Complete Genome Sequences of Two Klebsiella pneumoniae Phages Isolated as Part of an International Effort

We report the genomic sequences of phages KpCHEMY26 and KpGranit, isolated in Israel during a worldwide effort against a multidrug- and phage-resistant strain of Klebsiella pneumoniae from a patient in Finland. These results demonstrate the importance of an efficient worldwide network for collaborating in personalized therapy for infectious diseases.Peer reviewe

Defeating Antibiotic- and Phage-Resistant Enterococcus faecalis Using a Phage Cocktail in Vitro and in a Clot Model

The deteriorating effectiveness of antibiotics is propelling researchers worldwide towards alternative techniques such as phage therapy: curing infectious diseases using viruses of bacteria called bacteriophages. In a previous paper, we isolated phage EFDG1, highly effective against both planktonic and biofilm cultures of one of the most challenging pathogenic species, the vancomycin-resistant Enterococcus (VRE). Thus, it is a promising phage to be used in phage therapy. Further experimentation revealed the emergence of a mutant resistant to EFDG1 phage: EFDG1r. This kind of spontaneous resistance to antibiotics would be disastrous occurrence, however for phage-therapy it is only a minor hindrance. We quickly and successfully isolated a new phage, EFLK1, which proved effective against both the resistant mutant EFDG1r and its parental VRE, Enterococcus faecalis V583. Furthermore, combining both phages in a cocktail produced an additive effect against E. faecalis V583 strains regardless of their antibiotic or phage-resistance profile. An analysis of the differences in genome sequence, genes, mutations, and tRNA content of both phages is presented. This work is a proof-of-concept of one of the most significant advantages of phage therapy, namely the ability to easily overcome emerging resistant bacteria

Identification and Characterization of Fusolisin, the <i>Fusobacterium nucleatum</i> Autotransporter Serine Protease

<div><p><i>Fusobacterium nucleatum</i> is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61–65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in <i>F. nucleatum</i>. Here we describe the identification of this serine protease, named fusolisin, in three oral <i>F. nucleatum</i> sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55–101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the <i>F. nucleatum</i> fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96–113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55–65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In <i>F. nucleatum</i> ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of <i>F. nucleatum</i> in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.</p></div

Protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections

Summary: Phage therapy has re-emerged as a promising treatment for non-resolving infections. Given the lack of approved phage treatments, there is a need to establish a compassionate use pipeline. Here, we present a protocol for phage matching, treatment, and monitoring for compassionate bacteriophage use in non-resolving infections. We describe steps for consultation and request implementation, evaluating and comparing different aspects of phage activity, and phage production. We then detail procedures for multidisciplinary meetings, ethics approvals, phage therapy, and follow-up.For complete details on the use and execution of this protocol, please refer to Onallah et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Time course hydrolysis of FRETS-25-Thr and Fu-S-P by fusolisin.

<p>Purified fusolisin (1.2 µg) was incubated with 0.05 mM of Fu-S-P or 0.1 mM (blue) of FRETS-25-Thr (red) in TBS pH 8.0. Relative Fluorescent Units (RFU) were determined as described in materials and methods. *P<0.05 compared to control with heat inactivated fusolisin, determined with Bonferroni test for multiple comparisons using the SPSS 15.0 software.</p

Fu-S-P activity correlates with the number of <i>F. nucleatum</i> cells.

<p>Fu-S-P (0.03 mM) was incubated for 2 hrs with increasing numbers of washed <i>F. nucleatum</i> cells. Relative Fluorescent Units (RFU) were determined as described in Materials and Methods. No activity was observed with boiled cells.</p

Estimated molecular mass of <i>F. nucleatum</i> fusolisin detected in outer membrane vesicles or in growth medium.

a<p>Activity detected only in samples prepared from outer membrane vesicles.</p><p>Estimated molecular mass of <i>F. nucleatum</i> fusolisin detected in outer membrane vesicles or in growth medium.</p

PMSF inhibits growth of <i>F. nucleatum</i> but not of <i>E. coli</i>.

<p>(A) Growth of <i>F. nucleatum</i> 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by <i>P. gingivalis</i> supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of <i>E. coli</i> is not affected by PMSF, ruling out PMSF toxicity. *P<0.05 compared to PMSF-treated bacteria, determined with Bonferroni test for multiple comparisons using the SPSS 15.0 software.</p

Sequence alignment of fusolisin.

<p>ClustalW alignment of Fsp25586, the available partial sequence of the homologues serine protease Fsp23726, Fsp10953 and Fsp49256. The predicted catalytic triad Asp, His and Ser are highlighted.</p