5, 15, and 30 minutes (1 tube at each time point) after mixing by the double-agar plate method (the counting method was the same below), and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time. The experiment was repeated three times. (6) The host bacteria solution at a concentration of 1×109 CFU/mL (300 μL per tube) and the phage KP168 solution at a titer of 5×108 PFU/mL (60 μL per tube) were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time. The phage KP168 titer was measured 0 (immediately), 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 minutes after culture, and a one-step growth curve was drawn. The experiment was repeated three times. (7) The phage KP168 solution at a titer of 2.5×1010 PFU/mL was left to stand for 1 hour at 37, 40, 50, 60, or 70 ℃ (3 tubes at each time point, 1 mL per tube) for counting the plaques, and then the thermal stability curve was drawn. SM buffer at a pH values of 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, or 10.0 were added to the phage KP168 solution at a titer of 3.0×1010 PFU/mL, respectively. The mixed solution was left to stand for 1 hour at 37 ℃ (3 tubes of each pH, each tube containing 100 μL phage KP168 solution and 900 μL SM buffer), and then the plaques were counted, and an acid-base stability curve was drawn. (8) The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment (2). The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168. Nucleotide′s BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence, and Blastx function was used to translate the coding sequence into protein for its function prediction. The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded. (9) In a 96-well plate, at a MOI of 1.000, 0.100, 0.010 or 0.001 (3 wells per MOI), 20 μL phage KP168 solution at a initial titer of 5.8×1010 PFU/mL was added to 200 μL host bacteria solution at a concentration of 1.5×108 CFU/mL (the same concentration below) for co-cultivation for 48 hours. After 200 μL host bacteria solution was left to stand for 48 hours, 20 μL phage KP168 solution at a titer of 1×106, 1×107, 1×108, 1×109, or 1×1010 PFU/mL (3 wells per titer) was added respectively for action for 4 hours. In both experiments, 200 μL host bacteria solution added with 20 μL SM buffer (3 wells) acted as a negative control, and 220 μL LB culture medium (3 wells) acted as a blank control. Absorbance values were measured by a microplate reader, and inhibition/destruction rates of biofilm were calculated. The experiments were both repeated three times.@*Results@#(1) The plaques of phage KP168 successfully isolated and purified were transparent and round, and its diameter was approximately 1.5 mm. (2) The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail. (3) The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients, with a lysis rate of 65.0%. (4) When MOI was 1.000, the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours, which was the optimal MOI. (5) After the mixing of the phage KP168 with the host bacteria for 4 minutes, the percentage of the adsorbed phage reached the highest, which was the optimal adsorption time. (6) The one-step growth curve showed that during the lysis of the host bacteria by phage KP168, the incubation period was about 10 minutes, and the lysis period was about 40 minutes. (7) With the condition of 40 ℃ or pH 7.4, the number of plaques and the activity of phage KP168 reached the highest. (8) The genome of phage KP168 was a linear double-stranded DNA with a length of 40 114 bp. There were 48 possible coding sequences. It had the highest similarity to Klebsiella phage_vB_Kp1. The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions. No resistance genes or virulence factor genes were found. The GeneBank accession number was KT367885. (9) After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI, the inhibition rates of biofilm were similar, with an average of about 45%. After the phage KP168 with a titer of 1×109 PFU/mL acted on the biofilm formed by the host bacteria for 4 h, the destruction rate of biofilm was the highest, reaching an average of 42%.@*Conclusions@#In this study, a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient, phage KP168, is isolated from sewage, which belongs to the tailless phage. It has a wide host spectrum, short adsorption time, and short incubation period, with certain thermal and acid-base stability. Its genomic information is clear, and it does not contain resistance genes or virulence factor genes. It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.

Could bacteriophages isolated from the sewage be the solution to methicillin-resistant Staphylococcus aureus?

@#Introductions: The emergence of multidrug-resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA) complicates the treatment of the simplest infection. Although glycopeptides such as vancomycin still proves to be effective in treating MRSA infections, the emergence of vancomycin-resistant strains limits the long term use of this antibiotic. Bacteriophages are ubiquitous bacterial viruses which is capable of infecting and killing bacteria including its antibiotic-resistant strains. Bactericidal bacteriophages use mechanisms that is distinct from antibiotics and is not affected by the antibioticresistant phenotypes. Objectives: The study was undertaken to evaluate the possibility to isolate bacteriolytic bacteriophages against S.aureus from raw sewage water and examine their efficacy as antimicrobial agents in vitro. Methods: Bacteriophages were isolated from the raw sewage using the agar overlay method. Isolated bacteriophages were plaque purified to obtain homogenous bacteriophage isolates. The host range of the bacteriophages was determined using the spot test assay against the 25 MRSA and 36 MSSA isolates obtained from the Sarawak General Hospital. Staphylococcus saprophyticus, Staphylococcus sciuri and Staphylococcus xylosus were included as non-SA controls. The identity of the bacteriophages was identified via Transmission Electron Microscopy and genomic size analysis. Their stability at different pH and temperature were elucidated. Results: A total of 10 lytic bacteriophages infecting S.aureus were isolated and two of them namely ΦNUSA-1 and ΦNUSA-10 from the family of Myoviridae and Siphoviridae respectively exhibited exceptionally broad host range against >80% of MRSA and MSSA tested. Both bacteriophages were specific to S.aureus and stable at both physiologic pH and temperature. Conclusion: This study demonstrated the abundance of S.aureus specific bacteriophages in raw sewage. Their high virulence against both MSSA and MRSA is an excellent antimicrobial characteristic which can be exploited for bacteriophage therapy against MRSA.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display / 대한소화기학회지

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies.METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay.RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models.CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display / 대한소화기학회지

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

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.

Effect of phage lysin on infection and biofilm formation in rabbit after knee joint prosthesis implantation / 中华骨科杂志

Objective To investigate the effect of local injection of phage lyase LysGH 15 into rabbits' knee joint on the systemic inflammation,local infection around knee joint prosthesis and biofilm formation on the prosthesis surface after their knee joint prosthesis implantation surgeries.Methods Models of Staphylococcus aureus infection on rabbits' knee joint prosthesis after prosthesis implantation were built and divided into experimental group for intra-articular injection with lyase and control group for injection with saline into their joint cavity.The phage lysin LysGH15 was synthesized and purified.On the 1st,2nd and 3rd day after the inoculation with Staphylococcus aureus bacteria into the rabbits' knee joint cavity of the prosthesis implanted side,0.5 ml diluted solution of LysGH15 was injected into the knee joint cavity with infection around the prosthesis for the experimental group rabbits and 0.5 ml saline was injected into the corresponding joint cavity for control group rabbits as blank contrast.On the 1st,3rd,7th and 14th day,blood samples were collected from their ear vein to make plasma procalcitonin test for evaluation of rabbits' systemic infection.After the last time for collection of venous blood samples,these rabbits were killed instantly and their knee joints of prosthesis implantation side were dissociated.Tissue around the prosthesis was processed with HE staining to observe and evaluate the local infection and tissue necrosis around the prosthesis.The biofilm formation on the prosthesis surface was evaluated with semi quantitative method after the observation of samples under scanning electron microscope (SEM).Results After the injection of LysGH15 in experimental group,their serum procalcitonin level,which worked as the systemic inflammatory marker,decreased rapidly especially on the 3rd day after lysin injection.Compared with the control group,the infection degree of experimental group significantly decreased.In the experimental group,the infection and necrosis degree of the tissue surrounding the prosthesis were significantly lower in the experimental group than those in the control group.The semi quantitative scores were conducted for these samples and graded to make rank sum test.The difference between the two groups was statistically significant (U=2.4948,P=0.0126).There was a statistically significant difference in the rank sum test between the two groups in the quality of biofilm formation (U=2.2539,P=0.0242).Conclusion Phage lysin LysGH15 can alleviate the rabbits'systemic inflammation caused by Staphylococcus aureus bacteria after their knee joint prosthesis implantation,reduce the extent of damage caused by infection and inflammation to the tissue around the prosthesis,and inhibit the formation of bacterial biofilm on the surface of implanted prosthesis.

Study on the construction and virulence observation of Rv2346 c gene knockout strains of Mycobacterium tuberculosis mediated by bacteriophage / 中华传染病杂志

Objective To investigate the Rv2346c gene function through constructing Rv2346c gene knockout strains of Mycobacterium tuberculosis (M . tuberculosis) mediated by bacteriophage and observing its virulence after infecting mice lung tissue in vivo .Methods The affinal exchange sites (AES) of the target gene was built ,and then integrated into the phage genomes of M .tuberculosis for harvesting the phagemids .The phagemids was imparted into Mycobacterium smegmatis to get recombinant phages with the same AES .A high titer of the recombinant phages was harvested through amplification in vitro . The M .tuberculosis was transfected and coated on solid medium with hygromycin resistance and cultured for 4 weeks at 37℃ .Single clone was picked out and gene knock-out was confirmed by PCR . Then C57BL/6J mice were infected with either wild type strain (WT ) or knockout strain (KO ) of M . tuberculosis .Mice mortality ,lung tissue inflammation and colony-forming units (CFU ) counts in vitro were observed 6 to 8weeks post infection with different strains . Paired-samples t test was used for comparison between groups ,chi-square test was used for comparison of rates .Results The products of PCR and inserted fragment sizes were consisted with the expectation and confirmed to be the target gene . The target fragment of Rv2346c was removed successfully and the mice were infected for 6-8 weeks .Themice infected with Rv2346c KO strain had reduced mortality (53％ vs 20％ ,χ2 =6 .1112 ,P<0 .05) ,lung tissue inflammation (1040 ± 89 vs 1960 ± 56 ,t=7 .1016 ,P<0 .05) and CFU count in vitro (15 .0 ± 0 .8 vs 90 .0 ± 1 .5 ,t=23 .0361 , P<0 .05) compared with WT strain 6-8 weeks post infection .Conclusion Rv2346c gene knockout strains of M . tuberculosis mediated by bacteriophageis are successfully constructed ,which establishes the foundation for the future gene function study of Rv2346c .

Bacteriophage and its application in ocular diseases / 中华实验眼科杂志

Phage (bacteriophage) is a kind of viruses which can infect bacteria,actinomyces and spirochetes.The phage can only reproduced by host bacteria instead of living independently.The process of phage reproduction is also the process of sterilization.Nowadays,the "post-antibiotic era" that leading by global rise in bacterial resistance,urges scientists to explore alternative therapies for antibiotics.The phage therapy has attracted the attention of researchers.Several researchers has successfully treated bacterial keratitis,endophthalmitis and conjunctivitis by using bacteriophage and phage lyzyme.Phage not only kills pathogenic bacteria,but also maintains the structural integrity of eye.This article reviewed the phage structure and classification,the process of its discovery and development,the bactericidal mechanism of phage,its application and therapeutic characteristics in the treatment of ophthalmological diseases.

Influencia de la procedencia de los componentes del medio de cultivo en la calidad de los ensayos microbiológicos para la determinación de bacteriófagos / Influence of the origin of the components of the culture medium on the quality of the microbiological assays for the determination of bacteriophages

La contaminación por virus bacterianos es uno de los problemas más difíciles de evaluar en la industria biotecnológica moderna, sobre todo en las producciones de proteínas que están basadas en cepas recombinante de Escherichia coli. Para certificar que un banco de cepas está libre de bacteriófagos el método más recomendado sigue siendo la inducción de la fase del ciclo lítico sobre placas con medios semisólidos. En este trabajo nos propusimos evaluar medios de cultivos elaborados con bases de diferentes casas comerciales para la detección de bacteriófagos contaminantes. Para ello utilizamos un aislado de bacteriófago ambiental y la cepa sensible LE 392 y bases de las firmas OXOID, BioCen y Biolife. Después de estandarizar la metodología de ensayo se evaluó la habilidad de una preparación concentrada del aislado ambiental de formar placas de lisis en los medios elaborados con reactivos de las tres casas comerciales. La mayor cantidad de placas de lisis se obtuvo cuando se emplearon las bases provenientes del Centro Nacional de Biopreparados, el medio donde menos placas de lisis observamos fue empleando bases proveniente de la Casa comercial Biolife con dos órdenes de magnitud menos. El medio preparado con bases provenientes de la casa comercial OXOID fue el que mostró mejor consistencia entre lotes(AU)

Contamination by bacterial viruses is one of the most difficult problems to evaluate in the modern biotechnology industry, especially in the productions of recombinant proteins that are based on Escherichia coli strains. To certify that a strain bank is free of bacteriophage, the most recommended method is still the induction of the lytic cycle phase on plates with semisolid media. In this work we set out to evaluate means of cultures made with bases of different commercial houses for the detection of contaminating bacteriophages. For this purpose we used an environmental bacteriophage isolate and the LE392 sensitive strain and bases of the firms OXOID, BioCen and Biolife. After standardization of the test methodology, the ability of a concentrated preparation of the environmental isolate to form lysis plates in the media made with reagents from the three commercial houses was evaluated. The largest number of lysis plates was obtained when using the bases from the National Center of Biopreparates, the means where less lysis plates were observed was using bases from the Biolife Commercial House with two orders of magnitude less. The medium prepared with bases from the commercial house OXOID was the one that showed better lot to lot consistency(AU)

Effects of different titers of bacteriophage D29 on growth and function of airway epithelia cell 9HTE / 重庆医学

Objective To research the effects of different titers of bacteriophage D29 on growth and function of airway epithelial cell 9HTE.Methods Cell viability rates was analyzed after applying high(109 PFU/mL)and low(107 PFU/mL)titers of bacteriophage D29 and phage buffer respectively by MTT colorimetry.Additionally,the secretion levels of IL-6,IL-8 in cell culture supernatant were detected by ELISA.RT-PCR was performed to detect the expression of ICAM-1 mRNA.Cell apoptosis rate was analyzed by flow cytometry.Results There was no difference in cell growth,secretion levels of IL-6,IL-8,ICAM-1 mRNA and cell apoptosis rate between cells treated with high and low titers of D29 and phage buffer(P>0.05).Conclusion Neither high nor low titer of bacteriophage D29 exerts effect on growth and function of airway epithelial cell 9HTE in vitro.

Selection and identification of human monoantibody TRAb Fab fragment / 天津医药

Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6%, and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6%. Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.

Characterization of bacteriophage vB_AbaP_PD-AB9 infecting Acinetobacter baumannii with broad host range / 中华检验医学杂志

Objective To isolate bacteriophages against extensively-drug resistant Acinetobacter baumannii from hospital sewage and analyze their biological characteristics.Methods Extensively-drug resistant Acinetobacter baumannii isolated from several hospitals in Shanghai during December, 2013 to July, 2014 were used as host bacteria, adopting double-layer agar method to isolate bacteriophages from raw sewage of these hospitals.The phage with broad host range was selected for further study, including observation of electron microscopic morphology, examination of thermal stability, pH stability and the optimal MOI, drawing of the adsorption, one-step-growth and infection curves, as well as sequencing of the phage genome DNA. Results An extensively-drug resistant Acinetobacter baumannii bacteriophage vB_AbaP_PD-AB9 ( PD-AB9 for short) with broad host range was isolated, and electron microscopy revealed it belonged to Podoviridae family.The optimal MOI of PD-AB9 was 0.001.PD-AB9 remained stable among 4 ℃to 50 ℃and pH 4 to 11.In the adsorption experiment, the adsorption rate of PD-AB9 reached above 95%within 5 min.PD-AB9 had a latent period of 4 min and a burst size of 213.PD-AB9 could obviously restrain the host growth, with faster effect at the higher MOIs (MOI=1, 0.1, 0.01) than at the lower ones (MOI=0.001, 0.000 1).Furthermore, genome of PD-AB9 proved to be a double-stranded linear DNA with size of 40 938 bp and GC content of 39.34%.Conclusions PD-AB9 exhibits good thermal stability, wide pH tolerance range, very fast adsorption, a short latent period, a large burst size and it could quickly cause effective host lysis after infection.Therefore, PD-AB9 is promised to act as a new antimicrobial agent to control drug-resistant Acinetobacter baumannii infections and its bio information remains to be further studied.

El desafío de controlar las enfermedades transmitidas por alimentos: bacteriófagos como una nueva herramienta biotecnológica / The challenge of controlling foodborne diseases: bacteriophages as a new biotechnological tool

Foodborne diseases are an increasing public health issue, in which bacterial pathogens have a transcendental role. To face this situation, the food industry has implemented several control strategies, using in the last decade some biotechnological tools, such as direct application of bacteriophages on food, to effectively control bacterial pathogens. Their bactericidal and safe properties to humans and animals have been widely described in the literature, being nowadays some bacteriophage-based products commercially available. Despite this, there are so many factors that can interfere in their biocontrol effectiveness on food, therefore is essential to consider these factors before their application. Thus, the optimal bacterial reduction will be achieved, which would produce a safer food. This review discusses some factors to consider in the use of bacteriophages as biocontrol agents of foodborne pathogens, including historical background, taxonomy and biological description of bacteriophages, and also advantages, disadvantages, and considerations of food applications.

Las enfermedades transmitidas por alimentos son un creciente problema de salud pública, donde los agentes patógenos bacterianos juegan un rol trascendental. La industria alimentaria ha implementado diversas medidas de control para enfrentar esta situación, utilizando en la última década algunas herramientas biotecnológicas, como es la aplicación de bacteriófagos directamente en los alimentos. Sus propiedades exclusivamente bactericidas e inocuas para el hombre y los animales han sido descritas ampliamente en la literatura científica, existiendo a la fecha algunos productos comerciales disponibles en el mercado internacional. A pesar de esto, diversos son los factores que pueden influir en su efectividad bio-controladora en alimentos, por lo que conocer dichos factores resulta fundamental antes de considerar su aplicación. De esta manera, se logrará obtener la máxima actividad reductora de la carga bacteriana, generando así un alimento más seguro. Esta revisión aborda ciertos factores a considerar para el uso de bacteriófagos como agentes bio-controladores de patógenos alimentarios, incluyendo antecedentes históricos, taxonomía y descripción biológica de bacteriófagos, así como ventajas, desventajas y consideraciones de su aplicación en alimentos.