Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

Characterization and the host specificity of Pet-CM3-4, a new phage infecting Cronobacter and Enterobacter strains.

Bacteria belonging to Cronobacter and Enterobacter genera are opportunistic pathogens responsible for infections in immunocompromised patients including neonates. Phage therapy offers a safe method for pathogen elimination, however, phages must be well characterized before application. In the present study we isolated four closely related bacteriophages from the subfamily Tevenvirinae infecting Cronobacter and Enterobacter strains. Bacteriophage Pet-CM3-4 which was isolated on C. malonaticus strain possessed broader host specificity than other three phages with primary Enterobacter hosts. Based on genome sequences all these phages have been assigned to the genus Karamvirus. We also studied factors influencing the host specificity of Pet-CM3-4 phage and its host range mutant Pet-CM3-1 and observed that a lysine to glutamine substitution in the long tail fiber adhesin was the reason of the Pet-CM3-1 reduced host specificity. By characterization of phage-resistant mutants from transposon library of C. malonaticus KMB-72 strain we identified that LPS is the receptor of both phages. C. malonaticus O:3 antigen is the receptor of Pet-CM3-1 phage and the Pet-CM3-4 phage binds to structures of the LPS core region. Obtained results will contribute to our understanding of biology and evolution of Tevenvirinae phages.

Susceptibility of Staphylococcus aureus strains to commercial therapeutic phage preparations.

OBJECTIVES: The aim of this study was to determine the susceptibility of Staphylococcus aureus strains to commercial phage preparations. The strains were isolated from clinical patients as well as from nasal mucosa of healthy carriers. BACKGROUND: The elevating number of antibiotic-resistant S. aureus strains present a therapeutic challenge, especially in high-risk patients. One of the promising ways to solve this problem is phage therapy. METHODS: Susceptibility of 111 carrier strains of S. aureus (4 strains were methicillin-resistant; MRSA) and 81 clinical isolates from bloodstream or skin and soft tissue infections (28 were MRSA) to four commercial phage preparations was assessed in vitro by spot assay. The clonality of S. aureus strains was determined by spa typing. RESULTS: Spa typing revealed 97 distinct spa types. A proportion of 73-80 % of the tested S. aureus strains were revealed to have in vitro phage susceptibility, depending on the clonal affiliation of the strains and phage preparation tested. The susceptibility to phage preparations was significantly higher in MRSA strains (p < 0.001). CONCLUSIONS: In vitro results indicate a promising therapeutic potential of the tested commercial anti-staphylococcal phage preparations. They could be applied to a broad spectrum of bacterial clones, and have an excellent activity especially against MRSA strains (Tab. 2, Fig. 2, Ref. 43).

Characterization of Anti-Bacterial Effect of the Two New Phages against Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are among the events that most frequently need medical intervention. Uropathogenic Escherichia coli are frequently their causative agents and the infections are sometimes complicated by the presence of polyresistant nosocomial strains. Phage therapy is a tool that has good prospects for the treatment of these infections. In the present study, we isolated and characterized two bacteriophages with broad host specificity against a panel of local uropathogenic E. coli strains and combined them into a phage cocktail. According to genome sequencing, these phages were closely related and belonged to the Tequatrovirus genus. The newly isolated phages showed very good activity on a panel of local clinical E. coli strains from urinary tract infections. In the form of a two-phage cocktail, they were active on E. coli strains belonging to phylogroups B2 and D, with relatively lower activity in B1 and no response in phylogroup A. Our study is a preliminary step toward the establishment of a national phage bank containing local, well-characterized phages with therapeutic potential for patients in Slovakia.

Impact of DNA extraction methods on 16S rRNA-based profiling of bacterial communities in cheese.

Numerous factors associated with sample preparation, DNA extraction, primer choice, sequencing platform and data analysis can affect the accuracy of 16S rRNA sequencing results. The DNA extraction method is considered critical for the success of sequencing as it can be the source of considerable variations in the analysis of the microbiome. In this study, the impact of various DNA extraction methods on the results of analysis of bacterial communities in cheese was evaluated. DNA was isolated from Mozzarella as a model cheese using optimized bead-based homogenization followed by different extraction procedures. Five commercial kits and two open-formula DNA extraction protocols were evaluated for amplicon sequencing of a 16S rRNA fragment of ~1460 bp. In addition, model cheese samples artificially contaminated by defined concentrations of Listeria monocytogenes and Escherichia coli, as representatives of Gram positive and Gram negative bacteria, were analysed. Six out of seven DNA extraction procedures were found to be able to provide amplifiable bacterial DNA suitable for 16S rRNA sequence analysis, but individual extraction procedures led to variable results. In particular, lysis supported with bead-beating led to a higher proportion of G+ bacteria in relative abundance profiles, probably because of the more efficient cell wall disruption. Artificially added bacterial species were reliably detected with a quantitative response. The results demonstrated a risk in comparing the data on bacterial communities in cheese when different DNA extraction protocols are used and highlighted the need to choose a standardized approach when comparison across multiple sequencing runs is required.

Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese.

Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction-modification system, all strains carried 1-6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0-24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction-modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.