Juan A Ayala

Juan A Ayala
Centro De Biología Molecular Severo Ochoa · Microbiology and Virology

Ph. D.

About

163
Publications
41,231
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5,619
Citations
Additional affiliations
February 1986 - present
Centro De Biología Molecular Severo Ochoa
Position
  • Group Leader
Education
January 1974 - July 1978
Complutense University of Madrid
Field of study
  • Biochemistry

Publications

Publications (163)
Article
Full-text available
Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of pep...
Article
Full-text available
As part of this Special Issue of International Microbiology celebrating the 75th anniversary of the founding of the Spanish Society for Microbiology (SEM), Guest Editor Rafael Giraldo invited us to contribute an opinion article on the topic of 75 years of joint scientific ventures between Latin American microbiologists and Spanish microbiologists....
Article
Full-text available
The extensive use of β-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa , the mutation-driven AmpC β-lactamase hyperproduction stands out as the main resistan...
Article
Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to make biocatalytic processes more economically competitive with respect to chemical synthesis. In this work, we have applied selection by folding interference at high temperature in Thermus thermophilus to obta...
Article
Full-text available
The worldwide dissemination of metallo-β-lactamases (MBLs), mediating resistance to carbapenem antibiotics, is a major public health problem. The extent of dissemination of MBLs such as VIM-2, SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here, we...
Article
Full-text available
Con la finalidad de evaluar la patogenia en cepas de Salmonella Typhimurium con mutaciones en los genes invG/invE de la Isla de Patogenicidad de Salmonella 1 (SPI-1) y de los genes ssaJ/ssaK en la SPI-2, se evaluaron los modelos asa intestinal ligada de ratón asociado a la observación de los tejidos por microscopía electrónica de transmisión (MET)...
Article
Full-text available
Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using bioche...
Data
Protein detection in S. epidermidis biofilms. A proteinase K disruption assay shows the presence of a proteinaceous matrix in S. epidermidis biofilms grown in TSBg and PCs. Mean and standard deviation (SD) were calculated for each strain and growth environment (TSBg or PCs). (DOCX)
Data
Comparison of the peptidoglycan profiles of S. epidermidis ST10002 and AZ39 grown in PCs and in TSBg. (DOCX)
Data
CLSM signal intensities. For visualization of WGA-OG488, SYPRO-Ruby and DAPI staining, excitation lasers of 488 nm, 405 nm and 405 nm were used, respectively. For the TSBg samples, emission bands of 505–555 nm for WGA-OG488, 555–700 nm for SYPRO-Ruby, and 410–505 nm for DAPI were acquired using the GaAsP detectors and a Plan-Apochromat 40x NA 1.4 o...
Data
eDNA detection in S. epidermidis biofilms. A DNase I disruption assay shows values indicating the presence of eDNA in the matrix of S. epidermidis biofilms grown in TSBg. The difference between untreated and DNase-treated biofilms grown in TSBg is significant for all the tested strains (p<0.02) except for 9142 ΔicaA (p = 0.0775). No significant dif...
Data
CLSM signal intensities. Samples were visualized using excitation lasers of 488 nm and 640 nm for TOTO-1 and SYTO-60, respectively. An emission band of 450–630 nm was acquired for TOTO-1 using the Airyscan detector and an emission band of 656–700 nm was acquired for SYTO-60 using the GaAsP detector. Differential interference contrast microscopy (DI...
Preprint
Summary: The development of new and effective vaccines has been limited given the scarce availability of adjuvants. The particulate structures constitute very promising candidates given their immune-potentiating capacities and by antigen “in vivo” release. In this work new adjuvant formulations were evaluated based on proteoliposomes of less than...
Article
Full-text available
Bacterial cell division has been studied extensively under laboratory conditions. Despite being a key event in the bacterial cell cycle, cell division has not been explored in vivo in bacterial pathogens interacting with their hosts. We discovered in Salmonella enterica serovar Typhimurium a gene absent in nonpathogenic bacteria and encoding a pept...
Article
Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting...
Data
High-performance liquid chromatograms of peptidoglycan muropeptides of the indicated strains. Each series displays peaks corresponding to the common muropeptides in peptidoglycan of each strain. Each peak corresponds to a typical muropeptide whose name is indicated at the top. M3, disaccharide tripeptide; M4, disaccharide tetrapeptide; D43, cross-l...
Data
HPLC analysis of muropeptides prepared from the peptidoglycan of the PAO1 and derived knockout mutants. (DOCX)
Data
Activation of HEK-Blue hNOD1 cells with PAO1 and PA14-derived strains. 620 nm absorbance (proportional to NOD1 activation) after 20 h of stimulation with: A) heat-inactivated bacteria, MOI 1000; B) viable bacteria, MOI 250; C) cell-free supernatants (10% in detection medium) and D) purified peptidoglycans (PGN), 1 μg/well. 0.2 μg of C12-iE-DAP per...
Data
Strains and plasmids used in this work. (DOCX)
Data
Bactericidal activity of lysozyme (25 μg/mL) or colistin (0.1 μg/mL) treatments in PAO1 and derived knockout mutants, in the lysozyme assay buffer. The percentages of bacterial survival after treatments respect to the initial inoculum are shown; The bars represent the mean of 5 experiments and the error bars the SD. *Statistically significant, P<0....
Data
Activation of HEK-Blue hNOD2 cells with PAO1 and PA14-derived strains. 620 nm absorbance (proportional to NOD2 activation) after 20h of stimulation with: A) heat-inactivated bacteria MOI 500, B) viable bacteria, MOI 250; C) cell-free supernatants (10% in detection medium) and D) purified PGNs, 0.25 μg/well. 2 μg/mL of MDP were used as positive cont...
Article
Full-text available
Importance: Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and fu...
Article
Full-text available
Background Community and nosocomial infections by Pseudomonas aeruginosa still create a major therapeutic challenge. The resistance of this opportunist pathogen to β-lactam antibiotics is determined mainly by production of the inactivating enzyme AmpC, a class C cephalosporinase with a regulation system more complex than those found in members of t...
Article
Full-text available
L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to...
Data
Genetic changes in the stable L. monocytogenes L-form. The base positions refer to the position in the annoted NCBI L. monocytogenes EGDe reference sequence (NC_003210). The gene products are derived from the NCBI Gene database. SNP = single nucleotide polymorphism, Ins = insertion, Del = deletion, fs = frameshift. (DOC)
Data
The stable L. monocytogenes L-form was transformed with the following combinations of plasmids in order to test for reversion to the rod shape. Prha = rhamnose inducible promoter, Phelp = highly expressed, constitutive promoter. (DOC)
Data
Control experiments of the kinase assay. (a) Listeria L-form medium alone without any bacteria yields a strong GlcNAc-6P signal, when exposed to the kinase MurK in presence of radiolabeled ATP for 30 min. (b) Walled L. monocytogenes were analyzed for the presence of polymerized peptidoglycan strands. Untreated extracts of walled L. monocytogenes ex...
Article
This study aimed to characterize the role of Pseudomonas aeruginosa Low-Molecular-Mass penicillin-binding proteins (LMM-PBPs), namely PBP4 (DacB), PBP5 (DacC) and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance and ampC regulation. For this purpose, we constructed all single and combined mutants of dacB, dacC, pbpG and ampC in from w...
Presentation
Sala 2 12:00-12:20 Conferência (053) Avaliação imunotoxicológica pré-clínica de formulações vacinais baseadas em adjuvantes nanoparticulados. Evaluación inmunotoxicológica pre clínica de formulaciones vacunales basadas en adyuvantes nanoparticulados Introducción: Múltiples son las aplicaciones de la nanotecnología en la medicina, en particular las...
Article
The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodelling. We sought to characterize the changes that the cell w...
Article
La resistencia a la combinación de β-lactámico/inhibidor de β-lactamasa en enterobacterias es un problema creciente que no ha sido estudiado intensamente en Argentina. En el presente trabajo, 54/843 enterobacterias recolectadas en un hospital universitario de la ciudad de Buenos Aires fueron resistentes a ampicilina-sulbactama, pero se mantuvieron...
Article
Many bacteria are able to assume a transient cell wall-deficient (or L-form) state under favorable osmotic conditions. Cell wall stress such as exposure to β-lactam antibiotics can enforce the transition to and maintenance of this state. L-forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adop...
Article
Everyone working with bacterial genomics is familiar with the phrase ‘too much data’. In this Genome Update, we discuss two methods for helping to deal with this explosion of genomic information. First, we introduce the concept of calculating a quality score for each sequenced genome, and second, we describe a method to quickly sort through genomes...
Article
Full-text available
In this study, we incubated three strains of Shigella (Shigella sonnei (S1), Shigella boydii (S2) and Shigella flexneri (S3)) in seawater microcosms (at room temperature and at 4°C) for eight months and we studied the modification of the proteins secreted, cytosolic and inner membrane profiles and their composition in fatty acids. Plasmid profiles...
Article
Full-text available
Prc is a periplasmic protease involved in processing of penicillin-binding protein 3 (PBP3). Lack of Prc suppresses bile sensitivity in Dam-, Wec-, PhoP-, DamX-, and SeqA- mutants of Salmonella enterica, and increases bile resistance in the wild type. Changes in the activity of penicillin binding proteins PBP3, PBP4, PBP5/6 and PBP7 are detected in...
Article
The objectives of this study were to investigate clinical isolates of Salmonella enterica serovar Typhimurium resistant to b-lactam antibiotics, to characterise their mechanisms of antibiotic resistance and to evaluate the possible biological cost of expressing resistance genes. Two oxyimino- cephalosporin-resistant Salmonella isolates obtained fro...
Data
##Assembly-Data-START## Assembly Method :: Biolign v. 4.0.6.2 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
Data
##Assembly-Data-START## Assembly Method :: Biolign v. 4.0.6.2 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
Data
##Assembly-Data-START## Assembly Method :: Biolign v. 4.0.6.2 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
Article
In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spy operon). The invasion assays of S. Infantis isolat...
Article
Full-text available
Two multidrug-resistant Salmonella Infantis isolates behave like hypo-invasive strains but have high intracellular proliferation. In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was s...
Article
Desde los años 20 del pasado siglo, hasta el presente, en el mundo se han desarrollado y empleado vacunas de células enteras contra la leptospirosis que confieren una corta inmunidad; la mayoría no adyuvadas y dirigidas, fundamentalmente, contra los diferentes serogrupos de la especie Leptospira interrogans, contenidos en las preparaciones. Numeros...
Article
α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present...
Article
We studied a clinical isolate of Salmonella enterica serotype Enteritidis showing resistance to oxyiminocephalosporins. PCR analysis confirmed the presence of blaCTX-M-14 linked to IS903 in a 95-kb IncI1 conjugative plasmid. Such a plasmid is maintained on account of the presence of a pndAC addiction system. Multilocus sequence typing (MLST) analys...
Article
Full-text available
β-Lactamases and penicillin-binding proteins (PBPs) have evolved from a common ancestor. β-Lactamases are enzymes that degrade β-lactam antibiotics, whereas PBPs are involved in the synthesis and processing of peptidoglycan, which forms an elastic network in the bacterial cell wall. This study analyzed the interaction between β-lactamases and pepti...
Article
Whole cell Leptospirosis vaccines have been developed and used from the 20's of last century up today, most of them are not adjuvated and do not confer a long-lasting immunity. These vaccines are mainly against different serogroups of Leptospira interrogans contained in the preparation. Though several attempts to obtain a vaccinal formulation more...
Article
In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display dd-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related t...
Article
Full-text available
There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals i...
Article
The characterization of Bacteroides strains with regard to the cfxA gene, the MTn4555 mobilizable transposon, the role of penicillin-binding proteins (PBPs) and heterogeneous cefoxitin resistance. Eighty-four randomly selected and 11 heterogeneously or highly cefoxitin-resistant Bacteroides isolates were included. Agar dilution and Etest methods we...
Article
Full-text available
To analyse the prevalence of resistance to β-lactams and plasmid-mediated quinolone resistance in Enterobacteriaceae in the paediatric hospital of Uruguay. A total of 368 enterobacterial isolates collected between 1 May and 30 November 2009 were studied for the presence of extended-spectrum β-lactamases (ESBLs), qnr alleles and aac(6')Ib by phenoty...
Article
Full-text available
The main toxicity mechanism of Lysinibacillus sphaericus, which is used in the control of mosquitoes, is its binary toxin produced during sporulation; additionally the Mtx1, Mtx2 and Mtx 3 toxins are expressed in vegetative cells. Mosquito larvicidal potency of the S-layer protein that is expressed in vegetative cells has been determined. The prote...
Article
A method based on laser induced breakdown spectroscopy (LIBS) and neural networks (NNs) has been developed and applied to the identification and discrimination of specific bacteria strains (Pseudomonas aeroginosa, Escherichia coli and Salmonella typhimurium). Instant identification of the samples is achieved using a spectral library, which was obta...
Article
To understand the genetic basis of Salmonella pathogenicity mechanisms, various methods have been described to manipulate and generate mutant genomic DNA with specific characteristics. In this study we report the construction of mutants from several serotypes of S. enterica, substitution and inactivation of genes invG/invE in SPI-1 gene and ssaJ/ss...
Article
In this study, we searched for extended-spectrum β-lactamases (ESBLs), class 1 and 2 integrons, and qnrA, qnrB and qnrS genes in 56 oxyimino-cephalosporin and/or ciprofloxacin-resistant enterobacterial isolates obtained from the gastrointestinal tract of patients admitted in an Intensive Care Unit in Uruguay. ESBLs were detected in 11 isolates (6 C...
Article
Full-text available
Título en inglés: Construction of mutants of Salmonella enterica for inactivation of invG/invE y ssaJ/ssaK genes of pathogenicity islands 1 and 2 Resumen Para la comprensión de las bases genéticas de los mecanismos de patogenicidad de Salmonella se han descrito diversas metodologías para manipular el ADN genómico y generar mutantes con característ...
Article
Full-text available
Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent β-lactam derivatives both whole cells and membrane/cell enriched fractions. Analysis of the Listeria monocytogenes genome sequence predicted ten genes coding for putative PBPs, but not all of their products have been detected in studi...
Article
We have studied the mechanism by which beta-lactam challenge leads to beta-lactamase induction in Aeromonas hydrophila through transposon-insertion mutagenesis. Disruption of the dd-carboxypeptidases/endopeptidases, penicillin-binding protein 4 or BlrY leads to elevated monomer-disaccharide-pentapeptide levels in A. hydrophila peptidoglycan and con...
Article
Full-text available
Twenty-eight Klebsiella pneumoniae clinical isolates that exhibited an extended-spectrum cephalosporin-resistance profile from a city in the Northeast of Brazil were analysed by PCR and DNA sequencing in order to determine the occurrence of blaCTX-M genes and class 1 integrons. We determined the occurrence of the blaCTX-M-2 gene in six K. pneumonia...
Article
Full-text available
Alpha-Crystallin type heat shock protein (alpha-HSP) IbpA from Acholeplasma laidlawii was expressed in Escherichia coil and isolated from cell extract on Ni-sepharose column. Recombinant IbpA, like other alpha-HSPs, spontaneously formed oligomeres in vitro. High resolution electron microscopy revealed regular structures with 15 nm in diameter. Eval...
Article
OXA 101, a novel OXA-10 like enzyme, was found forming part of a class 1 integron located in a conjugative plasmid in three different species of Enterobacteriaceae. This beta-lactamase is related to OXA-35 and OXA-56 and displays a narrow substrate hydrolysis profile.
Article
To study the evolution of antibiotic resistance in isolates of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) obtained in Uruguay between the years 1976 and 2000, and to determine the incidence of class 1 and 2 integrons in the multi-resistant isolates. We studied 258 strains of Salmonella Typhimurium from vari...
Conference Paper
Background: Recently, three transferable mechanisms of quinolone resistance have been described, namely Qnr, QepA and Aac(6’)-Ib-cr. Lately, we reported in our country the presence of this last mechanism in an E. coli isolate. Thus we looked for the presence of qnr genes in gut-colonizing isolates from inpatients in an intensive care unit. Methods:...
Article
For growth and division of rod-shaped bacteria, the cylindrical part of the sacculus has to be elongated and two new cell poles have to be synthesized. The elongation is performed by a protein complex, the elongase that inserts disaccharidepentapeptide units at a limited number of discrete sites while using the cytoskeletal MreB helix as a tracking...