Biology subjects

Pacios, O.

Publications and source records attributed to Pacios, O..

4 recordsLinked to original sources

bioRxiv2023

In vitro and in vivo combination of lytic phages and octapeptin OPX10053 against B-lactamase-producing clinical isolates of Klebsiella pneumoniae

Backgroundnovel approaches to treat Klebsiella pneumoniae infections are desperately needed, such as the use of rationally designed combination therapies. Objectivesto evaluate the in vitro and in vivo therapeutic potential of lytic phages against K. pneumoniae in combination with octapeptin, a promising class of lipopeptides with broad spectrum Gram-negative activity. Methodswe determined the MICs to twenty-two lipopeptide compounds and chose one octapeptin (OPX10053) for evaluation of potential synergism in combination with lytic phages using checkerboard assays, optical density growth curves and time-kill (CFU enumeration). Toxicity and efficacy in vivo assays were conducted on Galleria mellonella larvae. Resultsthis study reports the synergy found in vitro between the octapeptin OPX10053 and two lytic phages previously characterized by our research group (vB_KpnM-VAC13 and vB_KpnM-VAC66) against clinical isolates of K. pneumoniae. This synergy was validated by the FIC index, OD growth curves and time-kill assay when OPX10053 was added following 4 hours of phage exposure. Preliminary evaluation of toxicity revealed that OPX10053, even at subinhibitory concentrations and in phage combinations, exerts a toxic effect on larvae, which requires further investigation. ConclusionsThe in vitro application of lytic phages in combination with octapeptin OPX10053 showed synergistic activity. Exposure of G. mellonella to the lytic phages was well tolerated, whereas combination treatment with subinhibitory concentrations of OPX10053 did not attenuate toxicity. Even so, this innovative approach of combining lytic phages could open the door to some interesting associations between chemically synthesized drugs and biological entities. Sequential or simultaneous application alongside time, dosing and stewardship warrants further research.

microbiology↗

bioRxiv2022

Molecular analysis of the interactions between phages and the bacterial host Klebsiella pneumoniae

Lytic phages are currently considered among the best options for treating infections caused by multi-drug resistant pathogens. Phages have some advantages over conventional antibiotics. For example, phages acquire modifications in accordance with their environment, and thus with the bacteria present, which has led to the co-evolution of both types of organism. Therefore, both 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 twenty-one novel lytic phages of Klebsiella pneumoniae in search of defence mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defence mechanisms of two clinical isolates of Klebsiella pneumoniae infected by phages. For this purpose, the twenty-one lytic phages were sequenced and de novo assembled using the Illumina-Miseq system and Spades V.3.15.2 respectively. Gene annotation was performed with Patric, Blast, Hhmer and Hhpred tools. The evolutionary relationships between phages were determined by RaxML. The host-range was determined in a collection of forty-seven clinical isolates of K. pneumoniae, revealing the variable infectivity capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the family Caudovirales. The size and GC content of the phages ranged from 39,371 to 178,532 bp and from 41.72 % to 53.76 %, respectively. 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 defence mechanisms against bacteria, including the restriction-modification (RM) system, the toxin-antitoxin (TA) system, evasion of DNA degradation, blocking of host RM, 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 defence mechanisms against phage infection (prophage, plasmid, defence/virulence/resistance and oxidative stress proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCEPhages, viral parasites of bacteria, have long protected the Earths biosphere against bacterial overgrowth and could now help in the fight against antimicrobial resistance. However, researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about the defence mechanisms in both viruses and bacteria. In this study, we analyzed the molecular mechanisms of viral and bacterial defence in phages infecting clinical isolates of Klebsiella pneumoniae. Viral defence mechanisms included RM system evasion, the Toxin-Antitoxin system, DNA degradation evasion, blocking of host RM and resistance to the abortive infection system (Abi), anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defence mechanisms, proteomic analysis revealed overexpression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defence/virulence/resistance (porins, efflux pumps, LPS, pili 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.

microbiology↗

bioRxiv2022

Application of RT-LAMP-CRISPR-Cas13a technology to the detection of OXA-48 producing Klebsiella pneumoniae

Carbapenem-resistant pathogens have been recognized as a health concern because of their ability to cause severe infections and because they are difficult to detect in laboratories. Researchers are making great efforts to develop a diagnostic technique with high levels of sensitivity and specificity, as accurate, early diagnosis is required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic techniques due to their high specificity: Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. This technology is usually combined with isothermal pre-amplification reactions in order to increase the sensitivity of diagnosis. We have developed an RT-LAMP-CRISPR-Cas13a-based assay for the detection of Klebsiella pneumoniae OXA-48 producer strains in clinical samples without the need for RNA extraction. The assay exhibited 100 % specificity, sensitivity, positive predictive value and negative predictive value.

microbiology↗

bioRxiv2022

Molecular characteristics of phages located in Carbapenemase-Producing Escherichia coli clinical isolates: New Phage-Like Plasmids

Escherichia coli normally inhabits the gastrointestinal tract of humans and animals. Most E. coli bacteria do not cause problems, but the acquisition of different resistance and virulence genes encoded by mobile plasmids or phages by different bacterial isolates has been associated with the appearance of successful high-risk clones of multidrug-resistant (MDR) E. coli such as ST131 or ST405. In the present study, 50 temperate bacteriophages present in 21 clinical isolates of carbapenemase-producing E. coli of sequence types (STs) ST38, ST131, ST167, ST405 and ST410 were analysed. These phages were classified in the three families of the order Caudovirales: 24 within the family Siphoviridae, 23 in Myoviridae and 3 in Podoviridae. The size of the phages studied ranged from 11 to 95 Kb. Phylogenetic analysis of the terminase large subunit allowed us to classify these phages into different groups showing similarity with the phage sequences deposited in the Microbe Versus Phage (MVP) database and which belonged to clusters 229, 604, 2503 and 2725. On the other hand, bioinformatic study revealed that most of the identified proteins exerted a structural function (26.73%) but also functions involved in lysis/lysogeny (6.70%) or regulation (5.20%) among others. In addition, the ParA-ParB partitioning system and the type II toxin-antitoxin Phd-Doc system were also found in two of the phages studied, which could indicate the presence of plasmid-prophages. Host range testing revealed that two isolates were more susceptible to infection than the other isolates. IMPORTANCEEscherichia coli is one of the pathogens that causes most problems in human health, as it presents multiple resistances to different antibiotics. The study of bacteriophages located in different isolates of this species is important for the development of new anti-infective therapies. Currently, antibiotic resistance is a major problem, but more and more studies are pointing to experimental treatments with bacteriophages as a possible solution.

bioinformatics↗