Felipe Piedade Gonçalves Neves

Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

ABSTRACT Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.

Nasopharyngeal carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among children from Brazil before the introduction of the 10-valent conjugate vaccine

BackgroundStreptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide. Nasopharyngeal colonization plays an important role in the development and transmission of pneumococcal diseases, and infants and young children are considered to be the main reservoir of this pathogen. The aim of this study was to evaluate the rates and characteristics associated with nasopharyngeal carriage, the distribution of serotypes and the antimicrobial resistance profiles of Streptococcus pneumoniae among children in a large metropolitan area in Brazil before the introduction of the 10-valent pneumococcal conjugate vaccine.MethodsBetween March and June 2010, nasopharyngeal swabs were collected from 242 children aged <6 years attending one day care center and the emergency room of a pediatric hospital. Pneumococcal isolates were identified by conventional methods and serotypes were determined by a sequential multiplex PCR assay and/or the Quellung reaction. The antimicrobial susceptibilities of the pneumococci were assessed by the disk diffusion method. MICs for erythromycin and penicillin were also performed. Erythromycin resistance genes were investigated by PCR.ResultsThe overall colonization rate was 49.2% and it was considerably higher among children in the day care center. Pneumococcal carriage was more common among day care attenders and cohabitants with young siblings. The most prevalent serotypes were 6B, 19F, 6A, 14, 15C and 23F, which accounted for 61.2% of the isolates. All isolates were susceptible to clindamycin, levofloxacin, rifampicin and vancomycin. The highest rate of non-susceptibility was observed for sulphamethoxazole-trimethoprim (51.2%). Penicillin non-susceptible pneumococci (PNSP) accounted for 27.3% of the isolates (MICs of 0.12-4 μg/ml). Penicillin non-susceptibility was strongly associated with serotypes 14 and 23F. Hospital attendance and the presence of respiratory or general symptoms were frequently associated with PNSP carriage. The two erythromycin-resistant isolates (MICs of 2 and 4 μg/ml) belonged to serotype 6A, presented the M phenotype and harbored the mef(A/E) gene.ConclusionsCorrelations between serotypes, settings and penicillin non-susceptibility were observed. Serotypes coverage projected for the 10-valent pneumococcal conjugate vaccine was low (45.5%), but pointed out the potential reduction of PNSP nasopharyngeal colonization by nearly 20%.

Characteristics related to antimicrobial resistance and biofilm formation of widespread methicillin-resistant Staphylococcus epidermidis ST2 and ST23 lineages in Rio de Janeiro hospitals, Brazil.

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in seriously ill or immunocompromised patients. Currently, the characteristics of methicillin-resistant S. epidermidis (MRSE) isolates from Rio de Janeiro hospitals are unknown. In this study, staphylococcal chromosomal cassette mec (SCCmec) types, antimicrobial susceptibility profiles, biofilm formation genes, and multilocus sequence types (MLST) were investigated in 35 MRSE clinical isolates. The collection of isolates was previously well characterized by pulsed-field gel electrophoresis (PFGE) into 2 main genotypes (A and B, 22 isolates) and 10 sporadic genotypes (13 isolates). MLST revealed a total of 8 different sequence types (STs), but ST2 and ST23, which were icaAB-positive, represented the majority (71.4%) of MRSE isolates tested. Almost all isolates (91.4%) belonged to clonal complex 2. SCCmec types III and IV were identified among 71.4% of the isolates, while the remaining was nontypeable. The predominant MRSE genotypes were defined as SCCmec type III/ST2 (PFGE type A) and SCCmec type IV/ST23 (PFGE type B) isolates, which were both associated with high antimicrobial resistance and presence of biofilm-related genes.

Molecular Characterization of Quinolone-Resistant Neisseria gonorrhoeae Isolates from Brazil

ABSTRACT Despite the rapid spread of antibiotic resistance among gonococci worldwide, limited reports are available from Brazilian locations. In the present study, 25 quinolone-resistant Neisseria gonorrhoeae (QRNG) strains isolated in Rio de Janeiro, Brazil, were characterized by phenotypic and molecular methods, including analysis of mutations in the gyrA and parC genes. They represented 16.5% of the N. gonorrhoeae isolates obtained during a survey performed from 2006 to 2010. A trend for increasing resistance to ciprofloxacin was observed in the period investigated. The most prevalent pattern of mutation observed among QRNG isolates, Ser-91 to Phe and Asp-95 to Gly in gyrA and Ser-87 to Arg in parC, was detected in 40% of the isolates exhibiting MICs ranging from 4 to >32 μg/ml. Rare types of mutations were found in the gyrA gene (Gln-102 to His [12%] and Asp-95 to Tyr [4%]) and in the parC gene (Ser-88 to Thr [4%]). The genetic relationship of the QRNG isolates, evaluated by pulsed-field gel electrophoresis, suggested that the increase in the frequencies of the QRNG isolates in Rio de Janeiro, Brazil, may have arisen as a result of simultaneous spread of two clonal groups. The results also indicate that fluoroquinolones may no longer be used as first line antibiotics for the treatment of gonorrhea in Rio de Janeiro, and that programs for antimicrobial susceptibility surveillance of N. gonorrhoeae should also be implemented in other regions of Brazil.

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.

Panton-Valentine leukocidin (PVL) gene carriage among Staphylococcus aureus strains with SCCmec types I, III, IV, and V recovered from cystic fibrosis pediatric patients in Brazil

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) is increasing in patients with cystic fibrosis (CF). We report a molecular characterization, antimicrobial resistance, and Panton-Valentine leukocidin (PVL) toxin gene detection of MRSA strains from 28 Brazilian pediatric CF patients (1 strain per patient). A significant proportion (50%) of MRSA SCCmec IV isolates was observed. Nearly half of MRSA strains harboring the PVL genes distributed in all SCCmec types detected. Multilocus sequence typing (MLST) analyses showed majority (57.1%) of the isolates belonged to known epidemic lineages, such as UK/EMRSA-3, Pediatric/USA 800, Southwest Pacific clone, and Brazilian/Hungarian clone. To our knowledge, this is the first Brazilian study of molecular epidemiology based on MLST and SCCmec typing and the first description of PVL genes in MRSA from CF patients.

Phenotypic and Molecular Characterization of Optochin-Resistant Streptococcus pneumoniae Isolates from Brazil, with Description of Five Novel Mutations in the atpC Gene

ABSTRACT Optochin (Opt) susceptibility is used largely for the identification of Streptococcus pneumoniae in diagnostic laboratories. Opt-resistant (Optr) S. pneumoniae isolates have been reported, however, indicating the potential for misidentification of this important pathogen. Point mutations in the atpC gene have been associated with the emergence of Optr S. pneumoniae, but data on the characterization of such atypical variants of S. pneumoniae are still limited. The present report describes the results of a polyphasic approach to identifying and characterizing 26 Optr S. pneumoniae isolates recovered from patients or carriers living in Brazil. Sixteen isolates consisted of heterogeneous populations, and 10 isolates were homogeneously Optr. The isolates had different serotypes and antimicrobial susceptibility profiles. They also presented diverse genetic characteristics, as indicated by pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem-repeat analysis (MLVA), and pspA gene typing. Except for Opt MICs (4- to 64-fold higher among Optr variants), Optr and Opt-susceptible (Opts) subpopulations originating from the same culture had identical characteristics. Sequencing of the atpC gene of the Optr variants revealed 13 different nucleotide changes distributed among eight different codons. Changes in codon 49 were the most frequent, suggesting that this might be a hot spot for optochin resistance-conferring mutations. On the other hand, five novel types of mutations in the atpC gene (Met13Ile, Gly18Ser, Gly20Ala, Ala31Val, and Ala49Gly) were identified. In silico prediction modeling indicated that the atpC gene mutations corresponded to alterations in the transmembrane region of the ATPase, leading to a higher hydrophobicity profile in α-helix 1 and to a lower hydrophobicity profile in α-helix 2.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

Antimicrobial susceptibility and genetic diversity of Streptococcus agalactiae recovered from newborns and pregnant women in Brazil

Abstract Background: Streptococcus agalactiae is known to be the major cause of neonatal infections and also causes complications during pregnancy. Methods: One hundred and six strains of Streptococcus agalactiae recovered from clinical specimens of newborns (n = 18) and pregnant women (n = 88) were submitted to antimicrobial susceptibility testing and investigation of genetic determinants of macrolide resistance, capsular type, and virulence factors. Genetic diversity was evaluated by pulsed-field gel electrophoresis (PFGE) analysis. Results: Strains were susceptible to ceftriaxone, levofloxacin, penicillin G, and vancomycin and resistant to tetracycline (85.8%) and erythromycin (4.7%). Erythromycin-resistant strains presented iMLSB phenotype, harbored the ermA gene, and were closely related by PFGE. Both bac and bca genes were found in low frequencies. PFGE analysis yielded 11 DNA restriction profiles among 35 selected isolates. The major clonal group, designated as A, was composed predominantly of strains belonging to capsular type Ia. Clonal group B was composed predominantly of strains with capsular type V, including all erythromycin-resistant isolates. Conclusions: Although low levels of erythromycin resistance have been observed, this is a fact of concern because this phenotype also confers resistance to clindamycin, an alternative agent for intrapartum prophylaxis. Despite the diversity of capsular types, Ia and V were among the most common and were significantly associated with distinct clonal groups. In a few cases, different capsular types were clustered into a single clonal group, which may be related to capsular switching.

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.