Biology subjects

Martinez-Martinez, L.

Publications and source records attributed to Martinez-Martinez, L..

4 recordsLinked to original sources

bioRxiv2023

Molecular and phylogenetic characterization of the monkeypox outbreak in the South of Spain

Until the May 2022 Monkeypox outbreak, which spread rapidly to many non-endemic countries, the virus was considered a viral zoonosis limited to some African countries. The Andalusian circuit of genomic surveillance was rapidly applied to characterize the Monkeypox outbreak in the South of Spain. Whole genome sequencing was used to obtain the genomic profiles of samples collected across the south of Spain, representative of all the provinces of Andalusia. Phylogenetic analysis was used to study the relationship of the isolates and the available sequences of the 2022 outbreak. Whole genome sequencing of a total of 160 monkeypox viruses from the different provinces that reported cases were obtained. Interestingly, we report the sequences of monkeypox viruses obtained from two patients who died. While one of the isolates bore no noteworthy mutations that explain a potential heightened virulence, in another patient the second consecutive genome sequence, performed after the administration of tecovirimat, uncovered a mutation within the A0A7H0DN30 gene, known to be a prime target for tecovirimat in its Vaccinia counterpart. In general, a low number of mutations were observed in the sequences reported, which were very similar to the reference of the 2022 outbreak (OX044336), as expected from a DNA virus. The samples likely correspond to several introductions of the circulating monkeypox viruses from the last outbreak. The virus sequenced from one of the two patients that died presented a mutation in a gene that bears potential connections to drug resistance. This mutation was absent in the initial sequencing prior to treatment.

microbiology↗

bioRxiv2023

Molecular Studies of Phages-Klebsiella pneumoniae in a Mucoid Environment: Innovative use of mucolytic agents prior to the administration of lytic phages

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co- evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced these conditions using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co- evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.

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↗

bioRxiv2019

In vitro and in vivo efficacy of the combination of colistin and endolysins against clinical strains of Multi-Drug Resistant (MDR) pathogens

The multidrug resistance (MDR) among pathogenic bacteria is jeopardizing the worth of antimicrobials, which had previously changed medical sciences. In this study, we used bioinformatic tools to identify the endolysins ElyA1 and ElyA2 (GH108-PG3 family) present in the genome of bacteriophages Ab1051{Phi} and Ab1052{Phi}, respectively. The muralytic activity of these endolysins over MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. The minimal inhibitory concentrations (MICs) of endolysin, colistin and their combination were determined using the microdilution checkerboard method. The antimicrobial activity of the combinations was confirmed by time kill curves and in vivo assays in larvae of Galleria mellonella. Our results showed that ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. No activity was detected when assays were done with endolysin ElyA2. The combined antimicrobial activity of colistin and endolysin ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays. In conclusion, the combination of colistin with new endolysins such as ElyA1 could increase the bactericidal activity and reduce the MIC of the antibiotic, thus also reducing the associated toxicity.\n\nIMPORTANCEThe development of multiresistance by pathogen bacteria increases the necessity of the development of new antimicrobial strategies. In this work, we combined the effect of the colistin with a new endolysin, ElyA1, from a bacteriophage present in the clinical strain of Acinetobacter baumannii Ab105. ElyA1 is a lysozyme-like family (GH108-GP3), whose antimicrobial activity was described for first time in this work. Also, another endolysin, ElyA2, with the same origin and family, was characterized but in this case no activity was detected. ElyA1 presented lytic activity over a broad spectrum of strains from A. baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. When colistin was combined with ElyA1 an increase of the antimicrobial activity was observed with a reduced concentration of colistin, and this observation was also confirmed in vivo in Galleria mellonella larvae. The combination of colistin with new endolysins as ElyA1 could increase the bactericidal activity and lowering the MIC of the antibiotic, thus also reducing the associated toxicity.

microbiology↗