Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new ...means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 β-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD
) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an
infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both
and
The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 β-lactamase. Interestingly, the combinations decreased the emergence of
resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 β-lactamase.
Abstract
Objectives
To search for new means of combatting carbapenemase-producing strains of Klebsiella pneumoniae by repurposing the anti-helminth drug niclosamide as an antimicrobial agent and ...combining it with the efflux pump inhibitor (EPI) phenyl-arginine-β-naphthylamide (PaβN).
Methods
Niclosamide and PaβN MICs were determined for six clinical K. pneumoniae isolates harbouring different carbapenemases by broth microdilution and chequerboard assays. Time–kill curves in the presence of each drug alone and in combination were conducted. The viability of bacterial cells in the presence of repetitive exposures at 8 h to the treatment at the same concentration of niclosamide and/or PaβN (adapted isolates) was determined. The acrAB-tolC genes and their regulators were sequenced and quantitative RT–PCR was performed to assess whether the acrA gene was overexpressed in adapted isolates compared with non-adapted isolates. Finally, the MICs of several antimicrobials were determined for the adapted isolates.
Results
Niclosamide and PaβN had synergistic effects on the six isolates in vitro, but adaptation appeared when the treatment was applied to the medium every 8 h, with an increase of 6- to 12-fold in the MIC of PaβN. Sequencing revealed different mutations in the regulators of the tripartite AcrAB-TolC efflux pump (ramR and acrR) that may be responsible for the overexpression of the efflux pump and the adaptation to this combination. Co-resistance to different antimicrobials confirmed the overexpression of the AcrAB-TolC efflux pump.
Conclusions
Despite the synergistic effect that preliminary in vitro stages may suggest, the combinations of drugs and EPI may generate adapted phenotypes associated with antimicrobial resistance that must be taken into consideration.
CheckDen, a program to compute quantum molecular properties on a variety of spatial grids is presented. The program reads as unique input wavefunction files written by standard quantum packages and ...calculates the electron density
ρ(
r), promolecule and density difference function, gradient of
ρ(
r), Laplacian of
ρ(
r), information entropy, electrostatic potential, kinetic energy densities
G(
r) and
K(
r), electron localization function (ELF), and localized orbital locator (LOL) function. These properties can be calculated on a wide range of one-, two-, and three-dimensional grids that can be processed by widely used graphics programs to render high-resolution images.
CheckDen offers also other options as extracting separate atom contributions to the property computed, converting grid output data into CUBE and OpenDX volumetric data formats, and perform arithmetic combinations with grid files in all the recognized formats.
Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella ...pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and
assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order
s. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages.
Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.
is the clinically most important species within the genus
and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in ...the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of
belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order
(27 prophages belonging to the family
, 10 prophages belonging to the family
and 3 prophages belonging to the family
). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from ter
operon) and methyltransferase proteins.
We report one case of Fournier's gangrene secondary to urethral catheterization.
We describe the clinical case, in which the initial cause was identified, and perform a short bibliographic review.
...Although Fournier's gangrene was initially considered as idiopathic in etiology, currently it is possible to identify the entrance site of the infection. In the present case the insertion of a urethral catheter was the starting mechanism, associated with factors such as diabetes and alcoholism which favour its development. The patient was treated by surgical debridement and partial urethrectomy but finally died.
We want to point out that urethral instrumentation should be done by expert hands due to the severity of possible complications. We should insist in the need of precocious treatment with wide spectrum antibiotics, radical debridement and complete urologic evaluation.
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