Characterization of four novel bacteriophages targeting multi-drug resistant Klebsiella pneumoniae strains of sequence type 147 and 307

Introduction: the development of alternative antimicrobial strategies is deemed to be a high priority to deal with the challenge caused by the spread of multidrug-resistant (MDR) bacteria in clinical settings. according to several international organizations, phages or components thereof are one of these possible options that could be useful to treat bacterial infections. among the drug-resistant bacteria, carbapenem resistant Klebsiella pneumoniae (CR-Kp) are particularly worrisome, given the extensive MDR profiles, their pandemic dissemination and primary role in healthcare associated and life-threatening infections. In this study we isolated and characterised four lytic bacteriophages targeting two major high-risk clones of CR-Kp circulating in hospital environments, i.e., those belonging to Sequence Type (ST) 307 and ST147. Materials and methods: wastewater samples collected from hospitals located in central Italy were screened for the presence of phages by using a previously characterized collection of K. pneumoniae clinical isolates as hosts and the top-agar overlay technique. host specificity and infection efficiency was assessed by spot test and efficiency of plating, respectively. Dynamic of bacterial infections was determined by the one-step growth curve method. Phages were visualized through transmission electron microscopy (TEM) and their genomes were obtained and analysed by a Next Generation Sequencing approach followed by bioinformatics analysis. Results: four bacteriophages, named GP-1, GP-2, GP-4 and GP-5, have been isolated, purified and produced at high titres. collectively, two phages were able to selectively lyse 12/14 K. pneumoniae strains of ST307, while the other two were active only against all the tested K. pneumoniae strains of ST147 (n=12). phages maintain an overall good stability to temperature and pH changes and were characterized by infection cycles having latency periods ranging from 10 to 50 minutes and burst sizes of 10-100 PFU. results from TEM analysis and genome sequencing demonstrated that the four phages were of different families and allowed to rule out the presence of antibiotic resistance genes, virulence factors or toxins. Discussion and Conclusions: Considering their strictly lytic nature and their high selectivity towards two of the major high-risk clones of K. pneumoniae, the isolated phages can be considered as good candidates for their evaluation in animal models as members of cocktails for applications to treat severe infections caused by CR-Kp strains

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1β, and IL-6, while increasing IL-10 and TGF-β levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress

The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function

Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA-or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections

Monosodium urate crystals promote innate anti-mycobacterial immunity and improve BCG efficacy as a vaccine against tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination

First Case of Patient With Two Homozygous Mutations in MYD88 and CARD9 Genes Presenting With Pyogenic Bacterial Infections, Elevated IgE, and Persistent EBV Viremia

We described for the first time a female patient with the simultaneous presence of two homozygous mutations in MYD88 and CARD9 genes presenting with pyogenic bacterial infections, elevated IgE, and persistent EBV viremia. In addition to defective TLR/IL1R-signaling, we described novel functional alterations into the myeloid compartment. In particular, we demonstrated a defective production of reactive oxygen species exclusively in monocytes upon E. coli stimulation, the inability of immature mono-derived DCs (iDCs) to differentiate into mature DCs (mDCs) and the incapacity of mono-derived macrophages (MDMs) to resolve BCG infection in vitro. Our data do not provide any evidence for digenic inheritance in our patient, but rather for the association of two monogenic disorders. This case illustrates the importance of using next generation sequencing (NGS) to determine the most accurate and early diagnosis in atypical clinical and immunological phenotypes, and with particular concern in consanguineous families. Indeed, besides the increased susceptibility to recurrent invasive pyogenic bacterial infections due to MYD88 deficiency, the identification of CARD9 mutations underline the risk of developing invasive fungal infections emphasizing the careful monitoring for the occurrence of fungal infection and the opportunity of long-term antifungal prophylaxis

Fighting MDR-Klebsiella pneumoniae infections by a combined host- and pathogen-directed therapeutic approach

Klebsiella pneumoniae is an opportunistic pathogen that is very difficult to treat mainly due to its high propensity to acquire complex resistance traits. Notably, multidrug resistance (MDR)-Klebsiella pneumoniae (KP) infections are responsible for 22%–72% of mortality among hospitalized and immunocompromised patients. Although treatments with new drugs or with combined antibiotic therapies have some degree of success, there is still the urgency to investigate and develop an efficient approach against MDR-KP infections. In this study, we have evaluated, in an in vitro model of human macrophages, the efficacy of a combined treatment consisting of apoptotic body-like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) and φBO1E, a lytic phage specific for the major high-risk clone of KPC-positive MDR-KP. Results show that ABL/PI5P did not affect in a direct manner KKBO-1 viability, being able to reduce only the intracellular KKBO-1 bacterial load. As expected, φBO1E was effective mainly on reducing extracellular bacilli. Importantly, the combination of both treatments resulted in a simultaneous reduction of both intracellular and extracellular bacilli. Moreover, the combined treatment of KKBO-1-infected cells reduced proinflammatory TNF-α and IL-1β cytokines and increased anti-inflammatory TGF-β cytokine production. Overall, our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with MDR pathogens such as MDR-KP

Phosphatidylserine Liposomes Reduce Inflammatory Response, Mycobacterial Viability, and HIV Replication in Coinfected Human Macrophages

Chronic immune activation is the key pathogenetic event of Mycobacterium tuberculosis-human immunodeficiency virus (HIV) coinfection. We assessed the therapeutic value of phosphatidylserine-liposome (PS-L) in an in vitro model of M. tuberculosis-HIV coinfection. PS-L reduced nuclear factor-kappa B activation and the downstream production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 in bacille Calmette-Guerin-infected macrophages and of TNF-alpha and IL-1 beta in M. tuberculosis-infected and M. tuberculosis-HIV-coinfected macrophages. Importantly, a significant reduction of intracellular M. tuberculosis viability and HIV replication were also observed. These results support the further exploitation of PS-L as host-directed therapy for M. tuberculosis-HIV coinfection

Characterization of vB_StuS_MMDA13, a Newly Discovered Bacteriophage Infecting the Agar-Degrading Species Sphingomonas turrisvirgatae

Members of Sphingomonas genus have gained a notable interest for their use in a wide range of biotechnological applications, ranging from bioremediation to the production of valuable compounds of industrial interest. To date, knowledge on phages targeting Sphingomonas spp. are still scarce. Here, we describe and characterize a lytic bacteriophage, named vB_StuS_MMDA13, able to infect the Sphingomonas turrisvirgatae MCT13 type strain. Physiological characterization demonstrated that vB_StuS_MMDA13 has a narrow host range, a long latency period, a low burst size, and it is overall stable to both temperature and pH variations. The phage has a double-stranded DNA genome of 63,743 bp, with 89 open reading frames arranged in two opposite arms separated by a 1186 bp non-coding region and shows a very low global similarity to any other known phages. Interestingly, vB_StuS_MMDA13 is endowed with an original nucleotide modification biosynthetic gene cluster, which greatly differs from those of its most closely related phages of the Nipunavirus genus. vB_StuS_MMDA13 is the first characterized lytic bacteriophage of the Siphoviridae family infecting members of the Sphingomonas genus

Phage Resistance Is Associated with Decreased Virulence in KPC-Producing Klebsiella pneumoniae of the Clonal Group 258 Clade II Lineage

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage phiBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that phiBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host