Publications (22)104.44 Total impact
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Article: Differential Distribution of Plasmid Mediated Quinolone Resistance Genes in Clinical Enterobacteria with Unusual Phenotypes of Quinolone Susceptibility from Argentina.
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ABSTRACT: We studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid mediated quinolone resistance (PMQR) and oqx genes and their implications on quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca. However, the oqxA/oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 (57%) isolates harbored at least one PMQR gene (qnrB19, qnrB10, qnrB2, qnrB1, qnrS1 or aac(6')-Ib-cr), belong to 8 enterobacterial species, were disseminated throughout the country and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusion-based method to improve the phenotypic detection of aac(6')-Ib-cr. The most frequent PMQR genes in our collection (qnrB19, qnrB10, aac(6')-Ib-cr) were differentially distributed among enterobacterial species and two different epidemiological settings were evident. First, the species associated to community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated to an extended-spectrum-β-lactamase phenotype. Second, the species associated to hospital-acquired infections (Enterobacter spp., Klebsiella spp. and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class-1 integrons that may also have aac(6')-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated to an extended-spectrum-β-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or not to integrons.Antimicrobial Agents and Chemotherapy 03/2013; · 4.84 Impact Factor -
Article: BtaE, an adhesin that belongs to the trimeric autotransporter family, is required for full virulence and defines a specific adhesive pole of Brucella.
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ABSTRACT: Brucella is responsible for brucellosis, one of the most common zoonosis worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a non-adherent E. coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild type strain, and it was outcompeted by the wild type strain in the binding to HeLa cells. The knock-out btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that in Brucella the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.Infection and immunity 01/2013; · 4.21 Impact Factor -
Article: Internalization of Locked Nucleic Acids/DNA Hybrid Oligomers into Escherichia coli.
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ABSTRACT: Delivery inside the cells is essential for practical application of antisense technologies. The hybrid locked nucleic acid (LNA)/DNA CAAGTACTGTTCCACCA (LNA residues are underlined) was labeled by conjugation to Alexa Fluor 488 (fLNA/DNA) and tested to determine its ability to penetrate Escherichia coli cells and reach the cytoplasm. Flow cytometry analysis showed that the fLNA/DNA was associated with 14% of cells from a stationary phase culture, while association with a labeled isosequential oligodeoxynucleotide was negligible. Laser scanning confocal microscopy confirmed that the fLNA/DNA was located inside the cytoplasm.BioResearch open access. 10/2012; 1(5):260-3. -
Article: Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor.
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ABSTRACT: Rhizobium leguminosarum is a soil bacterium that infects root hairs and induces the formation of nitrogen-fixing nodules on leguminous plants. Light, oxygen, and voltage (LOV)-domain proteins are blue-light receptors found in higher plants and many algae, fungi, and bacteria. The genome of R. leguminosarum bv. viciae 3841, a pea-nodulating endosymbiont, encodes a sensor histidine kinase containing a LOV domain at the N-terminal end (R-LOV-HK). R-LOV-HK has a typical LOV domain absorption spectrum with broad bands in the blue and UV-A regions and shows a truncated photocycle. Here we show that the R-LOV-HK protein regulates attachment to an abiotic surface and production of flagellar proteins and exopolysaccharide in response to light. Also, illumination of bacterial cultures before inoculation of pea roots increases the number of nodules per plant and the number of intranodular bacteroids. The effects of light on nodulation are dependent on a functional lov gene. The results presented in this work suggest that light, sensed by R-LOV-HK, is an important environmental factor that controls adaptive responses and the symbiotic efficiency of R. leguminosarum.Proceedings of the National Academy of Sciences 07/2012; 109(30):12135-40. · 9.68 Impact Factor -
Article: BmaC, a novel autotransporter of Brucella suis, is involved in bacterial adhesion to host cells.
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ABSTRACT: Brucella is an intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucella survives and proliferates within several types of phagocytic and non-phagocytic cells. Like in other pathogens, adhesion of brucellae to host surfaces was proposed to be an important step in the infection process. Indeed, Brucella has the capacity to bind to culture human cells and key components of the extracellular matrix, such as fibronectin. However, little is known about the molecular bases of Brucella adherence. In an attempt to identify bacterial genes encoding adhesins, a phage display library of Brucella suis was panned against fibronectin. Three fibronectin-binding proteins of B. suis were identified using this approach. One of the candidates, designated BmaC was a very large protein of 340 kDa that is predicted to belong to the type I (monomeric) autotransporter family. Microscopy studies showed that BmaC is located at one pole on the bacterial surface. The phage displaying the fibronectin-binding peptide of BmaC inhibited the attachment of brucellae to both, HeLa cells and immobilized fibronectin in vitro. In addition, a bmaC deletion mutant was impaired in the ability of B. suis to attach to immobilized fibronectin and to the surface of HeLa and A549 cells and was out-competed by the wild-type strain in co-infection experiments. Finally, anti-fibronectin or anti-BmaC antibodies significantly inhibited the binding of wild-type bacteria to HeLa cells. Our results highlight the role of a novel monomeric autotransporter protein in the adhesion of B. suis to the extracellular matrix and non-phagocytic cells via fibronectin binding.Cellular Microbiology 02/2012; 14(6):965-82. · 5.46 Impact Factor -
Article: Small plasmids harboring qnrB19: a model for plasmid evolution mediated by site-specific recombination at oriT and Xer sites.
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ABSTRACT: Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site.Antimicrobial Agents and Chemotherapy 01/2012; 56(4):1821-7. · 4.84 Impact Factor -
Article: Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting ftsZ.
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ABSTRACT: EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ.PLoS ONE 01/2012; 7(10):e47690. · 4.09 Impact Factor -
Article: Extracellular DNA: a major proinflammatory component of Pseudomonas aeruginosa biofilms.
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ABSTRACT: We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1beta (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.The Journal of Immunology 06/2010; 184(11):6386-95. · 5.79 Impact Factor -
Article: Naturally competent Acinetobacter baumannii clinical isolate as a convenient model for genetic studies.
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ABSTRACT: Acinetobacter baumannii A118 was isolated from a patient's blood culture. It is susceptible to several antibiotics, is naturally competent, and supports replication and stable maintenance of four plasmid replicons. A. baumannii A118 took up a fluorophore-labeled oligonucleotide analog. These characteristics make this isolate a convenient model for genetic studies.Journal of clinical microbiology 02/2010; 48(4):1488-90. · 4.16 Impact Factor -
Article: Inhibition of aac(6')-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria.
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ABSTRACT: Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6')-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6')-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6')-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect.Proceedings of the National Academy of Sciences 08/2009; 106(32):13230-5. · 9.68 Impact Factor -
Article: The cin and rai quorum-sensing regulatory systems in Rhizobium leguminosarum are coordinated by ExpR and CinS, a small regulatory protein coexpressed with CinI.
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ABSTRACT: To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.Journal of bacteriology 04/2009; 191(9):3059-67. · 3.94 Impact Factor -
Article: Interplay between two RND systems mediating antimicrobial resistance in Brucella suis.
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ABSTRACT: The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis DeltabepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the DeltabepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the DeltabepE DeltabepG double mutant showed a more severe phenotype than the DeltabepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.Journal of bacteriology 03/2009; 191(8):2530-40. · 3.94 Impact Factor -
Article: Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection.
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ABSTRACT: The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterized. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one of these loci (gmsA) determines glucomannan synthesis and one (gelA) determines a gel-forming polysaccharide, but the role of the other locus (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both of these characteristics, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but it did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but it was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria but are absent from closely related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterium-plant interactions.Journal of bacteriology 08/2008; 190(13):4706-15. · 3.94 Impact Factor -
Article: Characterization of bacterial DNA binding to human neutrophil surface.
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ABSTRACT: Bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Neutrophil activation does not require DNA internalization, suggesting that it results from the interaction of bacterial DNA with a neutrophil surface receptor. The aim of this study was to characterize the interaction of bacterial DNA with the neutrophil surface. Bacterial DNA binding showed saturation and was inhibited by unlabeled DNA but not by other polyanions like yeast tRNA and poly-A. Resembling the conditions under which bacterial DNA triggers neutrophil activation, binding was not modified in the presence or absence of calcium, magnesium or serum. Treatment of neutrophils with proteases not only dramatically reduced bacterial DNA binding but also inhibited neutrophil activation induced by bacterial DNA. Experiments performed with DNA samples of different lengths obtained after digestion of bacterial DNA with DNase showed that only DNA fragments greater than approximately 170-180 nucleotides competed bacterial DNA binding and retained the ability to trigger cell activation. Treatment of neutrophils with chemoattractants or conventional agonists significantly increased bacterial DNA binding. Moreover, neutrophils that underwent transmigration through human endothelial cell monolayers even in the absence of chemoattractants, exhibited higher binding levels of bacterial DNA. Together, our findings provide evidence that binding of bacterial DNA to neutrophils is a receptor-mediated process that conditions the ability of DNA to trigger cell activation. We speculate that neutrophil recognition of bacterial DNA might be modulated by the balance of agonists present at inflammatory foci. This effect might be relevant in bacterial infections with a biofilm etiology, in which extracellular DNA could function as a potent neutrophil agonist.Laboratory Investigation 08/2008; 88(9):926-37. · 3.64 Impact Factor -
Article: Klebsiella pneumoniae multiresistance plasmid pMET1: similarity with the Yersinia pestis plasmid pCRY and integrative conjugative elements.
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ABSTRACT: Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria. The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31). The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1 )show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains.PLoS ONE 02/2008; 3(3):e1800. · 4.09 Impact Factor -
Article: Complex class 1 integrons with diverse variable regions, including aac(6')-Ib-cr, and a novel allele, qnrB10, associated with ISCR1 in clinical enterobacterial isolates from Argentina.
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ABSTRACT: Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6')-Ib-cr within the variable region of integrons. The three arrays differed from bla(CTX-M-2)-bearing integrons, which are broadly distributed in Argentina.Antimicrobial Agents and Chemotherapy 01/2008; 51(12):4466-70. · 4.84 Impact Factor -
Article: Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris.
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ABSTRACT: Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence.Environmental Microbiology 09/2007; 9(8):2101-9. · 5.84 Impact Factor -
Article: External guide sequences targeting the aac(6')-Ib mRNA induce inhibition of amikacin resistance.
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ABSTRACT: The dissemination of AAC(6')-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6')-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6')-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6')-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 mug of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 mug of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin.Antimicrobial Agents and Chemotherapy 07/2007; 51(6):1918-25. · 4.84 Impact Factor -
Article: Continuous nondestructive monitoring of Bordetella pertussis biofilms by Fourier transform infrared spectroscopy and other corroborative techniques.
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ABSTRACT: This work describes the application of several analytical techniques to characterize the development of Bordetella pertussis biofilms and to examine, in particular, the contribution of virulence factors in this development. Growth of surface-attached virulent and avirulent B. pertussis strains was monitored in continuous-flow chambers by techniques such as the crystal violet method, and nondestructive methodologies like fluorescence microscopy and Fourier transform (FT) IR spectroscopy. Additionally, B. pertussis virulent and avirulent strains expressing green fluorescent protein were grown adhered to the base of a glass chamber of 1-microm thickness. Three-dimensional images of mature biofilms, acquired by confocal laser scanning microscopy, were quantitatively analysed by means of the computer program COMSTAT. Our results indicate that only the virulent (Bvg(+)) phase of B. pertussis is able to attach to surfaces and develop a mature biofilm. In the virulent phase these bacteria are capable of producing a biofilm consisting of microcolonies of approximately 200 microm in diameter and 24 microm in depth. FTIR spectroscopy allowed us not only to follow the dynamics of biofilm growth through specific biomass and biofilm marker absorption bands, but also to monitor the maturation of the biofilm by means of the increase of the carbohydrate-to-protein ratio.Analytical and Bioanalytical Chemistry 04/2007; 387(5):1759-67. · 3.78 Impact Factor -
Article: The TolC homologue of Brucella suis is involved in resistance to antimicrobial compounds and virulence.
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ABSTRACT: Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other alpha-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.Infection and Immunity 02/2007; 75(1):379-89. · 4.16 Impact Factor
Top co-authors
Top Journals
Institutions
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2006–2013
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Fundación Instituto Leloir
Buenos Aires, Buenos Aires F.D., Argentina
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2012
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University of Buenos Aires
Buenos Aires, Buenos Aires F.D., Argentina
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2006–2012
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California State University, Fullerton
- • College of Natural Sciences and Mathematics
- • Department of Biological Science
Fullerton, CA, USA
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