Antón Ambroa

Complexo Hospitalario Universitario A Coruña

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Co-Authors

Lucía Blasco

Instituto de Investigación Biomédica de A Coruña

María Tomás

Instituto de Investigación Biomédica de A Coruña

Laura Fernández-García

Universidade da Coruña

Inés Bleriot

Instituto de Investigación Biomédica de A Coruña

María López

Hospital Universitario Infanta Sofía

Germán Bou

Complexo Hospitalario Universitario A Coruña

Felipe Fernández-Cuenca

Universidad de Sevilla

Olga Pacios

Instituto de Investigación Biomédica de A Coruña

ÁP

Álvaro Pascual

Hospital Universitario Virgen Macarena

Rocío Trastoy

Complejo Hospitalario Universitario de Santiago

Cooperative Institutions

Instituto de Salud Carlos III

Centro de Investigación Biomédica en Red

Red Espanola de Investigacion en Patologia Infecciosa

Fundación Seimc-Gesida

Instituto de Biomedicina de Sevilla

Complexo Hospitalario Universitario A Coruña

Universidad de Sevilla

Instituto de Investigación Biomédica de A Coruña

Universidade da Coruña

Hospital Universitario Virgen Macarena

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Relationship Between Quorum Sensing and Secretion Systems

Frontiers in Microbiology (2019)

Rocío Trastoy Lucía Blasco Antón Ambroa Bertha González‐Pedrajo Laura Fernández-García

Quorum sensing (QS) is a communication mechanism between bacteria that allows specific processes to be controlled, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms such as bacterial competition systems including secretion systems (SS).These SS have an important role in bacterial communication.SS are ubiquitous; they are present in both Gram-negative and Gram-positive bacteria and in Mycobacterium sp.To date, 8 types of SS have been described (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, and T9SS).They have global functions such as the transport of proteases, lipases, adhesins, heme-binding proteins, and amidases, and specific functions such as the synthesis of proteins in host cells, adaptation to the environment, the secretion of effectors to establish an infectious niche, transfer, absorption and release of DNA, translocation of effector proteins or DNA and autotransporter secretion.All of these functions can contribute to virulence and pathogenesis.In this review, we describe the known types of SS and discuss the ones that have been shown to be regulated by QS.Due to the large amount of information about this topic in some pathogens, we focus mainly on Pseudomonas aeruginosa and Vibrio spp.

10.3389/fmicb.2019.01100

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Citations (238)

Genomic analysis of 40 prophages located in the genomes of 16 carbapenemase-producing clinical strains of Klebsiella pneumoniae

Microbial Genomics (2020)

Inés Bleriot Rocío Trastoy Lucía Blasco Felipe Fernández-Cuenca Antón Ambroa

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella 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 K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae, 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae). 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 terZABCDE operon) and methyltransferase proteins.

Prophage

Siphoviridae

Multilocus sequence typing

10.1099/mgen.0.000369

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Citations (33)

The role of PemIK (PemK/PemI) type II TA system from Klebsiella pneumoniae clinical strains in lytic phage infection

Scientific Reports (2022)

Inés Bleriot Lucía Blasco Olga Pacios Laura Fernández-García Antón Ambroa

Abstract Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.

Lytic cycle

Antitoxin

Lysogenic cycle

10.1038/s41598-022-08111-5

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Citations (27)

Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection

Frontiers in Microbiology (2022)

Antón Ambroa Lucía Blasco María López Olga Pacios Inés Bleriot

To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction–modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction–modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.

Phage therapy

Acinetobacter baumannii

Comparative Genomics

10.3389/fmicb.2021.784949

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Citations (22)

In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens

Scientific Reports (2020)

Lucía Blasco Antón Ambroa Rocío Trastoy Inés Bleriot Miriam Moscoso

The emergence of multidrug resistant (MDR) pathogenic bacteria is jeopardizing the value of antimicrobials, which had previously changed the course of medical science. In this study, we identified endolysins ElyA1 and ElyA2 (GH108-PG3 family), present in the genome of bacteriophages Ab1051Φ and Ab1052Φ, respectively. The muralytic activity of these endolysins against MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. Minimal inhibitory concentrations (MICs) of endolysin, colistin and a combination of endolysin and colistin were determined, and the antimicrobial activity of each treatment was confirmed by time kill curves. Endolysin ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. Endolysin ElyA2 did not display any such activity. The combined antimicrobial activity of colistin and 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 and in murine skin and lung infection models. In conclusion, combining colistin (1/4 MIC) with the new endolysin ElyA1 (350 µg) enhanced the bactericidal activity of colistin in both in vitro and in vivo studies. This will potentially enable reduction of the dose of colistin used in clinical practice.

Colistin

Acinetobacter baumannii

Lysin

10.1038/s41598-020-64145-7

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Citations (77)

Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection

Research Square (Research Square) (2021)

Antón Ambroa Lucía Blasco María López Olga Pacios Inés Bleriot

Abstract Background In order to optimize phage therapy, we need to understand how bacteria evolve against phage attack. One of the main problems of the phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage resistant strains, that can be overcomed by the analysis of metadata provided by WGS. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strain belonging to the ST-2 clonal complex during a decade (Ab2000 vs 2010): 9 from 2000 and 9 from 2010. Results The presence of genes putatively associated to phage resistance were detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defence systems but with unknown function and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands (GIs) in the 2000 strains and 32% in 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. Conclusions A moderately higher presence of these genes in the strains of the 2010 in comparison to those of the 2000 were found, especially those related to the R-M system and CRISPR-Cas system. The presence of these genes in GIs in a higher rate in the strains of the 2010 compared to those of the 2000 was also detected . WGS and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in a possible phage therapy.

Acinetobacter baumannii

Phage therapy

10.21203/rs.3.rs-699717/v1

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Citations (0)

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

bioRxiv (Cold Spring Harbor Laboratory) (2022)

María López Inés Bleriot Olga Pacios Laura Fernández-García Lucía Blasco

ABSTRACT 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. IMPORTANCE Escherichia 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.

Siphoviridae

Prophage

Myoviridae

Lysogenic cycle

Podoviridae

Lytic cycle

10.1101/2022.06.29.498070

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Quorum and Light Signals Modulate Acetoin/Butanediol Catabolism in Acinetobacter spp.

Frontiers in Microbiology (2019)

Marisel R. Tuttobene Laura Fernández-García Lucía Blasco Pamela Cribb Antón Ambroa

Acinetobacter spp. are found in all environments on earth due to their extraordinary capacity to survive in the presence of physical and chemical stressors. In this study, we analyzed global gene expression in airborne Acinetobacter sp. strain 5-2Ac02 isolated from hospital environment in response to quorum network modulators, and found that they induced the expression of genes of the acetoin/butanediol catabolism, volatile compounds shown to mediate interkingdom interactions. Interestingly, the acoN gene, annotated as a putative transcriptional regulator, was truncated in the downstream regulatory region of the induced acetoin/butanediol cluster in Acinetobacter sp. strain 5-2Ac02, and its functioning as a negative regulator of this cluster integrating quorum signals was confirmed in A. baumannii ATCC 17978. Moreover, we show that the acetoin catabolism is also induced by light, and provide insights into the light transduction mechanism by showing that the photoreceptor BlsA interacts with and antagonizes the functioning of AcoN in A. baumannii, integrating also a temperature signal. The data support a model in which BlsA interacts with and likely sequesters AcoN at this condition, relieving acetoin catabolic genes from repression, and leading to better growth under blue light. This photoregulation depends on temperature, occurring at 23 but not at 30ºC. BlsA is thus a dual regulator, modulating different transcriptional regulators in the dark but also under blue light, representing thus a novel concept. The overall data shows that quorum modulators as well as light regulate the acetoin catabolic cluster, providing a better understanding of environmental as well as clinical bacteria.

Acetoin

Catabolism

2,3-Butanediol

Acinetobacter baumannii

Response regulator

Gene cluster

10.3389/fmicb.2019.01376

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Citations (17)

Mechanisms of Tolerance and Resistance to Chlorhexidine in Clinical Strains of Klebsiella pneumoniae Producers of Carbapenemase: Role of New Type II Toxin-Antitoxin System, PemIK

Toxins (2020)

Inés Bleriot Lucía Blasco Mercedes Delgado-Valverde Ana Gual-de-Torrella Antón Ambroa

Although the failure of antibiotic treatment is normally attributed to resistance, tolerance and persistence display a significant role in the lack of response to antibiotics. Due to the fact that several nosocomial pathogens show a high level of tolerance and/or resistance to chlorhexidine, in this study we analyzed the molecular mechanisms associated with chlorhexidine adaptation in two clinical strains of Klebsiella pneumoniae by phenotypic and transcriptomic studies. These two strains belong to ST258-KPC3 (high-risk clone carrying β-lactamase KPC3) and ST846-OXA48 (low-risk clone carrying β-lactamase OXA48). Our results showed that the K. pneumoniae ST258-KPC3CA and ST846-OXA48CA strains exhibited a different behavior under chlorhexidine (CHLX) pressure, adapting to this biocide through resistance and tolerance mechanisms, respectively. Furthermore, the appearance of cross-resistance to colistin was observed in the ST846-OXA48CA strain (tolerant to CHLX), using the broth microdilution method. Interestingly, this ST846-OXA48CA isolate contained a plasmid that encodes a novel type II toxin/antitoxin (TA) system, PemI/PemK. We characterized this PemI/PemK TA system by cloning both genes into the IPTG-inducible pCA24N plasmid, and found their role in persistence and biofilm formation. Accordingly, the ST846-OXA48CA strain showed a persistence biphasic curve in the presence of a chlorhexidine-imipenem combination, and these results were confirmed by the enzymatic assay (WST-1).

Antitoxin

Multidrug tolerance

10.3390/toxins12090566

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Citations (24)

Relationship Between the Quorum Network (Sensing/Quenching) and Clinical Features of Pneumonia and Bacteraemia Caused by A. baumannii

Frontiers in Microbiology (2018)

Laura Fernández-García Antón Ambroa Lucía Blasco Inés Bleriot María López

Acinetobacter baumannii (Ab) is one of the most important pathogens associated with nosocomial infections, especially pneumonia. Interest in the Quorum network, i.e., Quorum Sensing (QS)/Quorum Quenching (QQ), in this pathogen has grown in recent years. The Quorum network plays an important role in regulating diverse virulence factors such as surface motility and bacterial competition through the type VI secretion system (T6SS), which is associated with bacterial invasiveness. In the present study, we investigated 30 clinical strains of A. baumannii isolated in the "II Spanish Study of A. baumannii GEIH-REIPI 2000-2010" (Genbank Umbrella Bioproject PRJNA422585), a multicentre study describing the relationship between the Quorum network in A. baumannii and the development of pneumonia and associated bacteraemia. Expression of the aidA gene (encoding the AidA protein, QQ enzyme) was lower (P < 0.001) in strains of A. baumannii isolated from patients with bacteraemic pneumonia than in strains isolated from patients with non-bacteraemic pneumonia. Moreover, aidA expression in the first type of strain was not regulated in the presence of environmental stress factors such as the 3-oxo-C12-HSL molecule (substrate of AidA protein, QQ activation) or H2O2 (inhibitor of AidA protein, QS activation). However, in the A. baumannii strains isolated from patients with non-bacteraemic pneumonia, aidA gene expression was regulated by stressors such as 3-oxo-C12-HSL and H2O2. In an in vivo Galleria mellonella model of A. baumannii infection, the A. baumannii ATCC 17978 strain was associated with higher mortality (100% at 24 h) than the mutant, abaI-deficient, strain (carrying a synthetase enzyme of Acyl homoserine lactone molecules) (70% at 24 h). These data suggest that the QS (abaR and abaI genes)/QQ (aidA gene) network affects the development of secondary bacteraemia in pneumonia patients and also the virulence of A. baumannii.

Acinetobacter baumannii

Quorum Quenching

Galleria mellonella

Autoinducer

10.3389/fmicb.2018.03105

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Citations (17)