Rosana R. Barros

Phenotypic and Genotypic Characterization of Pediococcus Strains Isolated from Human Clinical Sources

ABSTRACT Seventy-two strains of pediococci isolated from human clinical sources were characterized by conventional physiological tests, chromogenic enzymatic tests, analysis of whole-cell protein profiles (WCPP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analysis of chromosomal DNA restriction profiles by pulsed-field gel electrophoresis (PFGE). Conventional tests allowed identification of 67 isolates: 52 strains were identified as Pediococcus acidilactici, 15 strains were identified as Pediococcus pentosaceus, and 5 strains were not identified because of atypical reactions. Analysis of WCPP identified all isolates since each species had a unique WCPP. By the WCPP method, the atypical strains were identified as P. acidilactici (two strains) andP. pentosaceus (three strains). The chromogenic substrate test with o-nitrophenyl-β-d-glucopyranoside differentiated all 54 strains of P. acidilactici (negative reactions) and 13 (72%) of 18 strains of P. pentosaceus(positive reactions). Isolates of both species were shown to be nonclonal as revealed by the genetic diversity when chromosomal DNA was analyzed by PFGE. Using WCPP as the definitive identification procedure, P. acidilactici (28 of 54 strains; 51.8%) was more likely than P. pentosaceus (4 of 18 strains; 22.3%) to be isolated from blood cultures.

Phenotypic and Genotypic Characteristics of Streptococcus porcinus Isolated from Human Sources

ABSTRACT The phenotypic and genotypic characteristics of 25 Streptococcus porcinus isolates recovered from human sources were investigated and compared to the characteristics of 17 reference strains obtained from nonhuman sources. All of the S. porcinus isolates were beta-hemolytic (wide zones), susceptible to vancomycin, gave positive results for the leucine aminopeptidase and l-pyrrolidonylarylamidase tests, and produced acids from mannitol and sorbitol. Most of them were positive for the CAMP test and resistant to bacitracin. The isolates were susceptible to most of the 14 antimicrobials tested, except for tetracycline, for which 80% of the human isolates and 35.2% of the nonhuman strains were resistant. The tet(M) and the tet(O) genes were detected in 23 (88.5%) and 8 (30.8%) of the 26 tetracycline-resistant isolates, respectively. Analysis of whole-cell protein profiles obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high similarity among the profiles. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI and by random(ly) amplified polymorphic DNA (RAPD)-PCR using primer 1254. Analysis of SmaI-restricted genomic DNA revealed the substantial genetic diversity among S. porcinus isolates from nonhuman sources, which were also serologically more diverse. Most of the human isolates belonged to serogroup NG1 and shared highly related PFGE profiles that were distinct from profiles of isolates from nonhuman sources. These results were in agreement with those obtained by analysis of amplicons after RAPD-PCR, indicating the potential ability of these techniques for typing S. porcinus and suggesting the occurrence of a few clonal groups of S. porcinus strains adapted to the human host.

Genetic and Phenotypic Features of Streptococcus pyogenes Strains Isolated in Brazil That Harbor New emm Sequences

ABSTRACT In the present study, 37 group A Streptococcus (GAS) strains belonging to 13 new emm sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated st204, st211,st213, st809, st833,st854, st2904, st2911,st2917, st2926, st3757,st3765, and st6735. All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the speB gene, and 94.6% produced detectable SpeB. Most strains belonging to a given emmtype had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some emm types. Unlike the other isolates, all type st213 isolates were speA positive and produced SpeA. Strains belonging to st3765 were T6 and opacity factor (OF) negative. Individual isolates within OF-positive emm types were associated with uniquesof gene sequence types, while OF-negative isolates weresof negative by PCR. This report provides information on new emm sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.

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%.

Antimicrobial Susceptibility and Survey of Macrolide Resistance Mechanisms among Streptococcus pyogenes Isolated in Rio de Janeiro, Brazil

A total of 357 clinical Streptococcus pyogenes isolates collected between 1994 and 1999 in Rio de Janeiro city were tested for susceptibility to 10 antimicrobial drugs by agar-diffusion tests. All isolates were susceptible to penicillin, cephems, and vancomycin. High resistance rates were observed for tetracycline (43.1%) and trimethoprim/sulfamethoxazole (77.9%). Three isolates (0.8%) were resistant to erythromycin, and three exhibited intermediate susceptibility. Determination of the erythromycin MICs by the agar dilution method, showed 1.6% of erythromycin resistant isolates (the three erythromycin-resistant and the three erythromycin-intermediate isolates found by agar-diffusion test). Of the erythromycin-resistant isolates subjected to the double-disc diffusion test for erythromycin and clindamycin, three isolates expressed the iMLSB and three the M phenotype. The resistance phenotypes were confirmed by comparing the clindamycin MICs determined under normal testing conditions and those determined after induction by pre-growth in 0.06 microg/ml of erythromycin. Three ermTR and three mefA-containing isolates were detected by PCR. In strains belonging to the iMLSB phenotype, two clones were identified by PFGE following restriction with SmaI. M phenotype isolates could not be restricted with SmaI. Our results indicate a low rate of erythromycin resistance among S. pyogenes isolated in Rio de Janeiro, Brazil, and pointed to the presence of both resistance mechanisms found in streptococci.

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.

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.

Molecular characterization of the first fluoroquinolone resistant strains of Streptococcus agalactiae isolated in Brazil

Three isolates of Streptococcus agalactiae, recovered from residents of the metropolitan area of Rio de Janeiro with significant bacteriuria, were found to be resistant to levofloxacin. Determination of the minimal inhibitory concentration (MIC) confirmed one isolate as intermediate and two as resistant to levofloxacin. No reduction in levofloxacin MIC was observed with reserpine, indicating that resistance was not caused by an efflux mechanism. Typical point mutations were observed in the quinolone resistance determinant region of gyrA and parC. Other point mutations in parC generated novel altered codons: Ser80→Pro in the intermediate resistance isolate, and Gly128→Asp in a resistant isolate. Through molecular modeling, it was possible to observe that these novel substitutions might not play a role in resistance, since these amino acids were not involved in the antibiotic binding site. Pulsed field gel electrophoresis profiles revealed a non-clonal trend among these isolates. This is the first report of genetic characterization of levofloxacin-resistant S. agalactiae strains in Brazil.

Streptococcus pneumoniae Serotypes 9 and 14 Circulating in Brazil over a 23-Year Period Prior to Introduction of the 10-Valent Pneumococcal Conjugate Vaccine: Role of International Clones in the Evolution of Antimicrobial Resistance and Description of a Novel Genotype

ABSTRACT Antimicrobial-resistant pneumococcal strains have been detected worldwide since the 1960s. In Brazil, the first penicillin-nonsusceptible pneumococci (PNSP) were reported in the 1980s, and their emergence and dissemination have been mainly attributed to serogroup 9 and serotype 14 strains, especially those highly related to recognized international clones. In the present study, antimicrobial susceptibility testing and multilocus sequence typing were performed on 315 pneumococcal isolates belonging to serogroup 9 (n = 99) or serotype 14 (n = 216), recovered from patients or asymptomatic carriers between 1988 and 2011 in Brazil, in order to trace changes in antimicrobial resistance and genotypes prior to the full introduction of the pneumococcal conjugate vaccine in the country. Over the 23-year study period, the PNSP levels increased, and four clonal complexes (CC156, CC66, CC15, and CC5401) have played important roles in the evolution and dissemination of pneumococcal isolates belonging to serogroup 9 and serotype 14, as well as in the emergence of antimicrobial resistance, in the pre-pneumococcal-vaccination era. The earliest PNSP strains detected in this study belonged to serotype 9N/ST66 and were single locus variants of the international clone Tennessee14-18 ST67 (CC66). The first serotype 14 PNSP isolates were identified in 1990 and were related to the England14-9 ST9 (CC15) clone. Serotype 14 PNSP variants of the Spain9V-3 ST156 clone with elevated penicillin MICs and nonsusceptibility to other beta-lactams were detected in 1995 and showed an increasing trend over the years. The results also indicated that introduction of ST156 in our region was preceded by the emergence of trimethoprim-sulfamethoxazole resistance and by the dissemination of ST162. In addition to the presence of successful international clones, a novel regional serotype 14 genotype (CC5401) has emerged in 1996.