<p>Antimicrobial resistant bacteria pose a serious threat to global public health. With the development of antibioticresistance far outpacing the discovery of new antibiotics, there is a need to  develop  alternative  strategies  to  control  bacterial  infections without increasing  antibiotic resistance. This study focused on developing bacteriophage therapy as a non-antibiotic means to control colibacillosis in poultry. Avian colibacillosis causes significant mortality and economic loss to poultry industries around the world. The etiological agent is Avian Pathogenic<em> E.  coli</em> (APEC), with serotypes are O78, O1, O2 and O5 most often associated with infections. Here, seven bacteriophages (AHP, MP1, MP2, AKA, MKA, AHC and MIA) were isolated from human and poultry wastewater samples to target these four APEC serotypes.</p> <p>The host-spectrum analysis of these phages revealed that all seven phages lysed at least two different APEC strains, with four phages lysing four or more distinct APEC serotypes. Taken together, the isolated phages covered 90% (9/10) of O-serotyped APEC strains targeted in our study. When co-cultured with the targeted APEC strain, bacterial concentrations in phage treated APEC cultures (average OD₆₀₀= 0.09) were significantly (P < 0.05) lower than those of untreated cultures  (average  OD₆₀₀=  1.22)  after  4  hr  incubation.  However,  exposure  of  the  phages  to simulated gastric fluid (pH 2.2–2.5) reduced viability of three of the seven phages by 2.35 –4.01 log PFU/mL after 90 min and to undetectable levels after 5 min for other four phages. In contrast, phage viability was not impacted by simulated intestinal fluid (SIF) with no reductions in phage concentrations after exposure to SIF for 3 hr. All seven bacteriophages were encapsulated in sodium  alginate  microcapsules  with  encapsulation  efficiencies  between  94.4%  to  98.9%.  In contrast to unprotected phages, viability of encapsulated phages was reduced by only0.74 –1.21  log PFU/mL when exposed to SGF for 90 min. Incubating the encapsulated phages sequentially in SGF (1 hr) and SIF (3 hr) indicated that 85% -90% of phages were released from the microcapsules after1 hr incubation in SIF with the maximum release of phages from the microcapsules occurring after 3 hr of incubation in SIF. To assess the in vivoefficacy of the phage treatment, broiler chicks were challenged with APEC and treated with a mixture of unprotected and encapsulated phages. Concentrations of the APEC in the ceca of phage treated birds (2.79 log CFU/g) were significantly lower (P < 0.05) than those of untreated birds (6.18 log CFU/g) by 4 d post-challenge. Additionally, in  most  cases,  APEC  was  not  recovered  from  the  lungs  of  phage  treated  birds  whereas concentrations of APEC in lungs of untreated birds was 4.81 log CFU/g. Hence, these results indicate that phage treatmenteffectively controlled APEC colonization and replication in the ceca and lungs of APEC-challenged chickens and provide further evidence of the viability of phage-based treatments as a non-antibiotic means of controlling bacterial infections in chickens.</p>

E. coli isoladas de lesões de celulite aviária em frangos de corte produzem uma citotoxina, denominada ECVF (E. coli Vacuolating Factor), que causa intensa vacuolização citoplasmática em células aviárias, mas não em células mamárias. A importância de ECVF na patogenia da celulite foi avaliada neste estudo. ECVF purificado foi inoculado subcutaneamente em frangos de corte, e induziu sinais de inflamação nos tecidos subcutâneo, adiposo e conjuntivo. Em ensaios de citotoxicidade, foi verificado que ECVF induz alterações citoplasmáticas e nucleares que podem afetar diretamente o metabolismo celular, entre elas condensação da cromatina e fragmentação nuclear, intensa vacuolização citoplasmática e desorganização do citoesqueleto, conduzindo à apoptose. Também foi verificada interação de ECVF com proteínas de células aviárias, em detrimento das de células de mamíferos, sugerindo uma especificidade da toxina a este tipo celular. Nossos resultados, apoiados por dados de estudos anteriores, permitem sugerir um importante papel de ECVF na patogenia da celulite aviária. / E. coli isolated from cellulitis lesions in broiler chickens produce a citotoxin, called ECVF (Escherichia coli Vacuolating Factor), which causes intense cytoplasm vacuolization in avian cells, but not in mammalian cells. The importance of ECVF in the pathogenesis of avian cellulitis was assessed in this study. Purified ECVF was inoculated subcutaneously in broiler chickens, and induced signs of inflammation on subcutaneous, adipose and connective tissues. In citotoxicity assays, we verified that ECVF induced cytoplasmic and nuclear alterations, which can affect cellular metabolism directly, such as chromatin condensation and nuclear fragmentation, intense cytoplasm vacuolization, and disorganization of cytoskeleton, leading to apoptosis. It was also verified the interaction of ECVF with proteins of avian cells, instead of those from mammalian cells, suggesting the specificity of this toxin to this cells. Our results, supported by data from previous studies, suggest an important role of ECVF in the pathogenesis of avian cellulitis.

E. coli isoladas de lesões de celulite aviária em frangos de corte produzem uma citotoxina, denominada ECVF (E. coli Vacuolating Factor), que causa intensa vacuolização citoplasmática em células aviárias, mas não em células mamárias. A importância de ECVF na patogenia da celulite foi avaliada neste estudo. ECVF purificado foi inoculado subcutaneamente em frangos de corte, e induziu sinais de inflamação nos tecidos subcutâneo, adiposo e conjuntivo. Em ensaios de citotoxicidade, foi verificado que ECVF induz alterações citoplasmáticas e nucleares que podem afetar diretamente o metabolismo celular, entre elas condensação da cromatina e fragmentação nuclear, intensa vacuolização citoplasmática e desorganização do citoesqueleto, conduzindo à apoptose. Também foi verificada interação de ECVF com proteínas de células aviárias, em detrimento das de células de mamíferos, sugerindo uma especificidade da toxina a este tipo celular. Nossos resultados, apoiados por dados de estudos anteriores, permitem sugerir um importante papel de ECVF na patogenia da celulite aviária. / E. coli isolated from cellulitis lesions in broiler chickens produce a citotoxin, called ECVF (Escherichia coli Vacuolating Factor), which causes intense cytoplasm vacuolization in avian cells, but not in mammalian cells. The importance of ECVF in the pathogenesis of avian cellulitis was assessed in this study. Purified ECVF was inoculated subcutaneously in broiler chickens, and induced signs of inflammation on subcutaneous, adipose and connective tissues. In citotoxicity assays, we verified that ECVF induced cytoplasmic and nuclear alterations, which can affect cellular metabolism directly, such as chromatin condensation and nuclear fragmentation, intense cytoplasm vacuolization, and disorganization of cytoskeleton, leading to apoptosis. It was also verified the interaction of ECVF with proteins of avian cells, instead of those from mammalian cells, suggesting the specificity of this toxin to this cells. Our results, supported by data from previous studies, suggest an important role of ECVF in the pathogenesis of avian cellulitis.

Aviäre pathogene E. coli (APEC) sind extraintestinale Pathogene, die beim Huhn systemische Infektionskrankheiten hervorrufen. Zur Identifizierung Gene, die an der Kolonisierung des Wirtes beteiligt sind, wurde ein Lungen-Infektionsmodell in 5 Wochen alten SPF Hühnern etabliert. In dem Modell wurden 1.800 mittels Signature-tagged-Mutagenese (STM) hergestellten Mutanten des APEC Stamms IMT5155 (O2:K1:H5; ST-Komplex 95) auf ihre Fähigkeit zur Kolonisierung getestet. Die Untersuchung führte zur Identifizierung Gene, einschließlich Adhäsin-, LPS- und Kapsel-bildenden Genen, sowie Genen mit putativer Funktion. Die STM-Analyse erlaubte zudem die Identifizierung eines zuvor nicht charakterisierten putativen Fimbrien-bildenden Adhäsins (Yqi). Das Genprodukt wurde vorläufig als ExPEC Adhäsin I (EA/I) bezeichnet. Eine Deletion des EA/I-Gens führte zu einer Reduzierung der Adhäsionsfähigkeit des Stammes IMT5155 in vitro und in vivo. Eine Komplementierung des EA/I-Gens in trans resultierte in einer Wiederherstellung des Adhäsions¬vermögens in vitro. Das EA/I-Protein (~39 kDa) wurde als Fusionsprotein in vitro exprimiert, und mittels SDS-PAGE und Western Blot nachgewiesen. Durch Überexpression des EA/I-Operons in dem Fimbrien-negativen E. coli-Stamm AAEC189 konnten mittels elektronenmikroskopischer Aufnahmen Fimbrien-bildende Strukturen auf der äußeren Membran dargestellt werden. Das Vorkommen des yqi in den untersuchten extraintestinal pathogenen E. coli (ExPEC), bei gleichzeitigem Fehlen in allen untersuchten intestinal pathogenen E. coli bestätigt die Bezeichnung ExPEC Adhäsin I. Die Prävalenz des EA/I-Gens war am stärksten assoziiert mit Stämmen der B2-Phylogenetische-Gruppe und des ST95-Komplexes des Multi-Lokus-Sequenz-Typisierungs (MLST)-Schemas. Sequenzanalysen ergaben zudem erste Hinweise auf eine positive Selektion des EA/I-Gens innerhalb dieses Komplexes. In der vorliegenden Arbeit gelang somit die Identifizierung und Charakterisierung des neuen ExPEC Adhäsin I. / The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry. To identify genes, involved adhesion and colonization, a lung colonization model of infection was established in 5-week old specific-pathogen free (SPF) chickens, and Signature-tagged mutagenesis (STM) was applied to this model by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; ST complex 95). This led to the identification of new genes of interest, including adhesins, genes involved in capsule and LPS formation, and genes of putative function. Among the many genes identified was one coding for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis. Its gene product was temporarily designated ExPEC Adhesin I (EA/I). Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein (~ 39 kDa) was expressed as a fusion protein in vitro as shown by SDS-PAGE and western blotting. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial like appendages protruding out of the bacterial outer membrane. We observed that the adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates and absent in all of the intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the E. coli phylogenetic group B2 and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I.