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Batteriofagi contro patogeni umani e vegetali

La resistenza agli antibiotici nei batteri è diventata un fenomeno così importante da aver giustificato la previsione di un’era “post-antibiotica” nel XXI secolo. Prudenti stime sull’impatto dell’antibiotico-resistenza riportano infatti che attualmente le morti attribuibili a tale causa siano 700.000/anno, e che questo numero sia destinato a salire a 10 milioni nel 2050. Ciò significa che, ad esempio, il numero di morti per questa causa potrebbero essere anche superiore alla somma di quelli attualmente registrati per patologie tumorali (8,2 milioni) e incidenti d’auto (1,2 milioni) (https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf). In aggiunta, la problematica legata ai batteri resistenti investe non solo il campo della salute umana e degli animali in genere, ma anche quello ambientale. Allo scopo di limitare quanto più possibile l’utilizzo di antibiotici e la pressione selettiva che favorisce lo scambio e la selezione delle resistenze, nella ricerca di soluzioni alternative ha acquistato nuova importanza la possibilità di impiegare i batteriofagi. In questo campo ci siamo impegnati nella ricerca di batteriofagi virulenti nei confronti di batteri sia di interesse medico, sia di interesse per l’agricoltura. Le ricerche hanno portato all’isolamento e alla caratterizzazione di batteriofagi attivi contro Pseudomonas syringae pathovar actinidiae e di Klebsiella pneumoniae.

Le ricerche in questo campo sono condotte principalmente da Marco Maria D’Andrea e Gustavo Di Lallo, con il prezioso contributo del Prof. Gian Maria Rossolini (Università di Firenze) e dei suoi collaboratori.

I risultati preliminari sui batteriofagi di K. pneumoniae sono stati premiati in occasione del congresso della Società Italiana di Microbiologia (2014, Torino) e del congresso Bacteriophage 2016 (London, UK).

 

D'Andrea M.M., Frezza D., Romano E., Marmo P., Henrici De Angelis L., Perini N., Thaller M.C. and Di Lallo G. (2019) - THE LYTIC BACTERIOPHAGE VB_EFAH_EF1TV, A NEW MEMBER OF THE HERELLEVIRIDAE FAMILY, DISRUPTS BIOFILM PRODUCED BY ENTEROCOCCUS FAECALIS CLINICAL STRAINS. Journal of Global Antimicrobial Resistance, PII S2213-7165(19)30273-5. DOI: 10.1016/j.jgar.2019.10.019

 

Objectives. The aim of this study is to characterize a new bacteriophage able to infect Enterococcus faecalis, and to evaluate its ability to disrupt biofilm.

Methods. The vB_EfaH_EF1TV (EF1TV) host-range was determined by spot test and efficiency of plating using a collection of 15 E. faecalis clinical strains. The phage genome was sequenced with a next generation sequencing approach. Anti-biofilm activity was tested by crystal violet method and confocal laser scanning microscopy. Phage-resistant mutants were selected and sequenced to investigate receptors exploited by phage for infection.

Results. EF1TV is a newly discovered E. faecalis phage which belongs to the Herelleviridae family. EF1TV, whose genome is 98% identical to φEF24C, is characterized by a linear dsDNA genome of 143,507 bp with direct terminal repeats of 1,911 bp. The phage is able to infect E. faecalis and shows also the ability to degrade biofilm produced by strains of this species. The results were confirmed by confocal laser scanning microscopy analyzing the biofilm reduction in the same optical field before and after phage infection.

Conclusions. The EF1TV phage shows promising features such as an obligatory lytic nature, an anti-biofilm activity and the absence of integration-related proteins, antibiotic resistance determinants and virulence factors, and therefore could be a promising tool for therapeutic applications.

 

Ciacci N., D’Andrea M.M., Marmo P., Demattè E., Amisano F., Di Pilato V., Fraziano M., Lupetti P., Rossolini G.M. and Thaller M.C. (2018) - CHARACTERIZATION OF VB_KPN_F48, A NEWLY DISCOVERED LYTIC BACTERIOPHAGE FOR KLEBSIELLA PNEUMONIAE OF SEQUENCE TYPE 101. Viruses, 10(9): 482-497. DOI: 10.3390/v10090482

Resistance to carbapenems in Enterobacteriaceae, including Klebsiella pneumoniae, represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for K. pneumoniaeisolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of Myoviridae, order Caudovirales, on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the Tevenvirinae subfamily, which we tentatively named “F48virus”. Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications.

D'Andrea M., Marmo P., Henrici De Angelis L., Palmieri M., Ciacci N., Di Lallo G., Demattè E., Vannuccini E., Lupetti P., Rossolini G.M., Thaller M.C. (2017) - ΦBO1E, A NEWLY DISCOVERED LYTIC BACTERIOPHAGE TARGETING CARBAPENEMASE-PRODUCING KLEBSIELLA PNEUMONIAE OF THE PANDEMIC CLONAL GROUP 258 CLADE II LINEAGE. Scientific Reports 7(1): 2614. DOI: 10.1038/s41598-017-02788-9

The pandemic dissemination of KPC carbapenemase-producing Klebsiella pneumoniae (KPC-KP) represents a major public health problem, given their extensive multidrug resistance profiles and primary role in causing healthcare-associated infections. This phenomenon has largely been contributed by strains of Clonal Group (CG) 258, mostly of clade II, which in some areas represent the majority of KPC-KP isolates. Here we have characterized a newly discovered lytic Podoviridae, named φBO1E, targeting KPC-KP strains of clade II lineage of CG258. Genomic sequencing revealed that φBO1E belongs to the Kp34virus genus (87% nucleotide identity to vB_KpnP_SU552A). ΦBO1E was stable over a broad pH and temperature range, exhibited strict specificity for K. pneumoniae strains of clade II of CG258, and was unable to establish lysogeny. In a Galleria mellonella infection model, φBO1E was able to protect larvae from death following infection with KPC-KP strains of clade II of CG258, including one colistin resistant strain characterized by a hypermucoviscous phenotype. To our best knowledge φBO1E is the first characterized lytic phage targeting K. pneumoniae strains of this pandemic clonal lineage. As such, it could be of potential interest to develop new agents for treatment of KPC-KP infections and for decolonization of subjects chronically colonized by these resistant superbugs.