Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.

Background: Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms. Results: Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected. Conclusion: Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.

Improving phage therapy by evasion of phage resistance mechanisms.

Antibiotic failure is one of the most worrisome threats to global health. Among the new therapeutic efforts that are being explored, the use of bacteriophages (viruses that kill bacteria), also known as 'phages', is being extensively studied as a strategy to target bacterial pathogens. However, one of the main drawbacks of phage therapy is the plethora of defence mechanisms that bacteria use to defend themselves against phages. This review aims to summarize the therapeutic approaches that are being evaluated to overcome the bacterial defence systems, including the most innovative therapeutic approaches applied: circumvention of phage receptor mutations; modification of prophages; targeting of CRISPR-Cas systems and the biofilm matrix; engineering of safer and more efficacious phages; and inhibition of the anti-persister strategies used by bacteria.

Molecular studies of phages-Klebsiella pneumoniae in mucoid environment: innovative use of mucolytic agents prior to the administration of lytic phages.

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.

Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae.

Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.

Study of 32 new phage tail-like bacteriocins (pyocins) from a clinical collection of Pseudomonas aeruginosa and of their potential use as typing markers and antimicrobial agents.

Phage tail-like bacteriocins (PTLBs) are large proteomic structures similar to the tail phages. These structures function in bacterial competition by making pores in the membrane of their competitors. The PTLBs identified in Pseudomonas aeruginosa are known as R-type and F-type pyocins, which have a narrow spectrum of action. Their specificity is determined by the tail fiber and is closely related to the lipopolysaccharide type of the target competitor strain. In this study, the genome sequences of 32 clinical of P. aeruginosa clinical isolates were analysed to investigate the presence of R-type and F-type pyocins, and one was detected in all strains tested. The pyocins were classified into 4 groups on the basis of the tail fiber and also the homology, phylogeny and structure of the cluster components. A relationship was established between these groups and the sequence type and serotype of the strain of origin and finally the killing spectrum of the representative pyocins was determined showing a variable range of activity between 0 and 37.5%. The findings showed that these pyocins could potentially be used for typing of P. aeruginosa clinical isolates, on the basis of their genomic sequence and cluster structure, and also as antimicrobial agents.