Olga Cardoso

Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.

Metallo-β-lactamase VIM-2 in Pseudomonas aeruginosa isolates from a cystic fibrosis patient

The increased incidence of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) along with an increase in its multidrug resistance makes therapeutic management very problematic. Careful identification and accurate studies of susceptibility to antibiotics are critical for improving therapeutic measures and for facilitating our understanding of the epidemiology of this pathogen. Fifteen P. aeruginosa isolates obtained from five CF children in the Paediatric Hospital of Coimbra were studied. Isolates from a female patient were resistant to all agents tested except colistin. A VIM-2 enzyme inserted in integron In58 was detected, and this isolate presented a unique random amplified polymorphic DNA (RAPD) type. Others isolates were susceptible to beta-lactams, and each isolate had a different RAPD type. VIM-2 confers resistance to the majority of beta-lactams and is associated with other gene cassettes coding for enzymes that inactivate aminoglycosides. Person-to-person transmission of these isolates is not well understood, therefore it is important to design infection control policies to avoid acquisition and dissemination of multiresistant strains.

Helicobacter pylori antimicrobial resistance rates in the central region of Portugal

Helicobacter pylori resistance to antimicrobial agents is steadily increasing. It is extremely important to be aware of the local prevalence of antibiotic resistance so as to adjust treatment strategies. During this single-centre, prospective study, we aimed to determine primary and secondary resistance rates of H. pylori to antibiotics as well as host and bacterial factors associated with this problem. Overall, 180 patients (131 female; mean age 43.4±13.5 years; primary resistance 103; secondary resistance 77) with positive (13) C-urea breath test were submitted to upper endoscopy with gastric biopsies. Helicobacter pylori was cultured and antimicrobial susceptibility was determined by Etest and molecular methods. Clinical and microbiological characteristics associated with resistance were evaluated by logistic regression analysis. Among the 180 isolates 50% were resistant to clarithromycin (primary 21.4%; secondary 88.3%), 34.4% to metronidazole (primary 29.1%; secondary 41.6%), 33.9% to levofloxacin (primary 26.2%; secondary 44.2%), 0.6% to tetracycline and 0.6% to amoxicillin. Being female was an independent predictor of resistance to clarithromycin and metronidazole. Previous, failed, eradication treatments were also associated with a decrease in susceptibility to clarithromycin. History of frequent infections, first-degree relatives with gastric carcinoma and low education levels determined increased resistance to levofloxacin. Mutations in the 23S rRNA and gyrA genes were frequently found in isolates with resistance to clarithromycin and levofloxacin, respectively. This study revealed that resistance rates to clarithromycin, metronidazole and levofloxacin are very high and may compromise H. pylori eradication with first-line and second-line empiric triple treatments in Portugal.

Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy.

The aim of this work was the development of innovative levofloxacin-loaded swellable microspheres (MS) for the dry aerosol therapy of pulmonary chronicPseudomonas aeruginosainfections in Cystic Fibrosis patients. In a first step, a factorial design was applied to optimize formulations of chitosan-based MS with glutaraldehyde as crosslinker. After optimization, other crosslinkers (genipin, glutaric acid and glyceraldehyde) were tested. Analyses of MS included aerodynamic and swelling properties, morphology, drug loading, thermal and chemical characteristics,in vitroantibacterial activity and drug release studies. The prepared MS presented a drug content ranging from 39.8% to 50.8% of levofloxacin in an amorphous or dispersed state, antibacterial activity and fast release profiles. The highest degree of swelling was obtained for MS crosslinked with glutaric acid and genipin. These formulations also presented satisfactory aerodynamic properties, making them a promising alternative, in dry-powder inhalers, to levofloxacin solution for inhalation.

Triple Therapy with High-Dose Proton-Pump Inhibitor, Amoxicillin, and Doxycycline Is Useless for Helicobacter pylori Eradication: A Proof-of-Concept Study

Helicobacter pylori resistance to antibiotics is steadily increasing and multidrug‐resistant strains are common and difficult to eliminate, mainly in countries where bismuth, tetracycline, furazolidone, and rifabutin are unavailable.

Detection of Pseudomonas aeruginosa producing metallo-β-lactamase VIM-2 in a central hospital from Portugal

Pseudomonas aeruginosa remains one of the most important pathogens in the nosocomial setting [1]. P. aeruginosa exhibits intrinsic resistance to several antimicrobial agents. The antipseudomonal β-lactams represent a major weapon against Pseudomonas infections, either for monotherapy or for combination therapy, for which β-lactams almost invariably represent one of the components. Therefore, resistance to these agents constitutes a major challenge for anti-Pseudomonas chemotherapy. Several mechanisms can contribute to β-lactam resistance in P. aeruginosa, including β-lactamase production, outer membrane impermeability and active efflux mediated by RND-type efflux systems [1]. During the last decade, the metallo-β-lactamases (MBLs) have emerged as new threatening mechanisms of broad-spectrum β-lactam resistance in P. aeruginosa. In fact, these enzymes can efficiently degrade virtually all antipseudomonal β-lactams (except aztreonam), while they are not susceptible to therapeutic β-lactamases inhibitors [2]. Based on amino acid sequence homology, these MBLs have been classified into four major types: IMP, VIM, SPM and GIM. Clinical isolates harbouring the MBLs IMP and VIM have been increasingly reported worldwide, mostly in European and Asian countries [2]. This increase in occurrence, types and rate of dissemination of MBLs makes early detection very critical. The benefits of such treatment include the timely implementation of strict infection control practices, as well as clinical guidance. The aim of this study was to determine the presence of these enzymes in imipenem-resistant P. aeruginosa isolates collected at the Centro Hospitalar of Coimbra (CHC), during a two-year period (April 2003 to April 2005) and to ascertain their clonal relationship. CHC is a cluster formed by a central hospital and several specialised units located at distances of less than 8 km, namely, Hospital dos Covoes (central hospital), Instituto Maternal (maternity hospital) and Hospital Pediatrico (paediatric hospital). The Microbiology Laboratory also analysed samples from another hospital (Hospital de Pombal) located 30 km away. Bacterial identification and minimal inhibitory concentrations (MIC) were performed with the MicroScan WalkAway (Dadebehring) system according to the instructions of the manufacturer and API32GN or API PSE systems (bioMerieux). All intermediate strains were considered as nonsusceptible strains. The MICs of β-lactams were also determined by E-test in isolates that presented the enzyme VIM-2. The results were interpreted on the basis of CLSI recommended breakpoints [3]. The screening of MBLs was done by the double combined disk test [4]. Polymerase chain reaction (PCR) analyses for the detection of MBLs genes (blaIMP, blaVIM, blaGIM and blaSPM-1) were carried out for all strains in which the screening test gave positive results [5]. The amplicons were directly sequenced on both DNA strands on an ABI PRISM 377 automated sequencer. The nucleotide and deduced amino acid sequences were analysed with software available online (http://blast.ncbi.nlm.nih.gov/ Eur J Clin Microbiol Infect Dis (2008) 27:1269–1271 DOI 10.1007/s10096-008-0579-2

Multidrug and Extensive Drug Resistance in Pseudomonas aeruginosa Clinical Isolates from a Portuguese Central Hospital: 10-Year Survey

Multidrug-resistant (MDR) Pseudomonas aeruginosa isolates are increasing worldwide and greatly limit therapeutic options, particularly when considering extensively drug-resistant (XDR) or pandrug-resistant isolates. The resistance profile of P. aeruginosa isolates from a Portuguese central hospital was surveyed during 10 years (n=3,778). About 39.9% were classified as MDR and 2.9% as XDR. Statistical analysis (Mann-Whitney test and regression modeling) revealed a decrease in total MDR rates over time but an increase in XDR rates. This suggests a tendency for higher proportions of XDR isolates in the future, which is of great concern. Isolates of nosocomial origin presented similar results to total population but, when analyzing them according to the different wards of origin, it was still observed a trend of increase in MDR rates in some wards, particularly pneumology, neurology, and neurosurgery. Similar analysis considering the nosocomial specimen source revealed a negative trend of evolution in MDR rates of respiratory origin and a positive trend over time in XDR rates of isolates collected from urine. Regarding the association of antibiotic resistance to MDR and XDR profiles, it was observed a negative relation over time between imipenem resistance and MDR and gentamicin resistance and XDR, suggesting that resistance to these antibiotics may predict the absence of MDR or XDR in P. aeruginosa isolates, respectively. Similar studies in other European hospitals should be performed to give further information to physicians, important for their empirical antibiotherapy regimens.

Prevalence and Molecular Epidemiology of Imipenem-Resistant Pseudomonas aeruginosa Carrying Metallo-Beta-Lactamases from Two Central Hospitals in Portugal

Metallo-beta-lactamases (MBLs) can confer broad-spectrum beta-lactam resistance, including carbapenems. The aim of this work was to document the occurrence of MBLs in 122 imipenem-resistant Pseudomonas aeruginosa isolates collected in two Portuguese central hospitals, to determine their antimicrobial susceptibility, and to observe if there were intra- and interhospital epidemic spread. About 20.5% of these isolates presented blaVIM-2, which was found to be widespread in both hospitals. Clonal diversity was observed within hospitals, and no interhospital spread was observed. Ten of the blaVIM-2-positive isolates (44%), from both hospitals, presented one or two class 1 integrons. Two of those contained a VIM-2 gene, one from each hospital, which is indicative for the possibility of MBL gene transfer. No interhospital spread of integrons was observed. Regular screening and surveillance is needed to prevent spread of this worrisome resistance determinant.

Virulence factors as predictive tools for drug resistance in Pseudomonas aeruginosa.

P. aeruginosa is the fifth most frequent pathogen worldwide, the second in nosocomial pneumonia, the third in urinary infections, the forth in post-surgical infections and the seventh pathogen responsible for sepsis.1 It is also the major cause of mortality in cystic fibrosis patients and the most prevalent Gram-negative multidrug-resistant bacteria in the airway of mechanically ventilated patients2 and in pediatric patients hospitalized in intensive care units (ICU).1 Infections by P. aeruginosa are diverse and difficult to treat due to its intrinsic ability to develop resistance under antibiotic pressure and to produce a variety of virulence factors (VF), like adhesins, proteases, phenazines and exotoxins.3 Several studies report that the presence or expression of virulence traits is related to resistance to antibiotics.2,4,5,6 Some suggest these relations are antagonistic5,6 while others report enhanced resistance in isolates with high levels of VF.2,4 Also, literature indicates that inhibitors of specific VF may be good therapeutic alternatives to treat P. aeruginosa infections.7,8 With present work, we intend to address possible associations between VF and antibiotic resistance in P. aeruginosa, aiming to provide information that may be useful for the development of alternative therapies using VF inhibitors.

Trends in Pseudomonas aeruginosa Antibiotic Resistance: Case Study from a Portuguese Central Hospital

Aims: P. aeruginosa antibiotic resistance is increasing worldwide, greatly limiting therapeutic options, and is mainly associated to nosocomial infections, relating to high morbidity and mortality outcomes. Frequently updated regional guidelines, supported in statistically valid longitudinal information, are mandatory. Methodology: Resistance to 11 antibiotics used to treat P. aeruginosa infections were surveyed in clinical isolates from a Portuguese central during 10 years (n=3778) using the MicroScan WalkAway system. Statistical analysis (Mann-Whitney tests and regression modeling) were used to determine its time evolution according to origin, specimen samples and hospital wards. Data Article