Biology subjects

Tomas, M.

Publications and source records attributed to Tomas, M..

16 recordsLinked to original sources

bioRxiv2026

Variant emergence, not vaccine deployment, drives episodic positive selection on the SARS-CoV-2 spike at provincial scale in Canada

Mass immunization against SARS-CoV-2 created a heterogeneous landscape of antibody-mediated immune pressure, yet whether this pressure measurably altered episodic positive selection on spike remains unresolved. Using Canadian genomic surveillance data spanning the five major variants of concern (Alpha, Beta, Gamma, Delta, and Omicron), we inferred time-resolved phylogenies from spike-coding sequences and applied site- and branch-level episodic selection models to identify when and where adaptive change occurred. To evaluate whether vaccination intensity was associated with selection, we integrated these phylogenetic analyses with provincial vaccination time series using cross-correlation and lagged panel regression models that accounted for province and time effects, lineage prevalence, and sampling heterogeneity. Episodic positive selection was concentrated at a limited number of spike codons, especially within the N-terminal domain, receptor-binding domain, and furin cleavage region. However, these signals were dominated by substitutions associated with variant emergence, particularly during the Alpha-to-Delta transition, rather than by vaccination rollout. Whole-gene tests provided no evidence that vaccine intensity was associated with elevated episodic selection, and residualized vaccination trajectories did not predict selection at biologically plausible lags. Across provinces, the timing and distribution of selection events were inconsistent with a vaccine-driven escape model. Together, these results indicate that, at provincial resolution in Canada, episodic positive selection on SARS-CoV-2 spike was driven primarily by variant turnover rather than vaccine deployment. More broadly, this study provides a quantitative, VOC-resolved assessment of spike evolution in a structured epidemic and suggests that population-level vaccination intensity was not a detectable determinant of spike adaptation in the period examined.

evolutionary biology↗

bioRxiv2025

Identification and Functional Insights into New Phage Tail-Like Bacteriocins (PTLBs) Targeting Pseudomonas aeruginosa as new antimicrobials

The current health crisis caused by multidrug resistant (MDR) pathogens is one of the health problems of most concern globally. Infections caused by these pathogens, such as Pseudomonas aeruginosa, lead to high rates of complications, particularly in compromised patients such as cystic fibrosis (CF) patients. The need to counteract and minimize the forecast future impact has led to the rescue of phage therapy. The use of bacteriophages has important advantages, including highly specific targeting, self-amplification at the infection site, minimal disruption of the microbiome, safety and biocompatibility. However, the capacity of bacteria to escape these entities results in a form of resistance that compromises the effectiveness of the therapy. This involves the search for potential alternatives, such as the phage tail-like bacteriocins (PTLBs), also named as tailocins. These high molecular weight particles resemble the tail structure of bacteriophages and are characterized by the absence of genetic material, avoiding the development of resistance, one of the major handicaps associated with phage therapy. In this study, we detected 34 different PTLBs in 75 P. aeruginosa genomes, with different serotypes and sequence types, 11 of which were characterized as novel F-type PTLBs subtypes (F13-F24). Furthermore, we report that four selected PTLBs (R1, F15, F19, R3-F24) can deal with bacterial infection, with the R1 and the F15 PTLBs being the most efficient in clearing infection in vitro, yielding a survival rate of more than 75% in the Galleria mellonella larvae in vivo model. This reaffirms the potential of PTLBs to control P. aeruginosa infections, which can cause chronic infections in some patients, such as people with CF, due to its strong impact as a MDR bacterium. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/671207v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@76b60borg.highwire.dtl.DTLVardef@1a9cd0aorg.highwire.dtl.DTLVardef@d45dd0org.highwire.dtl.DTLVardef@5b345a_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS-The 75 Pseudomonas aeruginosa genomes from people with cystic fibrosis in the study collection included at least one Phage tail-like bacteriocins (PTLB) cluster. -From the 34 different PTLBs detected in the study collection, 7 were R-type, 10 were complex (R and F-type encoded) and 14 were F-type PTLBs. -11 new F-type PTLBs were described in the Pseudomonas aeruginosa collection under study. -An association between the O-antigen present on the surface of the Pseudomonas aeruginosa isolate and the encoded PTLB subtype was detected. -The R1 and F15 PTLBs subtypes display high antimicrobial activity both in vitro and in vivo (Galleria mellonella).

microbiology↗

bioRxiv2025

Proteo-transcriptomic reprogramming and resource reallocation define the aging mammalian brain

Brain aging is a major risk for neurodegeneration, yet the underlying molecular mechanisms remain poorly understood. Here we performed an integrative proteo-transcriptomic analysis of the aging mouse brain, uncovering molecular signatures of aging through the assessment of protein aggregation, mRNA relocalization, and comparative proteomics across eight models of premature aging and neurodegeneration. We identified dynamic changes in physiological aging highlighting differences in synaptic maintenance and energy-allocation. These were linked to changes associated with fundamental protein biochemical properties such as size and net charge. Network analysis highlighted a decrease in mitochondrial complex I proteins not compensated at the mRNA level. Aggregation of 60S ribosome subunits indicated deteriorating translation efficiency and was accompanied by mitochondrial and proteasomal imbalance. The analysis of the nine models revealed key similarities and differences between physiological aging and pathology. Overall, our study provides an extensive resource on molecular aging, and offers insights into mechanisms predisposing to neurodegeneration, easily accessible at our Brain Aging and Molecular Atlas Project (BrainAging-MAP) website.

neuroscience↗

bioRxiv2024

The LAMP-CRISPR-Cas13a technique for detecting CBASS-mechanism of phage resistance in bacteria

Antimicrobial resistance (AMR) is an important threat to public health that has led to the development of innovative alternative treatments for bacterial infections, such as phage therapy. However, one of the greatest disadvantages of phage therapy is the generation of phage-resistant bacterial mutants via bacterial defence mechanisms, which are mainly contained in genomic islands (GIs) and controlled by the quorum sensing (QS) network. In this study, 309 pathogenic islands (PAIs) harbouring a total of 22.1 % of proteins related to anti-phage defence (APD) were detected in the genome of 48 K. pneumoniae strains. Several type I and type II CBASS systems were also detected in the genome of the 48 K. pneumoniae strains, but only 2 type II CBASS systems were located in PAIs. We constructed a knockout K. pneumoniae strain, not expressing the cyclase gene from the type II CBASS system present in PAIs, to study the regulatory role of QS in expressing the gene. As the anti-phage CBASS system is an abortive infection (Abi) system, the role of the type II CBASS system in regulating cell viability was assessed. The knockout strain was confirmed by targeting the LAMP-CRISPR-Cas13a technique specifically to the cyclase gene, and the same protocol was also used to detect the gene of the main cyclase in these type I CBASS systems, i.e. APECO1. The study findings demonstrate the regulatory role of the QS network in anti-phage defence systems. Finally, this is the first work which development an innovative biotechnological application for the LAMP-CRISPR-Cas13a rapid-technique (<2 hours) in optimizing phage therapy by detecting bacterial resistance mechanisms, by predicting the potential inefficacy of a therapeutic phage and thus improving patient prognosis.

microbiology↗

bioRxiv2024

Studies in vitro e in vivo of Phage Therapy Medical Products (PTMPs) targeting clinical strains of Klebsiella pneumoniae belonging to the clone ST512

The widespread incidence of antimicrobial resistance has created renewed interest in the use of alternative antimicrobial treatments such as phage therapy. Phages are viruses that infect bacteria and generally have a narrow bacteria host-range. Combining phages with antibiotics can prevent the emergence of bacterial resistance. The aim of the present study was to develop phage therapy medical products (PTMPs) targeting clinical isolates of carbapenems-producing Klebsiella pneumoniae belonging to the high-risk clone ST512. From a collection of twenty-two seed of lytic phages sequenced belonging to MePRAM collection, four were used to generate PTMPs (CAC_Kpn1 and CAC_Kpn2). These PTMPs were partly active against three of the clinical strains of clone ST512 (A, B and C). The use of Appelmans method in the CAC_Kpn1_ad (adapted CAC_Kpn1) yielded a significant increase in the efficacy against strain A, while adapted CAC_Kpn2 (CAC_Kpn2_ad) only effectively reduced bacterial survival when combined with [1/2] x MIC {beta}-lactam antibiotic meropenem for 24 h in clinical strains B and C, showed after this time, resistance to PTMPs. In addition, the amounts of endotoxin released by the PTMPs were quantified and subsequently reduced in preparation for in vivo use of the PTMPs in Galleria mellonella infection model confirming the in vitro results from the CAC_Kpn1_ad and CAC_Kpn2_ad.

microbiology↗

bioRxiv2024

Involvement of Pf-like phages in resistance to phage infection in clinical isolates of P. aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa is a bacterial pathogen that is a major cause of lung infections in cystic fibrosis (CF) and other patients. Isolates of P. aeruginosa from CF patients commonly carry filamentous phages (Pf phages), a type of temperate phage known to be related to biofilm production and antibiotic sequestration. In this study, 12 new Pf-like phages were identified in a collection of clinical isolates of P. aeruginosa from CF patients. Analysis of the phage genomes revealed different anti-phage defence systems, described here for first time in these types of phages. Finally, relationships between resistance to phage infection and the presence of Pf-like phages and also between each defence system and resistance were observed. IMPORTANCEBacteria harbour a wide range of defence mechanisms to avoid phage infections. These mechanisms hamper the application of phage therapy because they can lead to the rapid acquisition of phage resistance. Temperate phages, including the filamentous phages, carry genes encoding virulence factors and also anti-phage defence mechanisms, as their survival depends on the host survival. In this study, we identified 12 new Pf-like phages encoding several different anti-phage defence mechanisms, some observed for the first time in this type of phage. A relationship between these phages and resistance to phage infection was also observed. The study findings are important as they provide information about newly discovered filamentous phages and their proteomes and also about the role of these phages in resistance to phage infections. Studying the genome of clinical isolates carrying these phages could help to improve phage therapy by targeting these phages or its genes.

microbiology↗

bioRxiv2023

Control of the phage defence mechanisms by Quorum sensing (QS) in clinical isolates of Klebsiella pneumoniae

Multidrug resistant (MDR) bacteria and the shortage of new antibiotics are a serious health problem and have increased the interest in bacteriophages, with great potential as antimicrobial agents but they can induce resistance. The objective of the present study was to reduce the development of phage resistance in K. pneumoniae strains by inhibiting the Quorum Sensing (QS). The QS inhibition by cinnamaldehyde (CAD) was confirmed indirectly by the reduction of biofilm production and directly by a proteomic analysis. Also, the infection assays showed that the phage resistance mechanisms of the bacteria were inhibited when phage-resistant K. pneumoniae strains were treated with a combination of phages with CAD. Finally, these results were confirmed by proteomic analysis as proteins related to the phage defence such as CBASS (bacterial cyclic oligonucleotide-based anti-phage signalling) and R-M systems as well as tail fiber proteins were present under phage treatment but not with the combination. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/570179v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@a97009org.highwire.dtl.DTLVardef@1fa7f02org.highwire.dtl.DTLVardef@a8909corg.highwire.dtl.DTLVardef@ae0284_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Phages Produce Persisters

Bacteria primarily encounter stress, and, arguably, their greatest threats are phages. It is often assumed that those bacteria that escape phage attack have mutated; however, another possibility is that a subpopulation forms the dormant persister state, in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defense systems more time to clear the phage infection. Here we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages. We found that bacteria in plaques can escape phage attack both by mutation (i.e., become resistant) and without mutation (i.e., become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g., causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially-growing cells. Phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

microbiology↗

bioRxiv2023

Single-Cell Analysis Reveals Cryptic Prophage Protease LfgB Protects Escherichia coli During Oxidative Stress by Cleaving Antitoxin MqsA

Although toxin/antitoxin (TA) systems are ubiquitous, beyond phage inhibition and mobile element stabilization, their role in host metabolism is obscure. One of the best-characterized TA systems is MqsR/MqsA of Escherichia coli, which has been linked previously to protecting this gastrointestinal species during the stress it encounters from the bile salt deoxycholate as it colonizes humans. However, some recent whole-population studies have challenged the role of toxins such as MqsR in bacterial physiology, since the mqsRA locus is induced over a hundred-fold during stress, but a phenotype was not found upon its deletion. Here, we investigate further the role MqsR/MqsA by utilizing single cells and demonstrate that upon oxidative stress, the TA system MqsR/MqsA has a heterogeneous effect on the transcriptome of single cells. Furthermore, we discovered that MqsR activation leads to induction of the poorly-characterized yfjXY ypjJ yfjZF operon of cryptic prophage CP4-57. Moreover, deletion of yfjY makes the cells sensitive to H2O2, acid, and heat stress, and this phenotype was complemented. Hence, we recommend yfjY be renamed to lfgB (less fatality gene B). Critically, MqsA represses lfgB by binding the operon promoter, and LfgB is a protease that degrades MqsA to derepress rpoS and facilitate the stress response. Therefore, the MqsR/MqsA TA system facilitates the stress response through cryptic phage protease LfgB.

molecular biology↗

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↗

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↗

bioRxiv2023

Toxin/Antitoxin Systems Induce Persistence and Work in Concert with Restriction/Modification Systems to Inhibit Phage

Myriad bacterial anti-phage systems have been described and often the mechanism of programmed cell death is invoked for phage inhibition. However, there is little evidence of suicide under physiological conditions for these systems. Instead of death to stop phage propagation, we show here that persister cells, i.e., transiently-tolerant, dormant, antibiotic-insensitive cells, are formed and survive using the Escherichia coli C496_10 tripartite toxin/antitoxin system MqsR/MqsA/MqsC to inhibit T2 phage. Specifically, MqsR/MqsA/MqsC inhibited T2 phage by one million-fold and reduced T2 titers by 500-fold. During T2 phage attack, in the presence of MqsR/MqsA/MqsC, evidence of persistence include the single-cell physiological change of reduced metabolism (via flow cytometry), increased spherical morphology (via transmission electron microscopy), and heterogeneous resuscitation. Critically, we found restriction-modification systems (primarily EcoK McrBC) work in concert with the toxin/antitoxin system to inactivate phage, likely while the cells are in the persister state. Phage attack also induces persistence in Klebsiella and Pseudomonas spp. Hence, phage attack invokes a stress response similar to antibiotics, starvation, and oxidation, which leads to persistence, and this dormant state likely allows restriction/modification systems to clear phage DNA.

molecular biology↗

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↗

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↗