In addition to specific antibiotic resistance, the formation of bacterial biofilm causes another level of complications in attempts to eradicate pathogenic or harmful bacteria, including difficult penetration of drugs through biofilm structures to bacterial cells, impairment of immunological response of the host, and accumulation of various bioactive compounds (enzymes and others) affecting host physiology and changing local pH values, which further influence various biological functions. In this review article, we provide an overview on the formation of bacterial biofilm and its properties, and then we focus on the possible use of phage-derived depolymerases to combat bacterial cells included in this complex structure. On the basis of the literature review, we conclude that, although these bacteriophage-encoded enzymes may be effective in destroying specific compounds involved in the formation of biofilm, they are rarely sufficient to eradicate all bacterial cells. Nevertheless, a combined therapy, employing depolymerases together with antibiotics and/or other antibacterial agents or factors, may provide an effective approach to treat infections caused by bacteria able to form biofilms.

Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis-encoded sRNAs (also called antisense RNA) and trans-acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression.

Phage therapy is one of main alternative option for antibiotic treatment of bacterial infections, particularly in the era of appearance of pathogenic strains revealing resistance to most or even all known antibiotics. Enterococcus faecalis is one of such pathogens causing serious human infections. In the light of high level of biodiversity of bacteriophages and specificity of phages to bacterial species or even strains, development of effective phage therapy depend, between others, on identification and characterization of a large collection of these viruses, including understanding of their interactions with host bacterial cells. Recently, isolation of molecular characterization of bacteriophage vB_EfaS-271, infecting E. faecalis strains have been reported. In this report, phage–host interactions are reported, including ability of vB_EfaS-271 to infect bacteria forming biofilms, efficiency of eliminating bacterial cells from cultures depending on multiplicity of infection (m.o.i.), toxicity of purified phage particles to mammalian cells, and efficiency of appearance of phage-resistant bacteria. The presented results indicate that vB_EfaS-271 can significantly decrease number of viable E. faecalis cells in biofilms and in liquid cultures and reveals no considerable toxicity to mammalian cells. Efficiency of formation of phage-resistant bacteria was dependent on m.o.i. and was higher when the virion-cell ratio was as high as 10 than at low (between 0.01 and 0.0001) m.o.i. values. We conclude that vB_EfaS-271 may be considered as a candidate for its further use in phage therapy.

A newly isolated bacteriophage infecting Enterococcus faecalis strains has been characterized, including determination of its molecular features. This phage, named vB_EfaS-271, has been classified as a Siphoviridae member, according to electron microscopy characterization of the virions, composed of a 50 nm-diameter head and a long, flexible, noncontractable tail (219 × 12.5 nm). Analysis of the whole dsDNA genome of this phage showed that it consists of 40,197 bp and functional modules containing genes coding for proteins that are involved in DNA replication (including DNA polymerase/primase), morphogenesis, packaging and cell lysis. Mass spectrometry analysis allowed us to identify several phage-encoded proteins. vB_EfaS-271 reveals a relatively narrow host range, as it is able to infect only a few E. faecalis strains. On the other hand, it is a virulent phage (unable to lysogenize host cells), effectively and quickly destroying cultures of sensitive host bacteria, with a latent period as short as 8 min and burst size of approximately 70 phages per cell at 37 °C. This phage was also able to destroy biofilms formed by E. faecalis. These results contribute to our understanding of the biodiversity of bacteriophages, confirming the high variability among these viruses and indicating specific genetic and functional features of vB_EfaS-271.

Apart from antibiotic resistance of pathogenic bacteria, the formation of biofilms is a feature that makes bacterial infections especially difficulty to treat. Shiga toxin-producing Escherichia coli (STEC) strains are dangerous pathogens, causing severe infections in humans, and capable of biofilm production. We have reported previously the identification and characterization of the vB_Eco4-M7 bacteriophage, infecting various STEC strains. It was suggested that this phage might be potentially used in phage therapy against these bacteria. Here, we tested the effects of vB_Eco4-M7 alone or in a phage cocktail with another STEC-infecting phage, and/or in a combination with different antibiotics (ciprofloxacin and rifampicin) on biofilm formed by a model STEC strain, named E. coli O157:H7 (ST2-8624). The vB_Eco4-M7 phage appeared effective in anti-biofilm action in all these experimental conditions (2–3-fold reduction of the biofilm density, and 2–3 orders of magnitude reduction of the number of bacterial cells). However, the highest efficiency in reducing a biofilm’s density and number of bacterial cells was observed when phage infection preceded antibiotic treatment (6-fold reduction of the biofilm density, and 5–6 orders of magnitude reduction of the number of bacterial cells). Previous reports indicated that the use of antibiotics to treat STEC-caused infections might be dangerous due to the induction of Shiga toxin-converting prophages from bacterial genomes under stress conditions caused by antibacterial agents. We found that ciprofloxacin was almost as efficient in inducing prophages from the E. coli O15:H7 (ST2-8624) genome as a classical inducer, mitomycin C, while no detectable prophage induction could be observed in rifampicin-treated STEC cells. Therefore, we conclude the latter antibiotic or similarly acting compounds might be candidate(s) as effective and safe drug(s) when used in combination with phage therapy to combat STEC-mediated infections.

Shiga toxin-converting bacteriophages (Stx phages) are present as prophages in Shiga toxin-producing Escherichia coli (STEC) strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m², their infectivity dropped by 1–3 log₁₀, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.