Eduardo Pérez-Roth

Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance

ABSTRACT In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance),ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S.aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S.aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.

Phenotypic and Molecular Characterization of Candida nivariensis sp. nov., a Possible New Opportunistic Fungus

ABSTRACT The new species Candida nivariensis, isolated from the clinical samples of three patients in Spain over a 3-year period, is presented here. This species can be easily differentiated from Candida glabrata, the closest genetic species, by different colony color on CHROMagar and by its ability to ferment trehalose. The analyses of the internal transcribed spacer region and the D1-D2 region of the 26S rRNA gene sequences support a new species designation.

Tracking Methicillin-Resistant Staphylococcus aureus Clones during a 5-Year Period (1998 to 2002) in a Spanish Hospital

ABSTRACT Three hundred seventy-five consecutive methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates recovered between 1998 and 2002 at the Nuestra Señora de Candelaria University Hospital in Tenerife, Spain, were analyzed by molecular fingerprinting techniques to determine MRSA clonal types and their prevalence over time. After determining antibiotic susceptibility, we used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize MRSA isolates and to establish PFGE types. Additionally, several selected isolates representative of each major PFGE type were tested by multilocus sequence typing (MLST) and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec), generating the corresponding sequence type (ST)-SCCmec types. Results of PFGE, supported by those of MLST and SCCmec typing, allowed us to identify six MRSA clones within the five major PFGE types and document temporal shifts in the prevalence of these MRSA clones from 1998 to 2002. Four of the clones were the pandemic “Iberian” (designated ST247-MRSA-IA), EMRSA-15 (ST22-MRSA-IV), EMRSA-16 (ST36-MRSA-II), and the so-called pediatric (ST5-MRSA-IV) clones, while the other two (ST125-MRSA-IVA and ST146-MRSA-IVA) clones could be derived from the pediatric one. The most striking temporal shift in the dominance of MRSA clones was the replacement of the multidrug-resistant and highly epidemic Iberian clone by the so-called British EMRSA-16 clone during the 5-year surveillance period. Our results are in accordance with previously stated findings showing the worldwide hospital dominance of relatively few pandemic and presumably virulent MRSA clones. We report for the first time the detection in Spain of the British EMRSA-15 and pediatric clones, as well as the abrupt replacement of the Iberian by the EMRSA-16 as the major MRSA clone.

High-Level Mupirocin Resistance within Methicillin-Resistant Staphylococcus aureus Pandemic Lineages

ABSTRACT The methicillin-resistant Staphylococcus aureus (MRSA) population in the Hospital Universitario Nuestra Señora de Candelaria over a 5-year period (1998 to 2002) was marked by shifts in the circulation of pandemic clones. Here, we investigated the emergence of high-level mupirocin resistance (Hi-Mupr). In addition to clonal spread, transfer of ileS2-carrying plasmids played a significant role in the dissemination of Hi-Mupr among pandemic MRSA lineages.

Antiproliferative activity of novel benzo[b][1,6]naphthyridines in human solid tumor cell lines.

A series of 2-substituted 1,2-dihydro-3-phenyl-1-(trichloromethyl)benzo[b][1,6]naphthyridines were synthesized and their in vitro antiproliferative activities were examined against human solid tumor cell lines and relevant strains of bacteria and Candida. The compounds induced considerably growth inhibition in all cancer cell lines, whilst showed inactive against microbial strains. Furthermore, we found analog 2-ethoxy-1H-pyrano[4,3-b]quinoline as selective inhibitor of microbial strains.

PCR Protocol for Specific Identification of Candida nivariensis, a Recently Described Pathogenic Yeast

ABSTRACT Candida nivariensis is a recently described pathogenic yeast closely related to Candida glabrata. We developed a specific set of oligonucleotide primers based on the internal transcribed spacer regions of the rRNA gene for the rapid identification of C. nivariensis. PCR with these primers amplified a 206-bp amplicon from C. nivariensis.

Association between the Presence of the Panton-Valentine Leukocidin-Encoding Gene and a Lower Rate of Survival among Hospitalized Pulmonary Patients with Staphylococcal Disease

Staphylococcus aureus is responsible for more than 2% of cases of community-acquired pneumonia and 10% of cases of nosocomial-acquired pneumonia. The lethality rate of such infections ranges from 30% to 80% ([6][1]). These infections are complicated by the fact that these bacteria have acquired

Anti-Inflammatory Activity of a Novel Family of Aryl Ureas Compounds in an Endotoxin-Induced Airway Epithelial Cell Injury Model

Background Despite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS. Methodology/Principal Findings After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings. Conclusions/Significance Using a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and sepsis-induced ALI/ARDS.

Antiproliferative activity of dmoPTA–Ru(II) complexes against human solid tumor cells

The biological evaluation of new Ru(II) complexes carrying dmoPTA (dmoPTA=3,7-dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) ligands is reported. The results on the biological activity revealed that the organometallic complexes are active against all cell lines with GI(50) values in the range 1.1-2.6 μM. When compared to the standard anticancer drug cisplatin, the bimetallic Ru(II) complexes showed a greater activity profile. The cell cycle analysis revealed that the new compounds induced arrest in G(1) phase. Contrary to cisplatin, these Ru(II) complexes do not interact with DNA. This result suggests that DNA might not be the key pharmacological target.

Mitotic arrest induced by a novel family of DNA topoisomerase II inhibitors.

A small structure-focused library of propargylic enol ethers was prepared by means of a modular and efficient chemodifferentiating organocatalyzed multicomponent reaction. The most active compound (GI(50) 0.25 microM) against solid tumor cells was selected as lead. Cell cycle analysis and immunoblotting demonstrated arrest at the metaphase, pointing out human topoisomerase II as plausible molecular target. In vitro assays were carried out, showing that the lead behaves as a catalytic inhibitor of the enzyme.