Suggestion for a new bacteriophage genus for the Klebsiella pneumoniae phage vB_KpnS-Carvaje.

This work describes the newly isolated Klebsiella pneumoniae phage vB_KpnS-Carvaje that presents unique features in relation to other phages reported to date. These findings provide new insights into the diversity and evolutionary pathways of Klebsiella phages. The genome characterization of the Carvaje phage revealed that its genome length is approximately 57 kb with 99 open reading frames (ORFs), 33 of which have assigned functions while 66 are unknown. This phage differs from other sequenced Klebsiella phages, showing the closest resemblance (up to 65.32%) with Salmonella phages belonging to the Nonanavirus and Sashavirus genera. Comparisons at the amino acid level and phylogeny analysis among homologous genomes indicate that the Klebsiella Carvaje phage forms a novel sister taxon within the node of the Nonanaviruses and Sashaviruses cluster. Due to the unique features of the Carvaje phage, we propose the constitution of a new genus within the Caudoviricetes class. Further studies include the exploitation of this phage and its identified proteins for the control of Klebsiella infections and as recognition molecules in diagnostic methods.

Intra- and Extra-Hospital Dissemination of IMP-22-Producing Klebsiella pneumoniae in Northern Portugal: The Breach of the Hospital Frontier Toward the Community.

The emergence of infections (and colonization) with Enterobacteriaceae-producing carbapenemases is a threatening public health problem. In the last decades, we watched an isolated case becoming a brutal outbreak, a sporadic description becoming an endemic problem. The present study aims to highlight the dissemination of IMP-22-producing Klebsiella pneumoniae in the North of Portugal, through the phenotypic and genotypic characterization of isolates collected from hospitalized patients (n=5) and out-patients of the emergency ward of the same acute care hospital (n=2), and isolates responsible for the intestinal colonization of residents in a Long-Term Care Facility (n=4). Pulsed-field gel electrophoresis (PFGE) results, associated with conjugation experiments pointed to a pattern of both vertical and horizontal dissemination. Overall, and complementing other studies that give relevance to IMP-22-producing K. pneumoniae in the clinical settings, here we show for the first time the public health threatening breach of the hospital frontier of this resistance threat, toward the community.

Pseudomonas aeruginosa PAO 1 In Vitro Time-Kill Kinetics Using Single Phages and Phage Formulations-Modulating Death, Adaptation, and Resistance.

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time-kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.

Characterization of MSlys, the endolysin of Streptococcus pneumoniae phage MS1.

Despite the use of pneumococcal conjugate vaccines, the number of infections related to Streptococcus pneumoniae continues to be alarming. Herein, we identified, characterized the MSlys endolysin encoded in the phage MS1. We further tested its antimicrobial efficacy against planktonic and biofilm cells, assessing the culturability of cells and biofilm structure by scanning electron microscopy, and confocal laser scanning microscopy. The modular MSlys endolysin consists of an amidase catalytic domain and a choline-binding domain. MSlys is active against isolates of children with otitis media, and conditions close to those found in the middle ear. Treatment with MSlys (2 h, 4 µM) reduced planktonic cultures by 3.5 log10 CFU/mL, and 24- and 48-h-old biofilms by 1.5 and 1.8 log10 CFU/mL, respectively. Imaging of the biofilms showed thinner and damaged structures compared to control samples. The recombinantly expressed MSlys may be a suitable candidate for treating pneumococcal infections, including otitis media.

Identification of genomic loci associated with genotypic and phenotypic variation among Pseudomonas aeruginosa clinical isolates from pneumonia.

In this work, a genotype-phenotype survey of a highly diversified Pseudomonas aeruginosa collection was conducted, aiming to detail pathogen-associated scenarios that clinicians face nowadays. Genetic relation based on RAPD-PCR of 705 isolates, retrieved from 424 patients and several clinical contexts, reported an almost isolate-specific molecular-pattern. Pneumonia-associated isolates HB13 and HB15, clustered in the same RAPD-PCR group, were selected to evaluate the genomic background underlying their contrasting antibiotic resistance and virulence. The HB13 genome harbors antibiotic-inactivating enzymes-coding genes (e.g. aac(3)-Ia, arr, blaVIM-2) and single-nucleotide variations (SNVs) in antibiotic targets, likely accounting for its pan-resistance, whereas HB15 susceptibility correlated to predicted dysfunctional alleles. Isolate HB13 showed the unprecedented rhl-cluster absence and variations in other pathogen competitiveness contributors. Conversely, HB15 genome comprises exoenzyme-coding genes and SNVs linked to increased virulence. Secretome analysis identified signatures features with unknown function as potential novel pathogenic (e.g. a MATE-protein in HB13, a protease in HB15) and antibiotic resistance (a HlyD-like secretion protein in HB13) determinants. Detection of active prophages, proteases (including protease IV and alkaline metalloproteinase), a porin and a peptidase in HB15 highlights the secreted arsenal likely essential for its virulent behavior. The presented phenotype-genome association will contribute to the current knowledge on Pseudomonas aeruginosa pathogenomics.

Use of newly isolated phages for control of Pseudomonas aeruginosa PAO1 and ATCC 10145 biofilms.

Pseudomonas aeruginosa is a relevant opportunistic pathogen involved in nosocomial infections that frequently shows low antibiotic susceptibility. One of its virulence factors is associated with the ability to adhere to surfaces and form virulent biofilms. This work describes the isolation and characterization of lytic phages capable of infecting antibiotic-resistant P. aeruginosa strains. In addition, characterization of P. aeruginosa biofilms and the potential of newly isolated phages for planktonic and biofilm control was accessed. According to the results, the isolated phages showed different spectra of activity and efficiency of lysis. Four broad lytic phages were selected for infection of planktonic cells; however, despite their broad range of activity, two of the selected phages failed to efficiently control planktonic cultures. Therefore, only two phages (phiIBB-PAA2 and phiIBB-PAP21), highly capable of causing strong biomass reduction of planktonic cells, were tested against 24 h biofilms using a m.o.i. of 1. Both phages reduced approximately 1-2 log the biofilm population after 2 h of infection and reduction was further enhanced after 6 h of biofilm infection. However, biofilm cells of P. aeruginosa PAO1 acquired resistance to phiIBB-PAP21; consequently, an increase in the number of cells after 24 h of treatment was observed. Conversely, phage phiIB-PAA2 for P. aeruginosa ATCC10145 continued to destroy biofilm cells, even after 24 h of infection. In these biofilms, phages caused a 3 log reduction in the number of viable counts of biofilm cells.

Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium.

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria.