Ana Alonso

Macrolide Resistance Genes in Enterococcus spp.

ABSTRACT Seventy-eight isolates of different Enterococcusspecies (E. faecalis, n = 27; E. faecium, n = 23; E. durans,n = 8; E. avium, n = 6;E. hirae, n = 9; E. gallinarum, n = 3; and E. casseliflavus, n = 2) with a variety of erythromycin resistance phenotypes were examined for the presence of macrolide resistance genes (ermA, ermB,ermC, ermTR, mefA/E, andmsrA). Positive PCR amplifications of ermB were obtained for 39 of 40 highly erythromycin-resistantEnterococcus isolates (MICs, >128 μg/ml) of different species; the remaining highly resistant E. faecium isolate was positive for PCR amplification of ermA but was negative for PCR amplification of the ermB and ermCgenes. For all enterococcal strains for which erythromycin MICs were ≤32 μg/ml PCRs were negative for erm methylase genes. For all E. faecium isolates PCR amplified products of the expected size of 400 bp were obtained when msrA primers were used, with the results being independent of the erythromycin resistance phenotype. All the other enterococcal species gave negative results by msrA PCRs. Sequencing of the msrAPCR products from either erythromycin-susceptible, low-level-resistant, or highly resistant E. faecium strains showed that the amplicons did not correspond to the msrA gene described forStaphylococcus epidermidis but corresponded to a new putative efflux determinant, which showed 62% identity with themsrA gene at the DNA level and 72% similarity at the amino acid level. This new gene was named msrC.

Stenotrophomonas maltophilia D457R Contains a Cluster of Genes from Gram-Positive Bacteria Involved in Antibiotic and Heavy Metal Resistance

ABSTRACT A cluster of genes involved in antibiotic and heavy metal resistance has been characterized from a clinical isolate of the gram-negative bacterium Stenotrophomonas maltophilia. These genes include a macrolide phosphotransferase (mphBM) and a cadmium efflux determinant (cadA), together with the genecadC coding for its transcriptional regulator. ThecadC cadA region is flanked by a truncated IS257 sequence and a region coding for a bin3invertase. Despite their presence in a gram-negative bacterium, these genetic elements share a common gram-positive origin. The possible origin of these determinants as a remnant composite transposon as well as the role of gene transfer between gram-positive and gram-negative bacteria for the acquisition of antibiotic resistance determinants in chronic, mixed infections is discussed.

Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the most important opportunistic pathogens involved in nosocomial infections, cystic fibrosis patients included. Hospital isolates frequently present multidrug-resistance (MDR) phenotypes as the consequence of constant antibiotic selective pressure. The kinetics of emergence of P. aeruginosa MDR mutants under antibiotic selective pressure indicated that long-term incubation in the presence of the bacteriostatic antibiotic tetracycline increases the mutation rate per cell per day of P. aeruginosa PAO1 by several orders of magnitude. The tetracycline-resistant mutants obtained were stable, showed decreased susceptibility to antibiotics belonging to different structural families, and contained an outer-membrane protein not present in the wild-type P. aeruginosa strain PAO1. These data are consistent with the hypothesis that incubation in the presence of tetracycline favours the emergence of MDR mutants in P. aeruginosa. The results are relevant for understanding the rapid emergence of antibiotic-resistant mutants among bacterial populations during infections. Their relationship to other models of increased mutagenesis under stress is discussed with respect to the adaptive mutation phenomenon.

Expression of Multidrug Efflux Pump SmeDEF by Clinical Isolates of Stenotrophomonas maltophilia

ABSTRACT The presence of the multidrug efflux pump SmeDEF was assessed in a collection of clinical isolates of Stenotrophomonas maltophilia. All isolates encoded this pump, as demonstrated by PCR. Forty-seven percent of the strains overproduced a protein of the same size that was immunoreactive against an anti-SmeF antibody, and 33% overexpressed the gene semD when they were tested by reverse transcription-PCR. A correlation between smeDEFoverexpression and antibiotic resistance was observed.

Cloning and Characterization of SmeT, a Repressor of the Stenotrophomonas maltophilia Multidrug Efflux Pump SmeDEF

ABSTRACT We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5′ end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PsmeT. The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.

Transcriptomics throughout the life cycle of Leishmania infantum: high down-regulation rate in the amastigote stage.

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in the Mediterranean Basin. The promastigote and amastigote stages alternate in the life cycle of the parasite, developing inside the sand-fly gut and inside mammalian phagocytic cells, respectively. High-throughput genomic and proteomic analyses have not focused their attention on promastigote development, although partial approaches have been made in Leishmania major and Leishmania braziliensis. For this reason we have studied the expression modulation of an etiological agent of visceral leishmaniasis throughout the life cycle, which has been performed by means of complete genomic microarrays. In the context of constitutive genome expression in Leishmania spp. described elsewhere and confirmed here (5.7%), we found a down-regulation rate of 68% in the amastigote stage, which has been contrasted by binomial tests and includes the down-regulation of genes involved in translation and ribosome biogenesis. These findings are consistent with the hypothesis of pre-adaptation of the parasite to intracellular survival at this stage.

Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

BackgroundThe extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.ResultsImmunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.ConclusionsThe specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.

Genome-wide analysis reveals increased levels of transcripts related with infectivity in peanut lectin non-agglutinated promastigotes of Leishmania infantum

Metacyclic promastigotes are transmitted during bloodmeals after development inside the gut of the sandfly vector. The isolation from axenic cultures of procyclic and metacyclic promastigotes by peanut lectin agglutination followed by differential centrifugation is controversial in Leishmania infantum. The purpose of this study has been to isolate both fractions simultaneously from the same population in stationary phase of axenic culture and compare their expression profiles by whole-genome shotgun DNA microarrays. The 317 genes found with meaningful values of stage-specific regulation demonstrate that negative selection of metacyclic promastigotes by PNA agglutination is feasible in L. infantum and both fractions can be isolated. This subpopulation up-regulates a cysteine peptidase A and several genes involved in lipophosphoglycan, proteophosphoglycan and glycoprotein biosynthesis, all related with infectivity. In fact, we have confirmed the increased infection rate of PNA(-) promastigotes by U937 human cell line infection experiments. These data support that metacyclic promastigotes are related with infectivity and the lack of agglutination with PNA is a phenotypic marker for this subpopulation.

Regulatory Regions of smeDEF in Stenotrophomonas maltophilia Strains Expressing Different Amounts of the Multidrug Efflux Pump SmeDEF

ABSTRACT The smeT-smeDEF region and the smeT gene, which encodes the smeDEF repressor, are highly polymorphic. Few changes in smeT might be associated with smeDEF overexpression. The results obtained with cellular extracts suggest that mutant SmeT proteins cannot bind to the operator and that other transcription factors besides SmeT are involved in the regulation of smeDEF expression.

Identification by Q-PCR of Trypanosoma cruzi lineage and determination of blood meal sources in triatomine gut samples in México

Triatomine vectors were collected on human dwellings in Michoacán México. Blood meal sources were identified by real time polymerase chain reaction (Q-PCR) using DNA extracted from triatomine guts. The assay was performed with one only specific primer set to amplify a fragment of the mitochondrial 12S ribosomal gene from vertebrate species. Also Trypanosoma cruzi parasites were detected in triatomine gut samples by microscopy and the positive infection was tested in mice. In addition T. cruzi discrete taxonomic units (DTUs) were identified by Q-PCR with two sets of primers that amplify the mini-circle region (miniexon) and 18S ribosomal mitochondrial gene. The sequences obtained from 18S ribosomal gene amplifications confirmed the presence of T. cruzi I and II lineages, and provide evidence of the presence of lineage TcIII and TcIV.