Ana Lúcia Peixoto de Freitas

High vancomycin resistance among biofilms produced by Staphylococcus species isolated from central venous catheters

Biofilm production is an important mechanism that allows microbes to escape host defences and antimicrobial therapy. Vancomycin has been used largely for the treatment of methicillin-resistant staphylococcal infections. Here, we determined the minimal inhibitory concentration (MIC) and minimal biofilm eradication concentration (MBEC) for 82 Staphylococcus species isolated from central venous catheters (CVC). Our results showed that the 41 strong and moderate-biofilm-producing isolates presented a higher MBEC/MIC ratio for vancomycin than the 24 weak-biofilm-producing isolates, illustrating the importance of biofilm production ability and the difficulty in treating biofilm-related infections. The MBEC was significantly higher in moderate-biofilm-producing isolates than in weak-biofilm-producing isolates (p < 0.001) and in strong-biofilm-producing isolates than in weak-biofilm-producing isolates (p = 0.001). The correlation between the MIC and the MBEC was poor. Based on our results, we recommend that bacterial biofilms be suspected in all cases of CVC infection.

Extended-spectrum beta-lactamases in Klebsiella spp and Escherichia coli obtained in a Brazilian teaching hospital: detection, prevalence and molecular typing

His study was performed to compare the methods of detection and to estimate the prevalence of extendedspectrum β-lactamases (ESBL) among Klebsiella spp and E. coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K. pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clinicas de Porto Alegre, using Kirby-Bauer zone diameter (KB), double-disk diffusion (DD), breakpoint for ceftazidime (MIC CAZ), increased zone diameter with clavulanate (CAZ/CAC) and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC). Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E. coli), contrasting with all the other methods (p < 0.05). The confirmatory methods (DD, MIC CAZ, CAZ/CAC and MIC CAZ/CAC) showed a range of ESBL production from 8 to 13% for E. coli and from 33 to 40% for Klebsiella species. Therefore, the KB method was useful only as a screening method as it provided several false positive results. Molecular typing of 17 ESBL K. pneumoniae indicated that the isolates had no clonal relation. We found a good correlation among the confirmatory methods for ESBL detection although the methods which evaluate inhibition of the β-lactamase by clavulanate appeared to be more specific. The high prevalence of ESBL Klebsiella in our hospital is probably due to individual selection of resistant strains rather than the transmission of a common strain.

Antibiotic resistance and molecular typing of Pseudomonas aeruginosa: focus on imipenem

Susceptibility tests by disk diffusion and by E-test and molecular typing by macrorestriction analysis were performed to determine the relatedness of Pseudomonas aeruginosa isolates from three distinct hospitals. The resistance profile of 124 isolates to 8 antimicrobial agents was determined in three different hospitals, in Porto Alegre, Brazil. Frequencies of susceptibility ranged from 43.9% for carbenicillin to 87.7% for ceftazidime. Cross-resistance data of imipenem-resistant isolates indicated that most (70%) were also resistant to carbenicillin, although 30% remained susceptible to ceftazidime and cefepime. In general, susceptibility profiles were not able to determine relatedness among isolates of P. aeruginosa. On the other hand, molecular typing by macrorestriction analysis demonstrated high discriminatory power and identified 66 strains among 72 isolates of P. aeruginosa. Imipenem-susceptible isolates were all different. However, identical clones of imipenem-resistant isolates were found in two of the hospitals, despite variable response to other antibiotics. No clustering of infection among the different medical centers was observed. In conclusion, clones of P. aeruginosa did not spread among the different hospitals in our city even though related isolates of imipenem-resistant P. aeruginosa were found.

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.

When the resistance gets clingy: Pseudomonas aeruginosa harboring metallo-β-lactamase gene shows high ability to produce biofilm

The ability to produce biofilm and the presence of metallo-β-lactamase (MBL) among Pseudomonas aeruginosa isolates were evaluated. A total of 91 isolates were recovered from sputa of patients with (CF, n = 44) and without (non-CF, n = 47) cystic fibrosis diagnosis. Seventy-nine (86.8%; 95% CI 78.3–92.3%) were biofilm producers. Interestingly, all isolates harboring MBL showed ability (most strong or moderate) to produce biofilm in vitro. We alert to an “overlapping of mechanisms” that together represent an even greater challenge for the treatment of pulmonary infections by P. aeruginosa.

Feasible identification of Staphylococcus epidermidis using desferrioxamine and fosfomycin disks

Coagulase‐negative Staphylococcus spp. (CoNS) have emerged as predominant pathogens in hospital‐acquired infections, as well as reservoirs of antimicrobial resistance, increasing the necessity of developing reliable methods for identification of the most frequent species. The aim of this study was to propose a simplified method for identification of Staphylococcus epidermidis. A total of 490 isolates of CoNS were identified by Bannermans method. Taking into account distinct approaches for identification of S. epidermidis, among CoNS, we proposed the use of only two disks: desferrioxamine for the initial trial, and fosfomycin to match the final identification. Of the 320 isolates susceptible to desferrioxamine, Bannermans method identified 238 S. epidermidis and 73 S. hominis, while we achieved identification of 239 S. epidermidis and 76 S. hominis. Compared to Bannermans method, the method proposed here obtained a sensitivity of 99.5%, and had a positive predictor value of 99.2%. We also used a genotypic method for identification of S. epidermidis by polymerase chain reaction (PCR) targeting the tuf gene. In conclusion, the method proposed here has proved to be useful for the identification of S. epidermidis, the most frequent species of CoNS isolated from blood cultures in clinical microbiology laboratories.

High prevalence of methicillin-resistant Staphylococcus aureus with SCCmec type III in cystic fibrosis patients in southern, Brazil

INTRODUCTION Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.

Identification, antimicrobial resistance and genotypic characterization of Enterococcus spp. isolated in Porto Alegre, Brazil

Nas ultimas duas decadas os membros do genero Enterococcus emergiram como importantes patogenos nosocomiais ao redor do mundo. No presente estudo, nos avaliamos a resistencia antimicrobiana e as caracteristicas genotipicas de 203 Enterococcus spp. obtidos de diferentes fontes clinicas em dois hospitais de Porto Alegre, Rio Grande do Sul, Brasil. As especies foram identificadas por testes bioquimicos convencionais e por um sistema automatizado. A diversidade genetica de E. faecalis demonstrando resistencia a altos niveis de aminoglicosideos (HLAR) foi avaliada atraves da analise do DNA cromossomico apos digestao com a enzima SmaI, seguido por eletroforese em campo pulsado. O E. faecalis foi a especie mais frequente (93,6%), seguido por E. faecium (4,4%). O perfil de resistencia antimicrobiana foi: 2,5% para ampicilina, 0,5% para vancomicina, 0,5% para teicoplanina, 33% para cloranfenicol, 2% para nitrofurantoina 66,1% para eritromicina, 66,5% para tetraciclina, 24,6% para rifampicina, 30% para ciprofloxacino e 87,2% para quinupristina-dalfopristina. Um total de 10,3% dos isolados apresentaram HLAR para ambos gentamicina e estreptomicina (HLR-ST/GE), sendo 23,6% resistentes somente a gentamicina (HLR-GE) e 37,4% somente a estreptomicina (HLR-ST). Um grupo clonal predominante foi encontrado em E. faecalis HLR-GE/ST. A prevalencia de resistencia a antibioticos ²-lactâmicos, e em particular aos glicopeptideos, foi muito baixa. Entretanto, neste estudo, houve um numero crescente de Enterococcus HLAR que podem estar se disseminando intra e interhospitais.

Evaluation of biofilm production by Pseudomonas Aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients.

Cystic fibrosis (CF) patients typically suffer of persistent and recurrent lung infections caused by Pseudomonas aeruginosa that many times possess ability for the biofilm production. Here, biofilm production among P. aeruginosa isolates recovered from sputum of CF and non-CF patients was evaluated. Most isolates were biofilm-producing independently of the patients condition.

High frequency of β-lactam susceptibility in CTX-M-type extended-spectrum-β-lactamase-producing Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis according to the new CLSI recommendations

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