George Eliava Institute of Bacteriophage, Microbiology and Virology

The emergence of antibiotic-resistant pathogens necessitates alternative therapies for treating microbial infections, especially in the oral cavity and upper respiratory tract. Our team has developed Phage Pastilles, a controlled-release formulation containing bacteriophages that target common pathogens, including Streptococcus pyogenes, Streptococcus salivarius, Staphylococcus aureus, Enterococcus faecalis, and E. coli. These pastilles incorporate eucalyptus oil and serve as a reservoir for gradually releasing bacteriophages, enhancing local therapeutic impact. This study focuses on the characterization of a novel lytic bacteriophage, vB_SPY_7, specifically targeting S. pyogenes which was incorporated into phage pastilles. Phage vB_SPY_7, isolated from wastewater, underwent host range analysis, plaque assays, adsorption kinetics, growth curve studies, electron microscopy, and whole-genome sequencing. Genomic analysis revealed that vB_SPY_7 is a member of the unclassified Caudoviricetes, with a 35,679 bp genome and 46 predicted ORFs, including essential structural and metabolic genes. Sequence analysis and biological characterization have demonstrated that vB_SPY_7 is a viable candidate for therapeutic use, and it is highly active when combined in a sustained-release format like Phage Pastilles.

Microbiomes are ecosystems, and their stability can impact the health of their hosts. Theory predicts that predators influence ecosystem stability. Phages are key predators of bacteria in microbiomes, but phages are unusual predators because many have lysogenic life cycles. It has been hypothesized that lysogeny can destabilize microbiomes, but lysogeny has no direct analog in classical ecological theory, and no formal theory exists. We studied the stability of computationally simulated microbiomes with different numbers of temperate (lysogenic) and virulent (obligate lytic) phage species. Bacterial populations were more likely to fluctuate over time when there were more temperate phages species. After disturbances, bacterial populations returned to their pre-disturbance densities more slowly when there were more temperate phage species, but cycles engendered by disturbances dampened more slowly when there were more virulent phage species. Our work offers the first formal theory linking lysogeny to microbiome stability.

Bacteroides fragilis is a commensal gut bacterium that is associated with a number of blood and tissue infections. It has not yet been recognized as one of the drug-resistant human pathogens, but cases of the refractory infections, caused by strains that are not susceptible to the common antibiotic regimes established for B. fragilis, have been more frequently reported. Bacteriophages (phages) were found to be a successful antibacterial alternative to antibiotic therapy in many cases of multidrug-resistant (MDR) bacterial infections. We have characterized the bacteriophage GEC_vB_Bfr_UZM3 (UZM3), which was used for the treatment of a patient with a chronic osteomyelitis caused by a B. fragilis mixed infection. Studied biological and morphological properties of UZM3 showed that it seems to represent a strictly lytic phage belonging to a siphovirus morphotype. It is characterized by high stability at body temperature and in pH environments for about 6 h. Whole genome sequencing analysis of the phage UZM3 showed that it does not harbor any known virulence genes and can be considered as a potential therapeutic phage to be used against B. fragilis infections.

Streptococcus agalactiae, also known as group B streptococci, was first isolated from cow's milk with mastitis, and it was first identified in 1930 by Rebecca Lancefield. GBS or streptococcus agalacticae is a gram-positive cocci, beta-hemolytic, facultative anaerobic, which is a harmless inhabitant of the normal human microflora. About 30% of the population is an asymptomatic carrier of this microbe. However, it was considered the leading cause of neonatal invasions upon its discovery. GBS is encapsulated in a polysaccharide capsule, which is a major virulence factor, and 10 serotypes of group B streptococci are known for their immunological activity. According to the existing studies, vaginal colonization is most often caused by type Ia, III, and V in other countries of the world. Today number of invasive GBS diseases continues to increase and it remains a significant pathogen among both infants and adults. The use of broad-spectrum antibiotics has certainly played a major role in the fight against infections, although the number of antibiotic-resistant strains has increased and thus modern medicine starts new insights into alternative antibacterials. Bacteriophages are often suggested as an alternative therapeutic agent against bacterial infections. In the present study, our aim was to isolate and study bacteriophages active against group B streptococci. Between March 2019 and March 2020, 257 anovaginal swabs were collected at "Nia Oniashvili Clinic" Tbilisi, Georgia, and Clinic "Curatio"by using standard bacterial identification procedures, in particular group B streptococcus identification guideline provided by CDC we identified 87 Streptococcus agalactiae strains. We used both standard and modified methods to isolate bacteriophages and study their life cycle, which were developed by the team. In this study, two bacteriophages active against Streptococcus agalactiae were identified: vB_GBS_1 (Syphoviridae) and vB_GBS_2 (Syphoviridae). The biological characteristics, morphology, adsorption, and host range were studied.

To investigate the potential role of bacteriophages in the treatment of surgical infections, we conducted a retrospective analysis of four surgical patients who have sought treatment at the Eliava Phage Therapy Center, Tbilisi, Georgia. Two patients had chronic osteomyelitis, one presented with a diabetic foot ulcer, and the fourth patient had developed a severe infectious complication after skin grafting surgery. Patients were treated with different combinations of bacteriophage preparations, based on the sensitivity of the isolated bacterial strain toward commercially available bacteriophages. The treatment lasted on average for 1 month, and positive results were obtained in all four cases: the wounds have healed, the general health status of the patients has improved. No allergic or adverse reactions have been observed throughout the treatment.

Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß-hemolytic due to the presence of an SLS-like gene cluster. However, the majority of strains do not display ß-hemolysis. To investigate ß-hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß-hemolytic and non-ß-hemolytic strains. All of the ß-hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß-hemolysis do not harbor sag genes. Phylogenetic analysis of the ß-hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß-hemolytic versus non-ß-hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non-ß-hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß-hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self-targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß-hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus.

GBS-Group B streptococcus or Streptococcus agalactiae is a Gram-positive coccus found in 20% of healthy women as part of normal gastrointestinal and genital tract flora. It is associated with pathogenicity in immunocompromised, elderly and pregnant adults as well as infants and neonates. GBS are encapsulated organisms and sub divided into ten antigenically distinct capsular serotypes. GBS case treatment mainly performed with Penicillin G but Erythromicyn, Vancomycin and Clindamycin are recommended for penicillin-allergic individuals. In this study between March 2019 and September 2019 an anovaginal swabs were collected from 80 women at 35-37 weeks of gestation and 39 were identified as str. agalactiae. 24 str. agalactiae strains provided by clinic "Curation" were isolated from urine, vagina and rectum. Culturing, identification of GBS were carried out according to standard microbiological methods. Antimicrobial susceptibility profiles were determined by disk diffusion method. Our results revealed that in total from 104 samples 29% were Str. agalactiae. All GBS strains were all sensitive to Penicillin, Ampicilin and Vancomycin. 25% were resistant to Erythromycin and 13% to Clindamycin. Our study has shown that the prevalence of Streptococcus agalactiae in Georgia quite high and especially with Erythromycin-resistant strains, which makes treatment of penicillin allergic patients problematic. In this case only hope is vancomycin. Fortunately, vancomycin-resistant strains have not been identified; however the problem is still relevant. Obtained data could be used as a base for further epidemiological studies and also managing targeted new approaches of treatment and prevention of GBS.