Juliana Caierão

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.

Serotypes and Genotypes of Invasive Streptococcus pneumoniae Before and After PCV10 Implementation in Southern Brazil

To reduce the burden of pneumococcal diseases, different formulations of pneumococcal conjugate vaccines (PCV) have been introduced in many countries. In Brazil, PCV10 has been available since 2010. We aimed to analyze the serotype and genetic composition of invasive pneumococci from Brazil in pre- and post- vaccination periods (2007–2012). Antibiotic susceptibility was determined and genotypes of macrolide and fluoroquinolone resistance were characterized. The genotypes of isolates of the most frequent serotypes were determined by multilocus sequence typing. The study included 325 isolates, which were primarily recovered from blood. The most common serotypes recovered were 14, 3, 4, 23F, 7F, 9V, 12F, 20, 19F, 8, 19A, and 5. Thirty-eight pneumococci (11.7%) were from children ≤5 years old. Considering the overall population, PCV10 and PCV13 serotype coverage was 50.1% and 64.9%, respectively. During the pre-vaccine period, isolates with serotypes belonging to the PVC10 represented 51.5% (100/194), whereas in the post vaccine they represented 48.0% (63/131). PCV13 serotypes represented 67.5% (131/194) and 59.2% (77/131) of total for pre- and post-vaccination periods, respectively. Seventy different sequence types [STs] were found, accounting for 9 clonal complexes [CCs] and 45 singletons. Eight STs (156, 180, 218, 8889, 53, 191, 770, and 4967) represented the majority (51.5%) of isolates. Fifty STs were associated with the pre-vaccination period (27 exclusive) and 43 (20 exclusive) with the post-vaccination period; 23 STs were identified in both periods. Some serotypes were particularly clonal (7F, 8, 12F, 20). Non-susceptibility to penicillin was associated with serotype 19A, CC320. Erythromycin resistance was heterogeneous when considering serotype and ST. A single serotype 23F (ST4967) isolate was resistant to levofloxacin. Continued surveillance is required to determine vaccine impact and to monitor changes in pneumococcal population biology post-PCV10 introduction in Brazil.

Automated systems in the identification and determination of methicillin resistance among coagulase negative staphylococci

Coagulase-negative staphylococci (CoNS) are an important cause of nosocomial bacteremia, specially in patients with indwelling devices or those submitted to invasive medical procedures. The identification of species and the accurate and rapid detection of methicillin resistance are directly dependent on the quality of the identification and susceptibility tests used, either manual or automated. The objective of this study was to evaluate the accuracy of two automated systems--MicroScan and Vitek--in the identification of CoNS species and determination of susceptibility to methicillin, considering as gold standard the biochemical tests and the characterization of the mecA gene by polymerase chain reaction, respectively. MicroScan presented better results in the identification of CoNS species (accuracy of 96.8 vs 78.8%, respectively); isolates from the following species had no precise identification: Staphylococcus haemolyticus, S. simulans, and S. capitis. Both systems were similar in the characterization of methicillin resistance. The higher discrepancies for gene mec detection were observed among species other than S. epidermidis (S. hominis, S. saprophyticus, S. sciuri, S. haemolyticus, S. warneri, S. cohnii), and those with borderline MICs.

Corynebacterium striatum infecting a malignant cutaneous lesion: the emergence of an opportunistic pathogen

We described a case of a 27-year old male patient with skin and soft tissue infection of a neoplastic lesion caused by Corynebacterium striatum, an organism which has been rarely described as a human pathogen. Identification was confirmed by DNA sequencing. Successful treatment with penicillin was achieved. The role of the C. striatum as an emerging opportunistic pathogen is discussed.

Correlation between biofilm formation and gelE, esp, and agg genes in Enterococcus spp. clinical isolates

Enterococci are a leading cause of nosocomial infections. Enterococcus faecalis is the species most frequently isolated, and it is commonly recovered from surgical wounds, intra-abdominal infections, the bloodstream, and especially urinary tract infections (UTIs).1,2

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.

Use of the D test method to detect inducible clindamycin resistance in coagulase negative staphylococci (CoNS)

According to the National Committee for Clinical Laboratory Standards (NCCLS, 2004), a method to evaluate the inducible clindamycin resistance in accordance with an approach of the disks of erythromycin and clindamycin--the D test--has been reported. We analyzed the performance of this method in 200 coagulase negative staphylococci (CoNS) strains obtained from blood cultures of hospitalized patients at a general hospital in Southern Brazil. Twenty-seven clinical isolates with suitable profile (erythromycin-resistant and clindamycin-susceptible) were evaluated for the D test realization. Thus, only 5 CoNS were D test positive. The D test method showed to be simple and an important technique in the detection of inducible clindamycin resistance.

Surveillance of impact of PCV-10 vaccine on pneumococcal meningitis in Mozambique, 2013 – 2015

Background Vaccination using the 10-valent conjugate vaccine (PCV-10) was introduced into the Extended Program on Immunization in Mozambique in March 2013, however its impact on pediatric pneumococcal meningitis is unknown. In this study, we assessed for the first time the impact of PCV10 on the burden of pneumococcal meningitis in children less than 5 years of age at the three largest hospitals in Mozambique. Method Between March 2013 and December 2015, a total of 744 cerebrospinal fluid (CSF) samples were collected from eligible children, of which 160 (21.5%) were positive for S. pneumoniae. Of these, only 86 samples met the criteria for serotyping and were subsequently serotyped using sequential multiplex PCR (SM-PCR), but 17 samples were non-typable. Results The proportion of cases of pneumococcal meningitis decreased from 33.6% (124 of 369) in 2013 to 1.9% (3 of 160) in 2015 (p < 0.001). The relative frequency of PCV10 serotype cases also decreased from 84.2% (48 of 57) in 2013 to 0% (0 of 3) in 2015 (p = 0.006). Between 2013 and 2015, serotype coverage of PCV-10 and PCV13 vaccine formulations was 66.7% and 81.2%, respectively. Conclusion Altogether, our findings shows that introduction of PCV-10 immunization resulted in rapid decline of pneumococcal meningitis children less than 5 years old in Mozambique. This decline was accompanied by substantial changes in the pattern of circulating pneumococcal serotypes.

EVALUATION OF METHODS IN DETECTING VANCOMYCIN MIC AMONG MRSA ISOLATES AND THE CHANGES IN ACCURACY RELATED TO DIFFERENT MIC VALUES

INTRODUCTION: Methicillin-Resistant Staphylococcus aureus (MRSA) presenting reduced susceptibility to vancomycin has been associated to therapeutic failure. Some methods used by clinical laboratories may not be sufficiently accurate to detect this phenotype, compromising results and the outcome of the patient. OBJECTIVES: To evaluate the performance of methods in the detection of vancomycin MIC values among clinical isolates of MRSA. MATERIAL AND METHODS: The Vancomycin Minimal Inhibitory Concentration was determined for 75 MRSA isolates from inpatients of Mãe de Deus Hospital, Porto Alegre, Brazil. The broth microdilution (BM) was used as the gold-standard technique, as well as the following methods: E-test® strips (BioMérieux), M.I.C.E® strips (Oxoid), PROBAC® commercial panel and the automated system MicroScan® (Siemens). Besides, the agar screening test was carried out with 3 µg/mL of vancomycin. RESULTS: All isolates presented MIC ≤ 2 µg/mL for BM. E-test® had higher concordance (40%) in terms of global agreement with the gold standard, and there was not statistical difference among E-test® and broth microdilution results. PROBAC® panels presented MICs, in general, lower than the gold-standard panels (58.66% major errors), while M.I.C.E.® MICs were higher (67.99% minor errors). CONCLUSIONS: For the population of MRSA in question, E-test® presented the best performance, although with a heterogeneous accuracy, depending on MIC values.

Pleural effusion with negative culture: a challenge for pneumococcal diagnosis in children.

BACKGROUND Pneumococcal parapneumonic effusion seems to be increasing in children in the postvaccine era and is frequently associated with negative culture. Due to the low yield of culture, culture-independent tools are evaluated. METHODS Culture-negative pleural fluid specimens from 38 children with parapneumonic effusion were examined for pneumococcal lytA by quantitative polymerase chain reaction (qPCR) and soluble antigen (C-polysaccharide) using an immunochromatographic test (BinaxNow Streptococcus pneumoniae). RESULTS In 81% (30/37) and 63% (24/38) of the specimens, a positive result was obtained by qPCR and antigen detection, respectively. Most mismatches were observed in specimens with low quantities of pneumococcal DNA and a negative antigen test. CONCLUSIONS Our results suggest an imperfect relationship between the 2 described methods. The immunochromatographic assay is a simple diagnostic tool, which can be used when resources are limited, and even after antibiotic use, but negative results may require confirmation through a more sensitive test, such as qPCR.