The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, πVLC5 and πVLC6, isolated from environmental samples. These phages belong to the genus Drulisvirus within the family Podoviridae. Both phages encode a similar tail spike protein with putative depolymerase activity, which is shared among other related phages and probably determines their ability to specifically infect K. pneumoniae capsular types K22 and K37. In addition, we found that phage πVLC6 also infects capsular type K13 and is capable of striping the capsules of K. pneumoniae KL2 and KL3, although the phage was not infectious in these two strains. Genome sequence analysis suggested that the extended tropism of phage πVLC6 is conferred by a second, divergent depolymerase. Phage πVLC5 encodes yet another putative depolymerase, but we found no activity of this phage against capsular types other than K22 and K37, after testing a panel of 77 reference strains. Overall, our results confirm that most phages productively infected one or few Klebsiella capsular types. This constitutes an important challenge for clinical applications.

Despite their simplicity, viruses can display social-like interactions such as cooperation, communication, and cheating. Focusing on bacteriophages, here we review features including viral product sharing, cooperative evasion of antiviral defenses, prudent host exploitation, superinfection exclusion, and inter-phage peptide-mediated signaling. We argue that, in order to achieve a better understanding of these processes, their mechanisms of action need to be considered in the context of social evolution theory, paying special attention to key population-level factors such as genetic relatedness and spatial structure.

Background: Klebsiella pneumoniae is an opportunistic pathogen that causes a wide range of infections worldwide. The emergence and spread of multidrug-resistant clones requires the implementation of novel therapeutics, and phages are a promising approach. Results: In this study, two Klebsiella phages, KpTDp1 and KpTDp2, were isolated from wastewater samples in Tunisia. These phages had a narrow host range and specifically targeted the hypervirulent K2 and K28 capsular types of K. pneumoniae. Both phages have double-stranded linear DNA genomes of 49,311 and 49,084 bp, respectively. Comparative genomic and phylogenetic analyses placed phage KpTDp2 in the genus Webervirus, while phage KpTDp1 showed some homology with members of the genus Jedunavirus, although its placement in a new undescribed genus may be reconsidered. The replication efficiency and lytic ability of these phages, combined with their high stability at temperatures up to 70 °C and pH values ranging from 3.5 to 8.2, highlight the potential of these phages as good candidates for the control of hypervirulent multidrug-resistant K. pneumoniae. Methods: Phage isolation, titration and multiplicity of infection were performed. The stability of KpTDp1 and KpTDp2 was tested at different pH and temperatures. Genomic characterization was done by genome sequencing, annotation and phylogenetic analysis. Conclusions: The ability of KpTDp1 and KpTDp2 to lyse one of the most virulent serotypes of K. pneumoniae, as well as the stability of their lytic activities to pH and temperature variations, make these phages promising candidates for antibacterial control.

The emergence of phage-resistant bacteria poses a significant challenge to the success of phage therapy. Although phage cocktails can delay resistance, their efficacy relies on the ability to target the full spectrum of resistant variants, which are often more diverse than those represented by clonal isolates. In this study, we investigated how phenotypic diversity within phage-resistant Klebsiella pneumoniae influences susceptibility to newly isolated phages, and the effectiveness of phage cocktails. We isolated phages using three cultures resistant to a capsule-dependent phage: a heterogeneous acapsular population, an acapsular mutant with a stable phenotype, and a capsule-reverted isolate that regained capsule expression upon removal of phage pressure. These phages differed in host tropism and in their capacity to delay resistance emergence when combined with the capsule-dependent phage. Phages targeting acapsular variants, particularly those isolated from the heterogeneous population, were the most effective, exhibiting strong synergy. Single-cell analyses further revealed that sustained selective pressure from the capsule-dependent phage prevents capsule reversion and maintains cocktail efficacy. Overall, our results highlight the importance of accounting for phenotypic heterogeneity when designing phage therapies, and support population-level approaches for optimizing phage cocktail composition. IMPORTANCE: Phage therapy is a promising alternative to antibiotics, but its success is often limited by the rapid emergence of phage-resistant bacteria. These resistant populations can be highly heterogeneous, comprising both stable mutants and variants with reversible, non-genetic resistance. In this study, we explore how this phenotypic diversity influences the effectiveness of phage cocktails. By isolating new phages and testing them in combination, we demonstrate that the selective pressure exerted by specific phages can prevent the reversion in transiently resistant variants, thereby sustaining treatment efficacy. Our findings highlight the need to consider not only the range of bacterial targets but also how phage pressure shapes bacterial population dynamics. This work offers a more refined strategy for designing phage cocktails with improved clinical potential.

The emergence of phage-resistant bacteria poses a significant challenge to the success of phage therapy. Although phage cocktails can delay resistance, their efficacy relies on the ability to target the full spectrum of resistant variants, which are often more diverse than that represented by clonal isolates. In this study, we investigated how phenotypic diversity within phage-resistant Klebsiella pneumoniae influences susceptibility to newly isolated phages and the effectiveness of phage cocktails. We isolated phages from three cultures resistant to a capsule-dependent phage: a heterogeneous acapsular population, an acapsular mutant with a stable phenotype, and a capsule-reverted isolate that regained capsule expression upon removal of phage pressure. These phages differed in host tropism and in their capacity to delay resistance emergence when combined with the capsule-dependent phage. Phages targeting acapsular variants, particularly those isolated from the heterogeneous population, were the most effective, exhibiting strong synergy. Single-cell analyses further revealed that sustained selective pressure from the capsule-dependent phage prevents capsule reversion and maintains cocktail efficacy. Overall, our results highlight the importance of accounting for phenotypic heterogeneity when designing phage therapies and support population-level approaches for optimizing phage cocktail composition. Importance: Phage therapy is a promising alternative to antibiotics, but its success is often limited by the rapid emergence of phage-resistant bacteria. These resistant populations can be highly heterogeneous, comprising both stable mutants and variants with reversible, non-genetic resistance. In this study, we explore how this phenotypic diversity influences the effectiveness of phage cocktails. By isolating new phages and testing them in combination, we demonstrate that the selective pressure exerted by specific phages can prevent the reversion in transiently resistant variants, thereby sustaining treatment efficacy. Our findings highlight the need to consider not only the range of bacterial targets but also how phage pressure shapes bacterial population dynamics. This work offers a more refined strategy for designing phage cocktails with improved clinical potential.