Showing papers by "José Melo-Cristino published in 2011"

Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Staphylococcus aureus

Abstract: Background Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones.

Analysis of Invasiveness of Pneumococcal Serotypes and Clones Circulating in Portugal before Widespread Use of Conjugate Vaccines Reveals Heterogeneous Behavior of Clones Expressing the Same Serotype

Abstract: To estimate the invasive disease potential of serotypes and clones circulating in Portugal before extensive use of the seven-valent pneumococcal conjugate vaccine, we analyzed 475 invasive isolates recovered from children and adults and 769 carriage isolates recovered from children between 2001 and 2003. Isolates were serotyped and genotyped by pulsed-field gel electrophoresis, and a selection of isolates were also characterized by multilocus sequence typing. We found that the diversities of serotypes and genotypes of pneumococci responsible for invasive infections and carriage were identical and that most carried clones could also be detected as causes of invasive disease. Their ability to do so, however, varied substantially. Serotypes 1, 3, 4, 5, 7F, 8, 9N, 9L, 12B, 14, 18C, and 20 were found to have an enhanced propensity to cause invasive disease, while serotypes 6A, 6B, 11A, 15B/C, 16F, 19F, 23F, 34, 35F, and 37 were associated with carriage. In addition, significant differences in invasive disease potential between clones sharing the same serotype were found among several serotypes, namely, 3, 6A, 6B, 11A, 14, 19A, 19F, 22F, 23F, 34, and NT. This heterogeneous behavior of the clones was found irrespective of the serotype's overall invasive disease potential. Our results highlight the importance of the genetic background when analyzing the invasive disease potential of certain serotypes and provide an important baseline for its monitoring following conjugate vaccine use. Continuous surveillance should be maintained, and current research should focus on uncovering the genetic determinants that contribute to the heterogeneity of invasive disease potential of clones sharing the same serotype.

Group B Streptococci Causing Neonatal Infections in Barcelona Are a Stable Clonal Population: 18-Year Surveillance

Abstract: We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years.

Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of S

Abstract: BackgroundAntimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones.ResultsAugmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds.ConclusionsThe results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role played by efflux systems in the development of resistance to fluoroquinolones in clinical isolates of S. aureus.

Limitation of malaria diagnosis with the Cell-Dyn® analyser: not all haemozoin-containing monocytes are detected or shown.

Abstract: Sir, When imported malaria is not suspected in a febrile patient, some laboratory results, like anaemia or thrombocytopenia detected during a full blood count, or raised LDH levels, may alert to the diagnosis, although these changes are rather unspecific. However, as pointed out in a recent review (Campuzano-Zuluaga, Hänscheid & Grobusch, 2010), several of the automated haematology analysers may be capable of detecting specific changes in samples from malarious patients. Malaria pigment (haemozoin) is birefringent and depolarizes the light used in flow cytometers. The Cell-Dyn instruments (Abbott, Santa Clara, CA, USA) have been shown to detect haemozoin-containing monocytes, found in a Side-Scatter versus depolarized Side-Scatter plot, often labelled as lobularity/granularity plot (Mendelow et al., 1999; Kramer et al., 2001). Although a review of the studies on the diagnostic accuracy of the Cell-Dyn instruments appear to show reassuring figures for sensitivity and specificity, results varied sometimes substantially between studies (Campuzano-Zuluaga, Hänscheid & Grobusch, 2010). One explanation may be the fact that no automated flag or alert exists. Laboratory staff usually have to screen for malaria-typical changes visually on the monitor, following criteria described in previous studies (CampuzanoZuluaga, Hänscheid & Grobusch, 2010). However, it has been noted that the graphic output may not represent all the events which the instrument analyses or detects (Hanscheid et al., 2008). Contrary to common flow cytometers, in the CellDyn instruments colours are used to designate different WBC populations and not frequency of detected cells. Thus, it remains unknown if any dot represents only one detected event, or several. Indeed, cells with little variation in lobularity and granularity may cause clustering, i.e. one coloured dot may represent many detected events, which is the case for monocytes. This observation was also made using a Cell-Dyn in a study in Lambaréné, Gabon. After analysing 153 samples, it was found that in the lobularity/ granularity plot only a mean of 5.6% of detected leukocytes and 4.3% of detected monocytes were represented (Hanscheid et al., 2008). This potential data loss may have the consequence that the number of Hz-containing monocytes is significantly underestimated, eventually causing false negative results. To estimate this potential data loss we compared the performance of a Cell-Dyn 3700, used at our Emergency Laboratory Services in Lisbon, with a flow cytometer which was modified to allow the detection of depolarizing leukocytes. EDTA anticoagulated blood samples from patients with confirmed Plasmodium falciparum malaria were routinely analysed with a Cell-Dyn 3700. Any purplecoloured dot above the diagonal separation line that divided neutrophils from eosinophils was considered to represent a Hz-containing monocyte; and the number of these dots was counted on-screen, as described elsewhere (Mendelow et al., 1999; Hanscheid, Melo-Cristino & Pinto, 2001). Blood from the same sample used for the Cell-Dyn was reanalysed within 12 h after marking the leukocytes with anti-CD14 (FITC) and anti-CD16 (PE) (Immunotools, Friesoythe, Germany). Erythrocytes were lysed prior to analysis with CyLyse solution (Partec, Münster, Germany). Thirty to 40 000 events were acquired and analysed on a modified bench top flow cytometer (CyFlow ; Partec). The CyFlow uses blue laser excitation (488 nm), and detects Forward-Scatter, Side-Scatter, green fluorescence (FL1) and orange-red fluorescence (FL3). The fluorescent detector (FL2) was turned into a detector of depolarized Side-Scatter. The gate for depolarizing events was set after gating on CD14 positive cells (monocytes) which showed depolarization above background, determined using blood from healthy volunteers. The resultant gate resembled the gate used in the Cell-Dyn analyser’s lobularity/granularity plot (Mendelow et al., 1999; Kramer et al., 2001) (Figures S1 and S2). For the comparison with the Cell-Dyn , any such depolarizing event was considered to be a true Hz-containing monocyte. Overall, the results from the Cell-Dyn instrument always detected or showed less Hz-containing monocytes LETTER TO THE EDITOR INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

INCF plasmids responsible by dissemination of blaKPC gene among enterobacteriaceae

Abstract: In recent years, carbapenem resistance has emerged among Enterobacteriaceae in many geographical locations due to Klebsiella pneumoniae producing KPC carbapenemase. The aim of this study was to characterize the genetic elements involved in blaKPC gene mobilization and diffusion.

Persistência de Klebsiella pneumoniae em doentes de unidades pediátricas do Hospital de Santa Maria, em Lisboa

Abstract: Introducao: A Klebsiella pneumoniae e responsavel por infeccoes nosocomiais e adquiridas na comunidade. A producao de β-lactamases de espectro alargado (ESBLs) e os factores de virulencia assumem particular importância nestas infeccoes.