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.

Analytical methods for vancomycin determination in biological fluids and in pharmaceuticals

Vancomycin is a glycopeptide antibiotic employed in the treatment of infections caused by certain methicillin-resistant staphylococci. It is indicated also for patients allergic to penicillin or when there is no response to penicillins or cephalosporins. The adequate vancomycin concentration levels in blood serum lies between 5 and 10 mg/L. Higher values are toxic, causing mainly nephrotoxicity and ototoxicity. Various analytical methods are described in the literature: spectrophotometric, immunologic, biologic and chromatographic methods. This paper reviews the main analytical methods for vancomycin determination in biological fluids and in pharmaceutical preparations.

Atividade antibacteriana de floroglucinóis e do extrato hexânico de Hypericum brasiliense Choysi

Three phloroglucinols were obtained from Hypericum brasiliense: japonicine A (1), isouliginosin B (2) and uliginosin B (3). Bioautography and disk diffusion methods were used to determine antibacterial activity of the hexanic extract. Strains of the Coagulase Negative Staphylococcus and American Methicillin Resistant Staphylococcus aureus clones showed a growth inhibition zone ranging from 10 to 12 mm and 7 to 15 mm, respectively. Minimal inhibitory concentration (MIC) values were used to measure antistaphylococcal activity for all phloroglucinols. Isouliginosin B and uliginosin B presented MIC values of 1.5 and 3.0 µg/mL, respectively, while japonicine A displayed MIC value of 50.0 µg/mL.

PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsed-field gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillin-resistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains.

In vitro antimicrobial activity of a new series of 1,4-naphthoquinones

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 µg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 µg/ml. All compounds presented a minimal bactericidal concentration higher than 500 µg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 µg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

Vancomycin-dependent Enterococcus faecium vanA: characterization of the first case isolated in a university hospital in Brazil

In this study, we report the characterization of a strain of Enterococcus faecium vanA, which grows only in the presence of vancomycin (VDEfm-UEL). The bacterium was isolated from the feces of a female patient who had undergone surgical treatment of Reinke’s edema and was receiving intravenous vancomycin therapy for infection with methicillin/oxacillin-resistant Staphylococcus aureus, a postoperative complication. Antimicrobial dependence was further confirmed by the vancomycin E-test. VDEfm-UEL was also shown to be resistant to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, levofloxacin, penicillin, rifampicin, and teicoplanin. The putative virulence genes efaA, gelE and esp were detected by PCR. The ddl gene from VDEfm-UEL was cloned and sequenced. Vancomycin dependence seems to be associated with the insertion of a nucleotide in that sequence, which results in a frame-shift mutation, introducing a premature stop codon. This is the first report of vancomycin-dependent E. faecium isolation in a university hospital in Brazil.

New multilocus sequence typing of MRSA in São Paulo, Brazil

An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE) profile and molecular sequence typing (MLST) genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5), showing a new yqiL allele gene, resulting in a new sequence typing (ST) (1176). Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

Clinical impact of Achromobacter xylosoxidans colonization/infection in patients with cystic fibrosis

The rate of diagnosis of colonization/infection of the airways with Achromobacter xylosoxidans has increased in cystic fibrosis patients, but its clinical significance is still controversial. This retrospective, case-control study aimed to evaluate the clinical impact of A. xylosoxidans colonization/infection in cystic fibrosis patients. Individuals who were chronically colonized/infected (n=10), intermittently colonized/infected (n=15), and never colonized/infected with A. xylosoxidans (n=18) were retrospectively evaluated during two periods that were 2 years apart. Demographic characteristics, clinical data, lung function, and chronic bacterial co-colonization data were evaluated. Of the total study population, 87% were pediatric patients and 65.1% were female. Individuals chronically colonized/infected with A. xylosoxidans had decreased forced expiratory volume in 1 s (51.7% in the chronic colonization/infection group vs 82.7% in the intermittent colonization/infection group vs 76% in the never colonized/infected group). Compared with the other two groups, the rate of co-colonization with methicillin-resistant Staphylococcus aureus was higher in individuals chronically colonized/infected with A. xylosoxidans (P=0.002). Changes in lung function over 2 years in the three groups were not significant, although a trend toward a greater decrease in lung function was observed in the chronically colonized/infected group. Compared with the other two groups, there was a greater number of annual hospitalizations in patients chronically colonized/infected with A. xylosoxidans (P=0.033). In cystic fibrosis patients, there was an increased frequency of A. xylosoxidans colonization/infection in children, and lung function was reduced in patients who were chronically colonized/infected with A. xylosoxidans. Additionally, there were no differences in clinical outcomes during the 2-year period, except for an increased number of hospitalizations in patients with A. xylosoxidans.