A Phage Therapy Model for the Prevention of Pseudomonas syringae pv. actinidiae Infection of Kiwifruit Plants.

Great efforts have been made with chemicals and pesticides to contain the spread of Pseudomonas syringae pv. actinidiae (Psa) responsible for kiwifruit canker. Unfortunately, only partial results were obtained for this bacterial pandemic, and alternative remedies were proposed to avoid soil pollution and the onset of antibiotic resistance. Among these, phage therapy represents a possible tool with low environmental impact and high specificity. Several phages have been isolated and tested for the capacity to kill Psa in vitro, but experiments to verify their efficacy in vivo are still lacking. In the present study, we demonstrated that the phage φPSA2 (previously characterized) contains the spread of Psa inside plant tissue and reduces the symptoms of the disease. Our data are a strong indication for the efficiency of this phage and open the possibility of developing a phage therapy based on φPSA2 to counteract the bacterial canker of kiwifruit.

Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker.

The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker of kiwifruit. In the last years, it has caused severe economic losses to Actinidia spp. cultivations, mainly in Italy and New Zealand. Conventional strategies adopted did not provide adequate control of infection. Phage therapy may be a realistic and safe answer to the urgent need for novel antibacterial agents aiming to control this bacterial pathogen. In this study, we described the isolation and characterization of two bacteriophages able to specifically infect Psa. φPSA1, a member of the Siphoviridae family, is a temperate phage with a narrow host range, a long latency, and a burst size of 178; φPSA2 is a lytic phage of Podoviridae family with a broader host range, a short latency, a burst size of 92 and a higher bactericidal activity as determined by the TOD value. The genomic sequence of φPSA1 has a length of 51,090 bp and a low sequence homology with the other siphophages, whereas φPSA2 has a length of 40 472 bp with a 98% homology with Pseudomonas putida bacteriophage gh-1. Of the two phages examined, φPSA2 may be considered as a candidate for phage therapy of kiwifruit disease, while φPSA1 seems specific toward the recent outbreak's isolates and could be useful for Psa typing.

Metallo-beta-lactamase production by Pseudomonas otitidis: a species-related trait.

Susceptibility to several ß-lactams and ß-lactamase production was investigated in a collection of 20 strains of Pseudomonas otitidis, a new Pseudomonas species that has been recently recognized in association with otic infections in humans. All strains appeared to be susceptible to piperacillin, cefotaxime, ceftazidime, and aztreonam, while resistance or decreased susceptibility to carbapenems was occasionally observed. All strains were found to express metallo-ß-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named bla(POM), that appeared to be highly conserved in this species. P. otitidis, therefore, is the first example of a pathogenic Pseudomonas species endowed with a resident MBL. The POM-1 protein from P. otitidis type strain MCC10330 exhibits the closest similarity (60 to 64%) to the L1 MBL of Stenotrophomonas maltophilia. Expression in Escherichia coli and Pseudomonas aeruginosa revealed that, similar to L1 and other subclass B3 MBLs, POM-1 confers decreased susceptibility or resistance to carbapenems, penicillins, and cephalosporins but not to aztreonam. Expression of the POM MBL in P. otitidis is apparently constitutive and, in most strains, does not confer a carbapenem-resistant phenotype. However, a strong inoculum size effect was observed for carbapenem MICs, and carbapenem-resistant mutants could be readily selected upon exposure to imipenem, suggesting that carbapenem-based regimens should be considered with caution for P. otitidis infections.

Analysis of Four New Enterococcus faecalis Phages and Modeling of a Hyaluronidase Catalytic Domain from Saphexavirus.

Background: Phage therapy (PT), as a method to treat bacterial infections, needs identification of bacteriophages targeting specific pathogenic host. Enterococcus faecalis, a Gram-positive coccus resident in the human gastrointestinal tract, may become pathogenic in hospitalized patients showing acquired resistance to vancomycin and thus representing a possible target for PT. Materials and Methods: We isolated four phages that infect E. faecalis and characterized them by host range screening, transmission electron microscopy, and genome sequencing. We also identified and three-dimensional modeled a new hyaluronidase enzyme. Results: The four phages belong to Siphoviridae family: three Efquatrovirus (namely vB_EfaS_TV51, vB_EfaS_TV54, and vB_EfaS_TV217) and one Saphexavirus (vB_EfaS_TV16). All of them are compatible with lytic cycle. vB_EfaS_TV16 moreover presents a gene encoding for a hyaluronidase enzyme. Conclusions: The identified phages show features suggesting their useful application in PT, particularly the Saphexavirus that may be of enhanced relevance in PT because of its potential biofilm-digestion capability.

Evidence of Another Anthropic Impact on Iguana delicatissima from the Lesser Antilles: The Presence of Antibiotic Resistant Enterobacteria.

The improper use of antibiotics by humans may promote the dissemination of resistance in wildlife. The persistence and spread of acquired antibiotic resistance and human-associated bacteria in the environment, while representing a threat to wildlife, can also be exploited as a tool to monitor the extent of human impact, particularly on endangered animal species. Hence, we investigated both the associated enterobacterial species and the presence of acquired resistance traits in the cloacal microbiota of the critically endangered lesser Antillean iguana (Iguana delicatissima), by comparing two separate populations living in similar climatic conditions but exposed to different anthropic pressures. A combination of techniques, including direct plating, DNA sequencing and antimicrobial susceptibility testing allowed us to characterize the dominant enterobacterial populations, the antibiotic resistant strains and their profiles. A higher frequency of Escherichia coli was found in the samples from the more anthropized site, where multi-drug resistant strains were also isolated. These results confirm how human-associated bacteria as well as their antibiotic-resistance determinants may be transferred to wildlife, which, in turn, may act as a reservoir of antibiotic resistance.

Therapeutic Options for Infections due to vanB Genotype Vancomycin-Resistant Enterococci.

Enterococci are ubiquitous, facultative, anaerobic Gram-positive bacteria that mainly reside, as part of the normal microbiota, in the gastrointestinal tracts of several animal species, including humans. These bacteria have the capability to turn from a normal gut commensal organism to an invasive pathogen in patients debilitated by prolonged hospitalization, concurrent illnesses, and/or exposed to broad-spectrum antibiotics. The majority of vancomycin-resistant enterococcus (VRE) infections are linked to the vanA genotype; however, outbreaks caused by vanB-type VREs have been increasingly reported, representing a new challenge for effective antimicrobial treatment. Teicoplanin, daptomycin, fosfomycin, and linezolid are useful antimicrobials for infections due to vanB enterococci. In addition, new drugs have been developed (e.g., dalbavancin, telavancin, and tedizolid), new molecules will soon be available (e.g., eravacycline, omadacycline, and oritavancin), and new treatment strategies are progressively being used in clinical practice (e.g., combination therapies and bacteriophages). The aim of this article is to discuss the pathogenesis of infections due to enterococci harboring the vanB operon (vanBVRE) and their therapeutic, state-of-the-art, and future treatment options and provide a comprehensive and easy to use review for clinical purposes.

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.

The lytic bacteriophage vB_EfaH_EF1TV, a new member of the Herelleviridae family, disrupts biofilm produced by Enterococcus faecalis clinical strains.

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 15E. 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 discoveredE. 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.

φBO1E, a newly discovered lytic bacteriophage targeting carbapenemase-producing Klebsiella pneumoniae of the pandemic Clonal Group 258 clade II lineage.

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.