Pedro Alves d’Azevedo

Evaluation of an automated system for the identification and antimicrobial susceptibility testing of enterococci

The performance of a new version of an automated system panel, the Positive Combo Panel Type 11 of MicroScan WalkAway 96 (WA96; Dade Behring) was evaluated and compared to that of reference methods for the identification and for antimicrobial susceptibility testing of the different enterococcal species. A total of 376 enterococcal isolates were tested. The MicroScan WA96 correctly identified 99.6% (266/267), 78.3% (18/23) and 68.6% (59/86) of Enterococcus faecalis, Enterococcus faecium and species other than E. faecalis and E. faecium, respectively. Although low probability of accurate identification was obtained for 37 (9.8%) strains, the system indicated that supplementary tests were necessary for precise identification of 8 (9.3%) among the 86 strains included in the non-faecalis/non-faecium group and of 3 (13.0%) among the E. faecium isolates. In comparison to the agar screening method, the percentage of agreement for detection of resistance markers by the automated system was 90.2% (37/41) for ampicillin, 90.6% (48/53) for high-level resistance to streptomycin (HLRS), 96.4% (80/83) for high-level resistance to gentamicin (HLRG), and 100% (14/14) for vancomycin. The results indicate that the MicroScan WA96 performed well for the identification of E. faecalis and typical E. faecium isolates, and for the detection of resistance to vancomycin and HLRG. However, the system still needs further improvement in order to provide reliable results for the characterization of the other enterococcal species, including atypical variants of E. faecium.

Community-acquired methicillin-resistant Staphylococcus aureus carrying SCCmec type IV in southern Brazil

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen commonly associated with nosocomial infections. However, it has also been associated with community-acquired skin and soft tissue infections (CA-MRSA). There are few data on the identification and prevalence of CA-MRSA infections in Brazil. METHODS This is a cross-sectional study of 104 patients with community-acquired skin infections attending two health care centers in Porto Alegre, southern Brazil. MRSA isolates were characterized by molecular methods, including detection of the mecA gene by PCR, gene SCCmec typing, Panton-Valentine leukocidin (PVL) detection, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). RESULTS From the 104 samples, 58 Staphylococcus aureus isolates were obtained, of which five (8.6%) had a CA-MRSA-resistant profile. All five isolates had the mecA gene and amplified to SCCmec type IV. Analysis of chromosomal DNA by PFGE revealed the presence of two clusters related to international clones (OSPC and USA 300), with a Dice similarity coefficient >80%. The study was complemented by MLST, which detected three different strains: ST30, ST8, and ST45, the latter not presenting any relation with the clones compared in PFGE. CONCLUSIONS The presence of CA-MRSA reveals an important change in the epidemiology of this pathogen and adds new elements to the knowledge of the molecular biology of infections by MRSA with SCCmec type IV in southern Brazil.

Sepse por Staphylococus aureus resistente à meticilina adquirida na comunidade no sul do Brasil

Methicillin-resistant Staphylococcus aureus was initially described as a typical microorganism acquired in nosocomial infections. However, over recent years, community-acquired methicillin-resistant Staphylococcus aureus has been a cause of skin and soft-tissue infections. Serious infections such as pneumonia and sepsis can also occur. This report describes a case of sepsis in a child that was complicated by pneumonia secondary to soft tissue lesions that were due to community-acquired methicillin-resistant Staphylococcus aureus in southern Brazil. The patient was attended at the Emergency Unit with a history of injury caused by lower-limb trauma that evolved to cellulitis, pneumonia and sepsis.

High biofilm production by invasive multiresistant staphylococci.

Reiter KC, Paim TGdS, de Oliveira CF, d’Azevedo PA. High biofilm production by invasive multiresistant staphylococci. APMIS 2011; 119: 776–81.

Susceptibility profiles and correlation with pneumococcal serotypes soon after implementation of the 10-valent pneumococcal conjugate vaccine in Brazil

OBJECTIVES To evaluate the susceptibility patterns among Streptococcus pneumoniae recovered during the years 2010-2012 and to correlate these with serotypes. METHODS Pneumococci from invasive sites were serotyped by sequential multiplex PCR and/or Quellung reaction. Etest strips were used to determine the minimal inhibitory concentrations, and the Clinical and Laboratory Standards Institute (CLSI) guidelines were used for interpretation. Genetic determinants of macrolide resistance were assessed by PCR, and the occurrence of the D phenotype was analyzed following the recommendations of the CLSI. RESULTS One hundred fifty-nine S. pneumoniae were studied; most were recovered from blood and were associated with serotypes 14, 3, 4, 23F, 20, 7F, 12F, 19A, and 19F. Pneumococcal conjugate vaccine PCV7, PCV10, and PCV13 and 23-valent polysaccharide vaccine serotypes represented 38.2%, 48.7%, 64.5%, and 85.5%, respectively. β-Lactam non-susceptibility (non-meningitis) was basically related to serotype 19A. For meningitis, it was observed in 21.4% (serotypes 14, 3, 9V, 23F, and 24F). Resistance to erythromycin occurred in 8.2% and mefA was the most common macrolide genetic determinant. One isolate was resistant to levofloxacin. Non-susceptibility to trimethoprim-sulfamethoxazole was 37.7% and to tetracycline was 22.0%. CONCLUSIONS Our population of pneumococci represents a transition era, soon after the introduction of PCV10. Non-susceptible patterns were found to be associated with classical PCV serotypes (especially serotype 14), which is still highly prevalent, and non-PCV10 ones (19A), which may disseminate, occupying the biological niche left by the vaccine serotypes.

Upregulation of icaA, atlE and aap genes by linezolid but not vancomycin in Staphylococcus epidermidis RP62A biofilms

Biofilms are complex bacterial structures protected by a self-produced polymer matrix that enables survival in hostile environments. Biofilms are more resistant to antibiotics than their planktonic counterparts and are therefore more difficult to eradicate. The aim of this study was to investigate the influence of vancomycin and linezolid on the maintenance of staphylococcal biofilms and their effect on the expression of biofilm-associated genes in Staphylococcus epidermidis. Pre-formed biofilms of S. epidermidis RP62A were challenged with linezolid and vancomycin at different concentrations as well as at their clinically relevant target concentration (15 mg/L) over time. Expression of icaA, atlE, aap, rnaIII, luxS, sarA, rsbU and icaR genes following 2h of exposure to these antibiotics was determined by quantitative PCR. Vancomycin did not significantly affect the biofilm under the tested conditions. However, linezolid affected the biofilm structure at concentrations of ≥ 2 mg/L (P<0.05); moreover, the exposure time to this antibiotic was a determinant for biofilm eradication. The level of transcription of icaA, aap and atlE increased by 5.18-, 2.58- and 3.06-fold, respectively, in biofilms exposed to linezolid, but no changes were observed for vancomycin. The other genes were not affected by these antibiotics. This study demonstrated that linezolid was effective in eradicating biofilms formed by S. epidermidis RP62A. Under the conditions tested, linezolid upregulated biofilm-associated genes probably due to the stress caused by low-dose antibiotic stimulation. In this study, linezolid showed better performance than vancomycin against staphylococcal biofilms.

Phenotypic, molecular and antimicrobial susceptibility assessment in isolates from chronic ulcers of cured leprosy patients: a case study in Southern Brazil

BACKGROUND One of the most stigmatizing physical sequelaeof leprosy in cured patients is the development of chronic lower extremity ulcers. The bacterial diversity present in ulcers is considered one of the factors that can delay the healing process, as well as serve as a focus for severe secondary infections. OBJECTIVE To identify the microbiota and antimicrobial resistance profile of bacteria isolated from skin ulcers in patients cured of leprosy. METHODS After obtaining informed consent, material was collected from ulcers of 16 patients treated at the Outpatient Public Health Dermatology Clinic of Rio Grande do Sul and Hospital Colônia Itapuã. Sampleswere collected during dressing, and the material sent to the Microbiology Laboratory of the Federal University of Health Sciences of Porto Alegre for microbiological culture. Methicillin-resistant Staphylococcus aureus (MRSA) was characterized by two molecular methods, including detection of the mecA gene by PCR and SCCmecgene typing. RESULTS Cultures revealed microorganisms in all ulcers: Gram-negative bacilli in 80%, Gram-positive cocci in 63%, and mixed microflora in 36%. Staphylococcus aureus and Pseudomonas aeruginosa were the most prevalent bacteria. Assessment of the antimicrobial resistance profile was notable for the presence of MRSA. Molecular analysis of this isolate revealed presence of the mecA gene contained in a type IV staphylococcal cassette chromosome mec (SCCmec). CONCLUSIONS In patients with leprosy, laboratory culture of skin ulcers is essential for correct antibiotic selection and to control emerging pathogens, such as MRSA carrying SCCmec type IV.

Enterococcus species diversity in fecal samples of wild marine species as determined by real-time PCR

Analyses using culture-independent molecular techniques have improved our understanding of microbial composition. The aim of this work was to identify and quantify enterococci in fecal samples of wild marine species using real-time quantitative PCR. Seven Enterococcus species were examined in fecal DNA of South American fur seals (Arctocephalus australis), Subantarctic fur seals (Arctocephalus tropicalis), green turtles (Chelonia mydas), Magellanic penguins (Spheniscus magellanicus), snowy-crowned tern (Sterna trudeaui), white-backed stilt (Himantopus melanurus), white-chinned petrels (Procellaria aequinoctialis), red knot (Calidris canutus), and black-browed albatross (Thalassarche melanophris). All Enterococcus species evaluated were detected in all fecal samples of wild marine species, with a concentration ranging between 106 and 1012 copies/ng of total DNA. Differences in the enterococci distribution were observed. Enterococcus faecalis and Enterococcus mundtii were most abundant in marine mammals. Enterococcus faecalis was frequent in green turtle, Magellanic penguin, snowy-crowned tern, red knot, and black-browed albatross. Enterococcus hirae and Enterococcus gallinarum showed elevated occurrence in white-backed stilt, and Enterococcus faecium in white-chinned petrel. This study showed highest diversity of enterococci in feces of wild marine species than currently available data, and reinforced the use of culture-independent analysis to help us to enhance our understanding of enterococci in gastrointestinal tracts of wild marine species.

Complete Genome Sequence of Staphylococcus aureus FCFHV36, a Methicillin-Resistant Strain Heterogeneously Resistant to Vancomycin

ABSTRACT We report here the sequence of the entire chromosome of Staphylococcus aureus strain FCFHV36, a methicillin-resistant strain heterogeneously intermediate to vancomycin, bearing a type II staphylococcal chromosome cassette mec element (SCCmec), belonging to multilocus sequence type (MLST) 105, and isolated from a vertebra of a patient with osteomyelitis.

Non-Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Causing Infection in Health-care Facilities in Southern Brazil

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is usually susceptible to a variety of non-beta-lactam drugs. These isolates commonly display SCCmecIV and are associated with community-acquired infections. More recently, CA-MRSA has been isolated from health-care-associated diseases. We categorize isolates resistant only to oxacillin or oxacillin plus no more than 3 non-beta-lactam antibiotics according to clinical and epidemiological characteristics, from a hospital in Porto Alegre, and performed a combination of molecular techniques including mecA, SCCmec, Panton-Valentine leukocidin (PVL) detection and Pulsed-field gel electrophoresis (PFGE). A total of twenty-five patients with non-multidrug-resistant MRSA were studied. Nineteen (76%) came from skin and soft tissue infections. All isolates presented SCCmec type IV (being 19/25 IVc) whereas the PFGE profile most frequently found was OSCP-like (15/25). The presence of international clones USA400, USA300 was also verified. Comparing the results of clonal type with source, origin, type of SCCmec, presence the PVL gene and antimicrobial resistance we observed that OSCP-like PFGE profile was associated with skin and soft tissue infections (P=0.0012) and that this clonal group was strongly associated with the presence of PVL gene (P<0.001). This study shows a clonal diversity of CA-MRSA and strengths the concept that these isolates emerged globally from different backgrounds.