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

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.

Actualización en fibrosis quística - fagoterapia: ¿es el futuro para tratar bacterias multirresistentes? / Phage therapy: is the future to treat multiresistant bacteria?

Las infecciones respiratorias representan una morbilidad y mortalidad significativas, con aumento progresivo de la resistencia a los antibióticos. La escasez de nuevos antibióticos disponibles y la pérdida de eficacia de los antiguos, ha impulsado a investigar otras alternativas de tratamiento. La terapia con bacteriófagos (fagos) representa uno de esos enfoques, la que ha demostrado ser eficaz contra una variedad de patógenos bacterianos, incluidas las cepas resistentes a los medicamentos. La administración puede ser tópica, intravenosa o inhalada, esta última requiere preparaciones estables de fagos y sistemas adecuados para proporcionar partículas que accedan al árbol respiratorio. En esta comunicación se revisan diversos aspectos de los bacteriófagos, los que podrían ser de gran utilidad para el tratamiento de las infecciones pulmonares en pacientes con diagnóstico de fibrosis quística.

Respiratory infections represent a significant morbidity and mortality, with a progressive increase in resistance to antibiotics. The scarcity of new antibiotics available and the loss of efficacy of the old ones has prompted investigation of other treatment alternatives. Bacteriophage (phage) therapy represents one such approach that has been shown to be effective against a variety of bacterial pathogens, including resistant strains to medications. Administration can be topical. Intravenous or inhaled, the latter requiring stable preparations of phages and adequate systems to provide particles that will access the respiratory tree. In this communication various aspects of bacteriophages and their clinical utility are reviewed, which could be very useful for the treatment of pulmonary infections in patients diagnosed with cystic fibrosis.

Screening of bacteriophages against different genotypes of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae Isolated from five hospitals in Cavite and Metro Manila, Philippines

Background@#Extended-spectrum β-lactamase (ESBL) K. pneumoniae infections are emerging health problems in the Philippines. Recently, bacteriophages have been explored to target several antibiotic-resistant bacteria as a potential alternative therapeutic option to conventional antibiotics. @*Objectives@#This study isolated extended-spectrum β-lactamase (ESBL) producing K. pneumoniae harboring different β-lactamase genes to evaluate the host range specificity of isolated bacteriophages.@*Methodology@#K. pneumoniae were isolated from five selected hospitals in Cavite and Metro Manila, Philippines and their ESBL-production was determined through the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). The identity of the isolates was then confirmed by amplification and sequencing of the 16 rRNA gene. The type of β-lactamase genes carried by the K. pneumoniae ESBL-positive strains was detected by amplification of the bla , bla , bla and bla genes. Meanwhile, bacteriophages were isolated from CTX-M TEM SHV OXA-1 water samples in Marikina River and their host range specificity was tested against the different ESBLproducing K. pneumoniae strains.@*Results@#From a total of 25 K. pneumoniae, 6 (24%) were found to be ESBL-producers by PCDDT. Genotyping of the β-lactamase genes showed that one of the phenotypically confirmed isolates contained the bla while CTX-M another possessed both the bla and bla genes. Furthermore, another isolate harbored the bla , bla , CTXM SHV CTX-M OXA-1 and bla genes while the remaining isolates contained all the four bla genes tested. Meanwhile, two virulent SHV phages namely, KP1 and KP2 that showed the largest clearing zones against K. pneumoniae were selected to determine their host range specificity against the different ESBL-positive K. pneumoniae strains. Both phages were able to infect and lyse all ESBL-positive K. pneumoniae regardless of the type or number of bla genes they possessed. Phage KP2, which showed the highest lytic capability, was then subjected to Transmission Electron Microscopy (TEM) and was found to belong to the Order Caudovirales under the Family Myoviridae. @*Conclusion@#This study showed that phage KP2 was host-specific to the different ESBL-producing K. pneumoniae harboring single or multiple bla genes suggesting that it might hold a great potential for possible phage therapy against ESBL-producing K. pneumoniae infections.

Phage therapy as an approach to control salmonella enterica serotype enteritidis infection in mice

Abstract INTRODUCTION: Salmonella enterica serotype Enteritidis (S. Enteritidis) is a cause of food-borne human illness. Given the prevalence of antibiotic resistance of Salmonella Enteritidis and the lack of antibiotic efficacy in future years, its replacement with other agents is necessary. One of the most useful agents is bacteriophages. METHODS S. Enteritidis was identified using a multiplex polymerase chain reaction assay. The effective bacteriophages were isolated from hospital wastewater samples. The effects of the bacteriophages were evaluated both in vitro and in vivo. RESULTS The phage SE20 belonged to the Podoviridae family, and the genome size was 40 kb. The evaluation of phage SE20 at variable pH ranges showed its susceptibility to pH < 3 and pH > 12. The animal model showed that mice infected with S. Enteritidis developed hepatomegaly and splenomegaly, but did not experience gastrointestinal complications after receiving the bacteriophages. CONCLUSIONS The results of this study suggest that phage SE20 is a promising candidate for controlling salmonellosis caused by Salmonella Enteritidis.

Bacteriophages in the control of pathogenic vibrios

Vibrios are common inhabitants of marine and estuarine environments. Some of them can be pathogenic to humans and/or marine animals using a broad repertory of virulence factors. Lately, several reports have indicated that the incidence of Vibrio infections in humans is rising and also in animals constitute a continuing threat for aquaculture. Moreover, the continuous use of antibiotics has been accompanied by an emergence of antibiotic resistance in Vibrio species, implying a necessity for efficient treatments. One promising alternative that emerges is the use of lytic bacteriophages; however, there are some drawbacks that should be overcome to make phage therapy a widely accepted method. In this work, we discuss about the major pathogenic Vibrio species and the progress, benefits and disadvantages that have been detected during the experimental use of bacteriophages to their control.

Efeito antimicrobiano do bacteriófago geneticamente modificado φEf11/φFL1C(Δ36)PnisA sobre cepas de E. faecalis em biofilme estático e em canais radiculares infectados / Antibacterial effect of a Genetically-Engineered Bacteriophage φEf11/φFL1C(Δ36)PnisA on static biofilms and on Dentin Infected with E. faecalis

A presença de micro-organismos no sistema de canais radiculares (SCR) tem sido apontada como uma das principais causas de insucesso da terapia endodôntica. A capacidade de formar biofilme e penetrar nos túbulos dentinários são fatores de sobrevivência que favorecem a perpetuação de micro-organismos no interior do SCR. Terapias que promovam a desorganizazão de biofilmes e eliminação de bactérias dentro dos túbulos dentinários são fundamentais para a desinfecção endodôntica. Uma terapia descoberta há um século, denominada de Bacteriofagoterapia, baseia-se na utilização de vírus capazes de infectar e matar bactérias. Esta abordagem antimicrobiana tem recebido bastante atenção atualmente por representar uma alternativa para o tratamento de doenças causadas por bactérias multirresistentes aos antibióticos. O objetivo deste estudo foi avaliar a eficácia de um bacteriófago modificado geneticamente, φEf11/φFL1C(Δ36)PnisA, para eliminar biofilmes de duas cepas de E. faecalis: JH2-2 (sensível à vancomicina e resistente ao ácido fusídico e à rifampicina) e V583 (resistente à vancomicina). Este estudo foi dividido em dois experimentos distintos. No primeiro experimento, biofilmes estáticos de 48 horas de cepas de E. faecalis JH2-2 (pMSP3535 nisR / K) ou V583 (pMSP3535 nisR / K) formados em lâminulas de vidro (coverslips) foram inoculados por suspensão do bacteriófago φEf11/φFL1C(Δ36)PnisA. Após 48 horas de incubação, a biomassa bacteriana foi fotografada por microscopia confocal e as células viáveis foram quantificadas por medição de unidades formadoras de colônias (UFC). No segundo experimento, segmentos radiculares de dentes humanos extraídos foram cimentados em dispositivos vedáveis de duas câmaras para formar modelos ex vivo com dentina infectada, contendo solução tampão na câmara inferior. Os modelos foram inoculados com uma suspensão de E. faecalis V583 ou E. faecalis JH2-2. Após sete dias de incubação a 37°C, adicionou-se ao canal de cada segmento de dentina infectada dos grupos 2 e 5 uma suspensão do fago geneticamente modificado, φEf11/φFL1C(Δ36)PnisA e manteve-se a incubação por mais 72 horas. Os segmentos de dentina foram instrumentados com Gates Glidden e a solução tampão foi aliquotada para semeadura e contagem de UFC e aferição do título residual de células de E. faecalis. Os resultados do primeiro experimento mostraram uma diminuição de 10-100 vezes (p≤ 0,05) das células viáveis (UFC / biofilme) após tratamento com bacteriófago, o que foi consistente com a comparação das imagens de biofilme tratado e não tratado visualizadas com projeções máximas da série Z. No segundo experimento a titulação de E. faecalis verificada após tratamento com o bacteriófago foi reduzida em 18% para os modelos infectados com JH2-2 e em 99% (p≤ 0,05) nos modelos infectados com V583. Com base nesses resultados, pode-se concluir que a biomassa dos biofilmes de E. faecalis, tanto sensíveis quanto resistentes à vancomicina, foi significantemente reduzida após a infecção pelo bacteriófago φEf11/φFL1C(Δ36)PnisA. Além disso, o tratamento da dentina infectada por E. faecalis com bacteriófago φEf11/φFL1C(Δ36)PnisA resultou em diminuição da população bacteriana residual de cepas sensíveis e resistentes à vancomicina, alcançando significância estatística no grupo que utilizou a cepa V583.

Residual microorganisms in the root canal system (RCS) have been identified as the main cause of endodontic therapy failure. The ability to form a biofilm and penetrate the dentin tubules are survival factors that favor the perpetuation of microorganisms within the RCS. Therapies that promote the disorganization of biofilms and elimination of bacteria within the dentinal tubules are essential for endodontic disinfection. A therapy discovered a century ago called Bacteriophage Therapy is based on the use of viruses capable of infecting and killing bacteria. Recently, this antimicrobial approach has been receiving considerable attention since it represents an alternative for the treatment of diseases caused by multiresistant antibiotic bacteria. The aim of this study was to evaluate the efficacy of a genetically engineered bacteriophage, φEf11/φFL1C(Δ36)PnisA, to disrupt biofilms of two Enterococcus faecalis strains: JH2-2 (vancomycin sensitive) and V583 (vancomycin resistant). This study was divided into two separate experiments. In the first experiment, 24 hour static biofilms of E. faecalis strains JH2-2(pMSP3535 nisR/K) and V583(pMSP3535 nisR/K) formed on cover slips were inoculated with bacteriophage φEf11/φFL1C(Δ36)PnisA. After 24 and 48 hours incubation, the bacterial biomass was imaged by confocal microscopy and viable cells were quantified by colony forming unit (CFU) measurement. In the second experiment, extracted human dentin root segments were cemented into sealable two-chamber devices, fabricated from syringe needle caps to form in vitro infected-dentin models. The models were inoculated with an overnight suspension of either E. faecalis V583 (vancomycin resistant strain) or E. faecalis JH2-2 (fusidic acid and rifampin resistant, vancomycin sensitive strain). After 7 days of incubation at 37°C, a suspension of a genetically engineered phage, φEf11/φFL1C(Δ36)PnisA, was added to the root canal of each infected dentin segment, and the incubation was continued for an additional 72-hours. Dentin was harvested from the walls of each root canal and assayed for the residual titer of E. faecalis cells. The results from the first experiment showed a 10-100-fold fewer decrease in viable cells (CFU/biofilm) after bacteriophage treatment, which was consistent with comparisons of treated and untreated biofilm images visualized as max projections of the Z-series. On the second experiment, the recovered E. faecalis titer was reduced by 18% for the JH2-2 infected models, and by 99% for the V583 infected models. These results suggest that the biomass of E. faecalis biofilms, both sensitive and resistant to vancomycin, was significantly reduced after infection by bacteriophage φEf11/φFL1C(Δ36)PnisA. In addition, treatment of E. faecalis-infected dentin with the phage resulted in the decrease of the residual bacterial population for both susceptible and vancomycin resistant strains, reaching statistical significance in strain V583 group.

Una mezcla de bacteriófagos reduce los recuentos de Salmonella enterica serovar. Enteritis en tejidos de salmón fresco y ahumado / Bacteriophage cocktail reduces Salmonella enterica serovar. Enteritis counts in raw and somoked salmon tissues

El uso de bacteriófagos en el biocontrol de patógenos está adquiriendo cada vez más aceptación. En este estudio se evaluó la efectividad de bacteriófagos en la reducción de los recuentos de Salmonella Enteritidis en salmón fresco y ahumado. Para ello, 25 muestras por grupo fueron contaminadas con S. Enteritidis, tratadas con una mezcla de bacteriófagos e incubadas durante 10 días a 18 °C o a 4 °C. A los días 3, 6 y 10 se obtuvo una reducción signifi cativa de los recuentos de S. Enteritidis en las muestras de salmón fresco incubadas a ambas temperaturas: la reducción fue de entre 0,75 y 3,19 log10 UFC/g a 18 °C y de entre 2,82 y 3,12 log10 UFC/g a 4 °C. En salmón ahumado las reducciones fueron menores (entre 1,02 y 1,96 log10 UFC/g a 18 °C y entre 0,50 y 1,16 log10 UFC/g a 4 °C). Los resultados indican que estos bacteriófagos constituyen una potencial herramienta de biocontrol de S. Enteritidis en tejidos de salmón fresco y ahumado. © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L. Todos los derechos reservados

The use of bacteriophages for the biocontrol of food-borne pathogens is increasingly gaining acceptance. In this study, the effectiveness of bacteriophages to reduce Salmonella Enteritidis counts was evaluated in raw and smoked salmon tissues. Groups of 25 samples each were contaminated with S. Enteritidis, treated with a phage mix and then incubated for ten days at 18 °C and 4 °C. A signifi cant bacterial reduction was obtained on days 3, 6 and 10 in raw salmon samples incubated at 18 °C (from 0.75 to 3.19 log10 CFU/g) and at 4 °C (from 2.82 to 3.12 log10 CFU/g), whereas in smoked salmon lower reductions were achieved (from 1.02 to 1.96 log10 CFU/g at 18°C and from 0.50 to 1.16 log10 CFU/g at 4 °C). These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent against S. Enteritidis in raw and smoked salmon tissues