Eradication of a Multidrug-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Isolate Following Oral and Intra-rectal Therapy With a Custom Made, Lytic Bacteriophage Preparation.

In July 2017, a patient presented colonization with a multidrug-resistant, carbapenemase (KPC-3)-producing Klebsiella pneumoniae isolate. A custom-made, lytic bacteriophage preparation was administered to the patient in December 2017, with subsequent eradication of the microorganism and without adverse effects.

Correlation of Host Range Expansion of Therapeutic Bacteriophage Sb-1 with Allele State at a Hypervariable Repeat Locus.

Staphylococci are frequent agents of health care-associated infections and include methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to first-line antibiotic treatments. Bacteriophage (phage) therapy is a promising alternative antibacterial option to treat MRSA infections. S. aureus-specific phage Sb-1 has been widely used in Georgia to treat a variety of human S. aureus infections. Sb-1 has a broad host range within S. aureus, including MRSA strains, and its host range can be further expanded by adaptation to previously resistant clinical isolates. The susceptibilities of a panel of 25 genetically diverse clinical MRSA isolates to Sb-1 phage were tested, and the phage had lytic activity against 23 strains (92%). The adapted phage stock (designated Sb-1A) was tested in comparison with the parental phage (designated Sb-1P). Sb-1P had lytic activity against 78/90 strains (87%) in an expanded panel of diverse global S. aureus isolates, while eight additional strains in this panel were susceptible to Sb-1A (lytic against 86/90 strains [96%]). The Sb-1A stock was shown to be a mixed population of phage clones, including approximately 4% expanded host range mutants, designated Sb-1M. In an effort to better understand the genetic basis for this host range expansion, we sequenced the complete genomes of the parental Sb-1P and two Sb-1M mutants. Comparative genomic analysis revealed a hypervariable complex repeat structure in the Sb-1 genome that had a distinct allele that correlated with the host range expansion. This hypervariable region was previously uncharacterized in Twort-like phages and represents a novel putative host range determinant.IMPORTANCE Because of limited therapeutic options, infections caused by methicillin-resistant Staphylococcus aureus represent a serious problem in both civilian and military health care settings. Phages have potential as alternative antibacterial agents that can be used in combination with antibiotic drugs. For decades, phage Sb-1 has been used in former Soviet Union countries for antistaphylococcal treatment in humans. The therapeutic spectrum of activity of Sb-1 can be increased by selecting mutants of the phage with expanded host ranges. In this work, the host range of phage Sb-1 was expanded in the laboratory, and a hypervariable region in its genome was identified with a distinct allele state that correlated with this host range expansion. These results provide a genetic basis for better understanding the mechanisms of phage host range expansion.

Whole genome sequence comparison of ten diagnostic brucellaphages propagated on two Brucella abortus hosts.

BACKGROUND: Recently the genome sequences of two brucellaphages, isolated in Georgia (Tb) and Mexico (Pr) were reported revealing pronounced sequence homogeneity and the presence of two major indels discriminating the two phages. Subsequent genome sequencing of six diagnostic brucellaphages: Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C phages identified three major genetic groups. However, the propensity for fine-scale genetic variability of diverse brucellaphages grown on multiple hosts within a single Brucella species remains unknown. METHODS: We sequenced the complete genomes of ten brucellaphages following initial propagation on B. abortus strain 141 and after subsequent propagation on B. abortus strain S19. All brucellaphages were isolated and propagated at the Eliava Institute including the original Tb phage. Genomic libraries were quantified using the Qbit and sheared on the Covaris M220. QC for fragmentation was performed on the BioAnalyzer 2100. DNA libraries were prepared using an Illumina Paired-End protocol and sequenced on the Illumina MiSeq. Sequence analysis was performed using Geneious and MEGA software. RESULTS: Comparative whole genome sequence analysis revealed genetic homogeneity consistent with previously published data as well as multiple nucleotide variations. Genomic changes as a result of passages were observed in similar genes and predominantly occurred at identical sites in separate phages. Multiple instances of within-sample genetic heterogeneity were observed often at shared genomics positions across phages. Positive selection was detected in the tail collar protein gene. We also identified a Staphylothermus marinus F1-like CRISPR spacer and sequences orthologous to both prophage antirepressors of Brucella spp. and intergenic sequences encoded by Ochrobactrum anthropi. CONCLUSION: We surveyed whole genome level diversity in phage lytic for B. abortus as they are propagated on alternate vaccine strains within the species. Our data extend previous results indicating select variable hotspots and broad genomic homogeneity as well as multiple common polymorphisms and within-sample variation. These data also provide additional genomes for future reference in comparative studies involving the molecular evolution and host specificity of brucellaphages.

Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae.

A 30-year-old bombing victim with a fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term (>700 days) antibiotic therapy is treated with a pre-adapted bacteriophage along with meropenem and colistin, followed by ceftazidime/avibactam. This salvage therapy results in objective clinical, microbiological and radiological improvement of the patient's wounds and overall condition. In support, the bacteriophage and antibiotic combination is highly effective against the patient's K. pneumoniae strain in vitro, in 7-day mature biofilms and in suspensions.

Phage Therapy Experience at the Eliava Phage Therapy Center: Three Cases of Bacterial Persistence.

In this retrospective descriptive study we focus on cases of three patients who underwent phage therapy procedures at Eliava Phage Therapy Center (EPTC) in Tbilisi, Georgia. Patients with chronic infectious diseases related to Pseudomonas aeruginosa (two patients, lower respiratory tract infection (LRTI)) and Klebsiella pneumoniae (one patient, urinary tract infection (UTI)) are among those very few EPTC patients whose pathogens persisted through phage therapy. By looking at bacterial strains and personalized phages used against them we tried to point towards possible adaptation strategies that are employed by these pathogens. Genome restriction-based Pulsed Field Gel Electrophoresis (PFGE) profiling of strains isolated before and after phage therapy hints towards two strategies of adaptation. In one patient case (Pseudomonas aeruginosa related lung infection) bacterial strains before and after phage therapy were indistinguishable according to their PFGE profiles, but differed in their phage susceptibility properties. On the other hand, in two other patient cases (Pseudomonas aeruginosa related LRTI and Klebsiella pneumoniae related UTI) bacterial adaptation strategy seemed to have resulted in diversification of infecting strains of the same species. With this work we want to attract more attention to phage resistance in general as well as to its role in phage therapy.

Efficacy of newly isolated and highly potent bacteriophages in a mouse model of extensively drug-resistant Acinetobacter baumannii bacteraemia.

OBJECTIVES: Bacteraemia can be caused by Acinetobacter baumannii (A. baumannii), with clinical manifestations ranging from transient bacteraemia to septic shock. Extensively drug-resistant A. baumannii (XDRAB) strains producing the New Delhi metallo-ß-lactamase, which confers resistance to all ß-lactams including carbapenems, have emerged. Infected patients suffer increased mortality, morbidity and length of hospitalisation. The lack of new antimicrobials has led to a renewed interest in phage therapy, the so-called forgotten cure. Accordingly, we tested new lytic bacteriophages in a Galleria mellonella and a mouse model of XDRAB-induced bacteraemia. METHODS: Galleria mellonella were challenged with 5.105 CFU of the XDRAB strain FER. Phages vB_AbaM_3054 and vB_AbaM_3090 were administrated alone or in combination 30min after bacterial challenge. Saline and imipenem were injected as controls. Mice were intraperitoneally (i.p.) challenged with 6.107 CFU of A. baumannii FER. vB_AbaM_3054 and vB_AbaM_3090 were administrated i.p. alone or in combination 2h after bacterial challenge. Saline and imipenem were injected as controls. Larvae and mice survival were followed for 7 days and compared with Log-Rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests. RESULTS: Phage-based treatments showed high efficacy in larvae (ca. 100% survival at 80h) and mice (ca. 100% survival at day 7) compared with the untreated controls (0% survival at 48h and 24h in larvae and mice, respectively). CONCLUSIONS: The present data reporting efficacy of phage therapy in a mouse model of bacteraemia support the development of phage-based drugs to manage infection due to multi-drug resistant A. baumannii and particularly XDRAB.

Evaluation of lytic activity of staphylococcal bacteriophage Sb-1 against freshly isolated clinical pathogens.

In recent decades the increase in antibiotic-resistant bacterial strains has become a serious threat to the treatment of infectious diseases. Drug resistance of Staphylococcus aureus has become a major problem in hospitals of many countries, including developed ones. Today the interest in alternative remedies to antibiotics, including bacteriophage treatment, is gaining new ground. Here, we describe the staphylococcal bacteriophage Sb-1 - a key component of therapeutic phage preparation that was successfully used against staphylococcal infections during many years in the Former Soviet Union. This phage still reveals a high spectrum of lytic activity in vitro against freshly isolated, genetically different clinical samples (including methicillin-resistant S. aureus) obtained from the local hospitals, as well as the clinics from different geographical areas. The sequence analyses of phage genome showed absence of bacterial virulence genes. A case report describes a promising clinical response after phage application in patient with cystic fibrosis and indicates the efficacy of usage of Sb-1 phage against various staphylococcal infections.