Structural Analysis of Jumbo Coliphage phAPEC6.

The phAPEC6 genome encodes 551 predicted gene products, with the vast majority (83%) of unknown function. Of these, 62 have been identified as virion-associated proteins by mass spectrometry (ESI-MS/MS), including the major capsid protein (Gp225; present in 1620 copies), which shows a HK97 capsid protein-based fold. Cryo-electron microscopy experiments showed that the 350-kbp DNA molecule of Escherichia coli virus phAPEC6 is packaged in at least 15 concentric layers in the phage capsid. A capsid inner body rod is also present, measuring about 91 nm by 18 nm and oriented along the portal axis. In the phAPEC6 contractile tail, 25 hexameric stacked rings can be distinguished, built of the identified tail sheath protein (Gp277). Cryo-EM reconstruction reveals the base of the unique hairy fibers observed during an initial transmission electron microscopy (TEM) analysis. These very unusual filaments are ordered at three annular positions along the contractile sheath, as well as around the capsid, and may be involved in host interaction.

Nanobodies targeting conserved epitopes on the major outer membrane protein of Campylobacter as potential tools for control of Campylobacter colonization.

Campylobacter infections are among the most prevalent foodborne infections in humans, resulting in a massive disease burden worldwide. Broilers have been identified as the major source of campylobacteriosis and reducing Campylobacter loads in the broiler caeca has been proposed as an effective measure to decrease the number of infections in humans. Failure of current methods to control Campylobacter in broilers stresses the urgency to develop novel mitigation measures. We obtained six nanobodies with a broad specificity, that recognize strains belonging to the two most relevant species, Campylobacter jejuni and Campylobacter coli. The target of the nanobodies was identified as the major outer membrane protein, a porin that contributes to bacterial virulence and viability. Multimerization of the nanobodies led to agglutination of C. jejuni cells, which may affect colonization in the chicken gut. These Campylobacter-specific nanobodies may be useful to develop a strategy for preserving chickens from Campylobacter colonization.

Curing a large endogenous plasmid by single substitution of a partitioning gene.

To investigate whether plasmid-free cells of pathogenic Escherichia coli can be isolated by disrupting a single gene in an endogenous plasmid without further treatment, the effect of the disruption of partitioning genes on the inheritance of the endogenous plasmid pUTI89 of the uropathogenic E. coli strain UTI89 was studied. We found that mutation of parB, which encodes a type Ib partitioning protein, could cause loss of the endogenous plasmid at a ratio of about 1%. Clones derived from parB mutants, identified by antibiotic sensitivity, were all plasmid free. Plasmid instability caused by the parB mutation was found to correlate with a negative effect on host cell growth. Thus, in this pathogenic E. coli, an endogenous plasmid as large as 114 kbp could be cured effectively by targeting a single type Ib partitioning gene followed by passaging, which may facilitate further investigations on the function of endogenous plasmids in their natural hosts.

Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.

NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the nadB gene of E. coli K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic E. coli O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.

Plasmid parB contributes to uropathogenic Escherichia coli colonization in vivo by acting on biofilm formation and global gene regulation.

The endogenous plasmid pUTI89 harbored by the uropathogenic Escherichia coli (UPEC) strain UTI89 plays an important role in the acute stage of infection. The partitioning gene parB is important for stable inheritance of pUTI89. However, the function of partitioning genes located on the plasmid in pathogenesis of UPEC still needs to be further investigated. In the present study, we observed that disruption of the parB gene leads to a deficiency in biofilm formation in vitro. Moreover, in a mixed infection with the wild type strain and the parB mutant, in an ascending UTI mouse model, the mutant displayed a lower bacterial burden in the bladder and kidneys, not only at the acute infection stage but also extending to 72 hours post infection. However, in the single infection test, the reduced colonization ability of the parB mutant was only observed at six hpi in the bladder, but not in the kidneys. The colonization capacity in vivo of the parB-complemented strain was recovered. qRT-PCR assay suggested that ParB could be a global regulator, influencing the expression of genes located on both the endogenous plasmid and chromosome, while the gene parA or the operon parAB could not. Our study demonstrates that parB contributes to the virulence of UPEC by influencing biofilm formation and proposes that the parB gene of the endogenous plasmid could regulate gene expression globally.

The dynamic transcriptome during maturation of biofilms formed by methicillin-resistant Staphylococcus aureus.

Background: Methicillin-resistant Staphylococcus aureus (MRSA), a leading cause of chronic infections, forms prolific biofilms which afford an escape route from antibiotic treatment and host immunity. However, MRSA clones are genetically diverse, and mechanisms underlying biofilm formation remain under-studied. Such studies form the basis for developing targeted therapeutics. Here, we studied the temporal changes in the biofilm transcriptome of three pandemic MRSA clones: USA300, HEMRSA-15, and ST239. Methods: Biofilm formation was assessed using a static model with one representative strain per clone. Total RNA was extracted from biofilm and planktonic cultures after 24, 48, and 72 h of growth, followed by rRNA depletion and sequencing (Illumina Inc., San Diego, CA, United States, NextSeq500, v2, 1 × 75 bp). Differentially expressed gene (DEG) analysis between phenotypes and among early (24 h), intermediate (48 h), and late (72 h) stages of biofilms was performed together with in silico co-expression network construction and compared between clones. To understand the influence of SCCmec and ACME on biofilm formation, isogenic mutants containing deletions of the entire elements or of single genes therein were constructed in USA300. Results: Genes involved in primarily core genome-encoded KEGG pathways (transporters and others) were upregulated in 24-h biofilm culture compared to 24-h planktonic culture. However, the number of affected pathways in the ST239 24 h biofilm (n = 11) was remarkably lower than that in USA300/EMRSA-15 biofilms (USA300: n = 27, HEMRSA-15: n = 58). The clfA gene, which encodes clumping factor A, was the single common DEG identified across the three clones in 24-h biofilm culture (2.2- to 2.66-fold). In intermediate (48 h) and late (72 h) stages of biofilms, decreased expression of central metabolic and fermentative pathways (glycolysis/gluconeogenesis, fatty acid biosynthesis), indicating a shift to anaerobic conditions, was already evident in USA300 and HEMRSA-15 in 48-h biofilm cultures; ST239 showed a similar profile at 72 h. Last, SCCmec+ACME deletion and opp3D disruption negatively affected USA300 biofilm formation. Conclusion: Our data show striking differences in gene expression during biofilm formation by three of the most important pandemic MRSA clones, USA300, HEMRSA-15, and ST239. The clfA gene was the only significantly upregulated gene across all three strains in 24-h biofilm cultures and exemplifies an important target to disrupt early biofilms. Furthermore, our data indicate a critical role for arginine catabolism pathways in early biofilm formation.

Agrobacterium strains and strain improvement: Present and outlook.

Almost 40 years ago the first transgenic plant was generated through Agrobacterium tumefaciens-mediated transformation, which, until now, remains the method of choice for gene delivery into plants. Ever since, optimized Agrobacterium strains have been developed with additional (genetic) modifications that were mostly aimed at enhancing the transformation efficiency, although an optimized strain also exists that reduces unwanted plasmid recombination. As a result, a collection of very useful strains has been created to transform a wide variety of plant species, but has also led to a confusing Agrobacterium strain nomenclature. The latter is often misleading for choosing the best-suited strain for one's transformation purposes. To overcome this issue, we provide a complete overview of the strain classification. We also indicate different strain modifications and their purposes, as well as the obtained results with regard to the transformation process sensu largo. Furthermore, we propose additional improvements of the Agrobacterium-mediated transformation process and consider several worthwhile modifications, for instance, by circumventing a defense response in planta. In this regard, we will discuss pattern-triggered immunity, pathogen-associated molecular pattern detection, hormone homeostasis and signaling, and reactive oxygen species in relationship to Agrobacterium transformation. We will also explore alterations that increase agrobacterial transformation efficiency, reduce plasmid recombination, and improve biocontainment. Finally, we recommend the use of a modular system to best utilize the available knowledge for successful plant transformation.

Expression of in vivo-inducible Salmonella enterica promoters during infection of Caenorhabditis elegans.

In vitro mimicking of the stimuli controlling in vivo-inducible bacterial promoters during infection of the host can be complex. Therefore, the use of the nematode Caenorhabditis elegans was evaluated, as a surrogate host to examine the expression of Salmonella enterica promoters. Green fluorescent protein (GFP+) was put under the control of the promoters of the pagC, mgtB, sseA, pgtE and fur genes of S. enterica. After infection of C. elegans with an S. enterica serovar Typhimurium vaccine strain expressing these constructs, clear bacterial expression of GFP+ was observed under the control of all five promoters, although significant expression was not always obtained in vitro. It is concluded that C. elegans constitutes a useful model system for the study of the in vivo expression of Salmonella promoters.

Inactivated Salmonella expressing the receptor-binding domain of bacterial adhesins elicit antibodies inhibiting hemagglutination.

We examined the potential of inactivated Salmonella strains to induce protective antibodies against two adhesins of pathogenic Escherichia coli. The receptor-binding domains of the F17a-G adhesin of F17a fimbriae and of the FimH adhesin of type 1 fimbriae were fused to the translocator domain of the autotransporter AIDA-I. An IgG response against F17a-G or FimH was induced after immunization of mice with acetone-inactivated Salmonella displaying the corresponding fimbrial receptor-binding domain. These sera inhibit in vitro agglutination of erythrocytes by E. coli carrying these fimbriae. Our results demonstrate that induced and subsequently acetone-inactivated Salmonella are useful delivery vehicles for the stimulation of an IgG antibody response against heterologous antigens.

A limited role for SsrA/B in persistent Salmonella Typhimurium infections in pigs.

Virulence genes regulated by the SsrA/B system are indispensable for systemic disease in BALB/c mice. The role of this regulating system in the pathogenesis of Salmonella Typhimurium infections in pigs is not documented. In the present study, the interactions of Salmonella Typhimurium and an ssrA/B mutant were compared in vitro and in vivo. The ssrA/B mutant strain displayed decreased Salmonella Pathogenicity Island 2 (SPI-2) expression levels, showed a replication defect in mouse macrophages and was attenuated in a mouse model after oral inoculation. Using real time qRT-PCR and a porcine ileal loop model, it was shown that the ssrA/B mutant strain was not significantly attenuated in overall virulence and SPI-1 expression in specific. Flowcytometric analysis demonstrated that the ssrA/B mutant strain was defective in intracellular replication in porcine macrophages. After oral inoculation of piglets with the wild type strain or the ssrA/B mutant strain, the animals of both groups excreted Salmonella and were colonized by Salmonella to the same extent. In an intravenous mixed infection model, the ssrA/B mutant strain was defective in the colonization of several internal organs. These results suggest that the ssrA/B gene of Salmonella Typhimurium plays a limited role in the persistent intestinal colonization of pigs.

Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300.

In methicillin-sensitive Staphylococcus aureus (MSSA), the tricarboxylic acid (TCA) cycle is known to negatively regulate production of the major biofilm-matrix exopolysaccharide, PIA/PNAG. However, methicillin-resistant S. aureus (MRSA) produce a primarily proteinaceous biofilm matrix, and contribution of the TCA-cycle therein remains unclear. Utilizing USA300-JE2 Tn-mutants (NARSA) in genes encoding TCA- and urea cycle enzymes for transduction into a prolific biofilm-forming USA300 strain (UAS391-Erys), we studied the contribution of the TCA- and urea cycle and of proteins, eDNA and PIA/PNAG, to the matrix. Genes targeted in the urea cycle encoded argininosuccinate lyase and arginase (argH::Tn and rocF::Tn), and in the TCA-cycle encoded succinyl-CoA synthetase, succinate dehydrogenase, aconitase, isocitrate dehydrogenase, fumarate hydratase class II, and citrate synthase II (sucC::Tn, sdhA/B::Tn, acnA::Tn, icd::Tn, fumC::Tn and gltA::Tn). Biofilm formation was significantly decreased under no flow and flow conditions by argH::Tn, fumC::Tn, and sdhA/B::Tn (range OD492 0.374-0.667; integrated densities 2.065-4.875) compared to UAS391-EryS (OD492 0.814; integrated density 10.676) (p ≤ 0.008). Cellular and matrix stains, enzymatic treatment (Proteinase K, DNase I), and reverse-transcriptase PCR-based gene-expression analysis of fibronectin-binding proteins (fnbA/B) and the staphylococcal accessory regulator (sarA) on pre-formed UAS391-Erys and Tn-mutant biofilms showed: (i) < 1% PIA/PNAG in the proteinaceous/eDNA matrix; (ii) increased proteins under no flow and flow in the matrix of Tn mutant biofilms (on average 50 and 51 (±11)%) compared to UAS391-Erys (on average 22 and 25 (±4)%) (p < 0.001); and (iii) down- and up-regulation of fnbA/B and sarA, respectively, in Tn-mutants compared to UAS391-EryS (0.62-, 0.57-, and 2.23-fold on average). In conclusion, we show that the biofilm matrix of MRSA-USA300 and the corresponding Tn mutants is PIA/PNAG-independent and are mainly composed of proteins and eDNA. The primary impact of TCA-cycle inactivation was on the protein component of the biofilm matrix of MRSA-USA300.

In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter.

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds.

Expression of garlic leaf lectin under the control of the phloem-specific promoter Asus1 from Arabidopsis thaliana protects tobacco plants against the tobacco aphid (Myzus nicotianae).

BACKGROUND: To check for correlation between the insecticidal properties and the specificity of lectins, a comparative study was made of the insecticidal activities of two garlic lectins with different biological activities. RESULTS: The insecticidal activity of the garlic (Allium sativum L.) leaf lectin ASAL and bulb lectin ASAII towards the tobacco aphid Myzus nicotianae Blackman was studied using bioassays with transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38). Bioassays were started with newborn nymphs of the tobacco aphid. Although during the first 7-8 days when nymphs developed to adults there were no apparent effects, part of the nymphal population was found to develop into winged (alate) forms. Later it became clear that transgenic plants expressing ASAL and ASAII had a significant effect on the reproduction capacity of the resulting adults, with a reduction of up to 40%. Different life table parameters such as prereproductive time, intrinsic rate of natural increase, generation time and doubling time were significantly affected (P < 0.05) in aphids grown on transgenic plant material expressing ASAL and ASAII. CONCLUSION: Bioassays with tobacco plants expressing ASAL and ASAII demonstrated a significant impact on the population growth of M. nicotianae. Therefore, both lectins can be considered as valuable candidate aphid control agents.

An orthologue of the host-defense protein psoriasin (S100A7) is expressed in frog skin.

Host-defense peptides and proteins are vital for first line protection against bacteria. Most host-defense peptides and proteins common in vertebrates have been studied primarily in mammals, while their orthologues in non-mammalian vertebrates received less attention. We found that the European Common Frog Rana temporaria expresses a protein in its skin that is evolutionarily related to the host-defense protein S100A7. This prompted us to test if the encoded protein, which is an important microbicidal protein in human skin, shows similar activity in frogs. The R. temporaria protein lacks the zinc-binding sites that are key to the antimicrobial activity of human S100A7 at neutral pH. However, despite being less potent, the R. temporaria protein does compromise bacterial membranes at low pH, similar to its human counterpart. We postulate that, while amphibian S100A7 likely serves other functions, the capacity to compromise bacterial cell membranes evolved early in tetrapod evolution.

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes.

BACKGROUND: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. RESULTS: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. CONCLUSION: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.

Salmonella Typhimurium SPI-1 genes promote intestinal but not tonsillar colonization in pigs.

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in several animal species. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections of pigs, however, is not well described. The interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants with disruptions in the SPI-1 genes hilA, sipA and sipB with porcine intestinal epithelial cells were characterized in vitro and in a ligated intestinal loop model in pigs. HilA and SipB were essential in the invasion of porcine intestinal epithelial cells in vitro. A sipA mutant was impaired for invasion using a polarized cell line, but fully invasive in a non-polarized cell line. All SPI-1 mutants induced a significant decrease in influx of neutrophils in the porcine intestinal loop model compared with the wild type strain. Pigs were orally inoculated with 10(8) colony forming units of both the wild type Salmonella Typhimurium strain and its isogenic sipB::kan mutant strain. The sipB mutant strain was significantly impaired to invade the intestinal, but not the tonsillar tissue, one day after inoculation and was unable to efficiently colonize the intestines and the GALT, but not the tonsils, 3 days after inoculation. This study shows that SPI-1 plays a crucial role in the invasion and colonization of the porcine gut and in the induction of influx of neutrophils towards the intestinal lumen, but not in the colonization of the tonsils.

The SlyB outer membrane lipoprotein of Burkholderia multivorans contributes to membrane integrity.

SlyB is a small lipoprotein of 158 amino acids which is conserved in different Gram-negative bacteria. In contrast to other bacteria, where slyB is monocistronic, in Burkholderia multivorans and in B. cenocepacia, slyB is the last gene of an operon comprising three open reading frames encoding a putative thiol peroxidase, a putative sugar kinase and SlyB. B. multivorans slyB mutants produced elongated cells and filaments which were never observed in cultures of wild-type or slyB-complemented cells. The slyB mutant also showed increased sensitivity to EDTA and SDS, and decreased siderophore production. Proteome analysis of a fraction enriched for membrane proteins suggested that SlyB, like the peptidoglycan-associated protein OpcL, is a major protein of the outer membrane. Taken together, these phenotypes suggest that SlyB contributes to the integrity of the cell envelope. By PCR amplification we were also able to demonstrate the conservation of slyB in all B. cepacia complex species tested.

The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome.

The plaque-forming VT2-encoding lambdoid bacteriophage varphi297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate. PCR walking, starting from the int gene of phage varphi297, demonstrated that the varphi297 prophage integrated in the yecE gene of a lysogenic E. coli K12 strain. This integration site, in E. coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site. The excisionase protein of phage varphi297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region. This can explain the different integration sites of both prophages. The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of varphi297, at the appropriate position in the E. coli chromosome.

Hurdles in bacteriophage therapy: deconstructing the parameters.

Bacterial infections in animals impact our food production, leading to economic losses due to food rejection and the need for preventive and curative measures. Since the onset of the antibiotic era, the rise of antibiotic-resistant pathogens is causing scares in health care and food producing facilities worldwide. In the search of new therapeutics, re-evaluation of bacteriophage (phage) therapy, using naturally occurring bacterial viruses to tackle infections, is gaining interest. Many studies report about phage therapy success, showing the value and power of these natural viruses. Although phages carry some interesting traits and their basic biology is now well understood, this review argues that phage therapy has not revealed all of its secrets and many parameters remain understudied, making the outcome of phage therapy highly variable depending on the disease incidence. These difficulties include poorly understood mechanisms of phage penetration and distribution throughout the body, the variable expression and accessibility of phage receptors on the bacterial host in in vivo conditions and the unusual (non-linear) phage pharmacokinetics. These parameters are not easily measured in realistic in vivo settings, but are nevertheless important hurdles to overcome the high variability of phage therapy trials. This critical approach is in accordance with Goethe's statement; "Difficulties increase the nearer we get to the goal". However, since the importance of the goal itself also rises, both difficulties and goal justify the need for additional in depth research in this domain.

A cocktail of in vitro efficient phages is not a guarantee for in vivo therapeutic results against avian colibacillosis.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, leading to important economic losses worldwide. To cure APEC-infected chickens, a cocktail of four different APEC-specific bacteriophages (phages) was composed and tested. Specific phages were selected from a collection of phages isolated in Belgium. The selection was based on their obligate lytic infection cycle, a broad host range, low cross-resistance and low frequency of development of resistant APEC mutants. Genome analysis of the phages indicated they were close relatives of T4 and N4, considered to be safe in vivo. Chickens were intratracheally infected with APEC strain CH2 (serogroup O78), causing a mortality of about 50% during the seven days following the infection. The phage cocktail was administered 2h after the infection, via three different ways: intratracheally, intra-esophageally or via the drinking water. Treated groups did not show a significant decrease in mortality, lesion scores or weight loss compared to untreated groups, although the APEC-specific phages could be re-isolated from the lung and heart of chickens that were euthanized. Moreover, the re-isolated bacteria from infected chickens had remained sensitive to the phage cocktail. Our results indicate that the efficiency of the phage cocktail used in treating CH2-infected chickens in vivo is negligible, even though in vitro, the phages in the cocktail were able to efficiently lyse the APEC strain CH2. Our results emphasize that the 'traditional' pathway of isolation, followed by phenotypical and genotypical characterization of phages composing the cocktail, does not lead to success in phage therapy in all cases.