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.

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.

Streptococcus agalactiae in Brazil: serotype distribution, virulence determinants and antimicrobial susceptibility.

BACKGROUND: Group B Streptococcus (GBS) remains a major cause of neonatal sepsis and is also associated with invasive and noninvasive infections in pregnant women and non-pregnant adults, elderly and patients with underlying medical conditions. Ten capsular serotypes have been recognized, and determination of their distribution within a specific population or geographical region is important as they are major targets for the development of vaccine strategies. We have evaluated the characteristics of GBS isolates recovered from individuals with infections or colonization by this microorganism, living in different geographic regions of Brazil. METHODS: A total of 434 isolates were identified and serotyped by conventional phenotypic tests. The determination of antimicrobial susceptibility was performed by the disk diffusion method. Genes associated with resistance to erythromycin (ermA, ermB, mefA) and tetracycline (tetK, tetL, tetM, tetO) as well as virulence-associated genes (bac, bca, lmb, scpB) were investigated using PCR. Pulsed-field gel electrophoresis (PFGE) was used to examine the genetic diversity of macrolide-resistant and of a number of selected macrolide-susceptible isolates. RESULTS: Overall, serotypes Ia (27.6%), II (19.1%), Ib (18.7%) and V (13.6%) were the most predominant, followed by serotypes IV (8.1%) and III (6.7%). All the isolates were susceptible to the beta-lactam antimicrobials tested and 97% were resistant to tetracycline. Resistance to erythromycin and clindamycin were found in 4.1% and 3% of the isolates, respectively. Among the resistance genes investigated, tetM (99.3%) and tetO (1.8%) were detected among tetracycline-resistant isolates and ermA (39%) and ermB (27.6%) were found among macrolide-resistant isolates. The lmb and scpB virulence genes were detected in all isolates, while bac and bca were detected in 57 (13.1%) and 237 (54.6%) isolates, respectively. Molecular typing by PFGE showed that resistance to erythromycin was associated with a variety of clones. CONCLUSION: These findings indicate that GBS isolates circulating in Brazil have a variety of phenotypic and genotypic characteristics, and suggest that macrolide-resistant isolates may arise by both clonal spread and independent acquisition of resistance genes.

Phenotypic and molecular characterization of optochin-resistant Streptococcus pneumoniae isolates from Brazil, with description of five novel mutations in the ATPC gene.

Optochin (Opt) susceptibility is used largely for the identification of Streptococcus pneumoniae in diagnostic laboratories. Opt-resistant (Opt(r)) 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 Opt(r) 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 Opt(r) S. pneumoniae isolates recovered from patients or carriers living in Brazil. Sixteen isolates consisted of heterogeneous populations, and 10 isolates were homogeneously Opt(r). 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 Opt(r) variants), Opt(r) and Opt-susceptible (Opt(s)) subpopulations originating from the same culture had identical characteristics. Sequencing of the atpC gene of the Opt(r) 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.

Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children.

Capsular serotype surveillance of clinical isolates of Streptococcus pneumoniae is essential for evaluation of the potential impact of introducing multivalent capsular serotype-based vaccines in Latin America. Here, a previously described sequential multiplex PCR method was revised for optimal targeting of prevalent serotypes in Latin America. The revised protocol successfully serotyped 139/147 pneumococci (94.6%) from Brazilian children, demonstrating a labour-efficient, accurate method requiring only conventional PCR capability.

Diversity of mutations in the atpC gene coding for the c Subunit of F0F1 ATPase in clinical isolates of optochin-resistant Streptococcus pneumoniae from Brazil.

We report the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase. Two isolates showed mutations in codons 23 (leading to the deduced amino acid substitution isoleucine instead of alanine) and 49 (serine instead of alanine, a novel type of mutation detected at this position), respectively. Two additional novel mutations, both located in codon 45, were detected in the other two isolates, corresponding to leucine or valine (instead of phenylalanine). The data indicate that three previously unrecognized alterations were detected in the atpC gene of S. pneumoniae and that Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group.

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 percent, 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.

Determination of penicillin resistance in Streptococcus pneumoniae isolates from southern Brazil by PCR.

OBJECTIVE: To demonstrate the potential clinical applicability of the PCR technique to the early detection of bacterial resistance in Streptococcus pneumoniae. METHODS: We studied 153 samples of S. pneumoniae, isolated from different anatomic sites, using polymerase chain reaction (PCR) for the detection of specific amplicons from genes that code for penicillin-binding proteins (PBP) 1a, 2b and 2x, which are responsible for penicillin resistance in this organism. The occurrence of these mutated genes was correlated with the minimum inhibitory concentration (MIC) of penicillin, determined by the agar dilution test. RESULTS: The rate of penicillin resistance in S. pneumoniae in Porto Alegre, Brazil was 22.8% (16.3% intermediate resistance and 6.5% high resistance). In a statistically significant proportion of cases (p < 0.05), penicillin-susceptible samples had no amplicons, intermediate samples had only one (generally from PBP 2x), and highly resistant samples had amplicons from all three PBPs investigated. CONCLUSION: These results suggest that penicillin resistance in S. pneumoniae in southern Brazil is on the increase, but is still lower than in other countries, and that PCR could be used for its early detection.

A reação em cadeia da polimerase na detecção da resistência à penicilina em Streptococcus pneumoniae / Polymerase chain reaction used to detect Streptococcus pneumoniae resistance to penicillin

INTRODUÇAO: O Streptococcus pneumoniae é o mais freqüente agente etiológico de infecções respiratórias adquiridas na comunidade e sua resistência aos antimicrobianos tem aumentado nos últimos anos. A determinação da resistência é feita rotineiramente por método lento que depende do crescimento em cultura e determinação da concentração inibitória mínima (CIM). A reação em cadeia da polimerase (PCR) detecta os genes responsáveis pela resistência do Streptococcus pneumoniae a penicilina em cerca de 8 horas. OBJETIVO: Comparar a PCR com o método da CIM no diagnóstico da resistência da Streptococcus pneumoniae a penicilina. MÉTODO: Foram estudadas 153 amostras de Streptococcus pneumoniae, isoladas de diferentes sítios anatômicos, usando-se para detecção de mutações nos genes que codificam as proteínas ligadoras de penicilina 1a, 2b e 2x, responsáveis pela resistência à penicilina. A ocorrência das mutações foi correlacionada com a CIM de penicilina, determinada pelo teste de difusão em ágar. RESULTADOS: A resistência global à penicilina do Streptococcus pneumoniae foi de 22,8 por cento (16,3 por cento de resistência intermediária e 6,5 por cento de resistência alta). Em proporções estatisticamente significativas, as amostras sensíveis à penicilina não tinham mutações, as intermediárias apenas uma, geralmente na proteína ligadora de penicilina 2x, e as altamente resistentes tinham mutações nas três proteínas investigadas. CONCLUSAO: A PCR é um método rápido para a detecção da resistência à penicilina do Streptococcus pneumoniae, que poderá vir a ser utilizado na prática clínica.

Laboratory tests in the detection of extended spectrum beta-lactamase production: National Committee for Clinical Laboratory Standards (NCCLS) screening test, the E-test, the double disk confirmatory test, and cefoxitin susceptibility testing

Extended spectrum beta-lactamase (ESBL) production by Klebsiella sp. and E. coli is an emerging problem. In this study, 107 clinical isolates (53 E. coli, 47 K. pneumoniae and 7 K. oxytoca) screened as ESBL producers by the NCCLS disk diffusion procedure were submitted to a double disk confirmatory test (DDT) and to the E-test double strip for confirmation of ESBL production by demonstration of clavulanic acid inhibition effect (CAIE). Only 72/107 (67 percent) of the isolates were confirmed as ESBL producers by DDT, with diverse results among species. By the E-test, 58/107 (54 percent) isolates were confirmed as ESBL producers, and 18/107 (17 percent) were not determinable. Susceptibility to cefoxitin was found in 57/68 (83 percent) of strains that did not show CAIE. ESBL detection remains a controversial issue and clinical laboratories are in need of a simple and effective way to recognize strains with this kind of resistance.

Laboratory tests in the detection of extended spectrum beta-lactamase production: National Committee for Clinical Laboratory Standards (NCCLS) screening test, the E-test, the double disk confirmatory test, and cefoxitin susceptibility testing.

Extended spectrum beta-lactamase (ESBL) production by Klebsiella sp. and E. coli is an emerging problem. In this study, 107 clinical isolates (53 E. coli, 47 K. pneumoniae and 7 K. oxytoca) screened as ESBL producers by the NCCLS disk diffusion procedure were submitted to a double disk confirmatory test (DDT) and to the E-test double strip for confirmation of ESBL production by demonstration of clavulanic acid inhibition effect (CAIE). Only 72/107 (67%) of the isolates were confirmed as ESBL producers by DDT, with diverse results among species. By the E-test, 58/107 (54%) isolates were confirmed as ESBL producers, and 18/107 (17%) were not determinable. Susceptibility to cefoxitin was found in 57/68 (83%) of strains that did not show CAIE. ESBL detection remains a controversial issue and clinical laboratories are in need of a simple and effective way to recognize strains with this kind of resistance.

Occurrence and Characteristics of Erythromycin-Resistant Streptococcus pneumoniae Strains Isolated in Three Major Brazilian States.

We investigated the occurrence and phenotypic and genotypic characteristics of erythromycin-resistant Streptococcus pneumoniae strains isolated in three major states in Brazil, from 1990 to 1999. Of the 931 pneumococcal strains evaluated, 40 (4.3%) were erythromycin-resistant (Ery-R). Among the 40 Ery-R strains, 90.0%, 80.0%, 27.5%, 5.0%, and 2.5% were resistant to tetracycline, trimethoprim-sulfamethoxazole, penicillin, chloramphenicol, and rifampin, respectively. None of the strains were resistant to ofloxacin or to vancomycin. Most [37 (92.5%)] of the 40 Ery-R isolates presented the MLS(B) phenotype and 3 (7.5%) strains showed the M phenotype. PCR testing indicated that all MLS(B) phenotype isolates harbored the erm(B) gene only, whereas the mef(A/E) gene was present in all isolates presenting the M phenotype. The tet(M) gene was the most frequent (86.1%) among Ery-R isolates that were also resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) analysis after SmaI digestion revealed the occurrence of clonal relationships within groups of strains belonging to serotypes 14, 19A, and 23F. All Ery-R isolates belonging to serotype 14 were susceptible to penicillin and were included in a single clonal group (named Ery(14)-A) related to the England(14-)9 internationally spread clone.

Antimicrobial resistance in respiratory pathogens isolated in Brazil during 1999-2000

The in vitro antimicrobial susceptibility of the respiratory pathogens Streptococcus Pneumoniae, Hemophilus influenzae, and Moraxella catarrhalis to commonly tested and prescribed agents was investigated during 1999-2000 and compared with results obtained during a previous 1997-1998 study. Of 448 isolates of S. Pneumoniae collected and tested in 1999-2000, 77.2 were susceptible, 19.9 were intermediate, and 2.9 were resistant to penicillin, demonstrating that there were no major changes in susceptibility to penicillin from 1997-1998 (77.1 susceptible, 18.7 intermediate, 4.2 resistant). All S. Pneumoniae isolates from 1999-2000 were susceptible to levofloxacin and vancomycin and >90 were susceptible to the B-lactams (amoxicillin-clavulanate, ceftriaxone, and cefuroxime) and macrolides (axithyromycin and clarithromycin), showing that susceptibility to these agents also remained unchanged since 1997-1998. The most notable increase in resistance between the two studies was demonstrated by trimethoprim-sulfamethoxazole, which increased from 23.4 to 38.6. Penicillin resistance correlated with resistance to B-lactams, macrolides, and trimethoprim-sulfamethoxazole in both studies. In H. influenzae, the prevalence of B-lactamase-producing isolates remained unchanged (10.6 in 1999-2000; 11.0 in 1997-1998). All H. influenzae isolates were susceptible to levofloxacine, ceftriaxone, cefuroxime, and azithromycin, and showed no change between the two studies. Trimethoprim-sulfamethoxazole resistance was present in 40.1 of isolates in 1999-2000, and in 45.2 in 1997-1998. In M. catarrhalis, the prevalence of B-lactamase-producing isolates was unchanged (97.9 in 1999-2000;98.0 in 1997-1998). The most active agents against M. catarrhalis were azithromycin (MIC(90),< or = 0.03 microg/ml) and levofloxacin (MIC(90),< or = 0.03 microg/ml). Overall, these results suggest that, in Brazil, between 1999-2000 and 1997-1998, there have been no significant changes in the susceptibility of respiratory pathogens to any of the commonly tested and prescribed agents with the exception of trimethoprim-sulfamethoxazole for S. Pneumoniae.

Isolation of Shiga toxin-producing Escherichia coli (STEC) serotype 091: H21 from a child with diarrhea in Porto Alegre city, RS, Brazil

We describe the isolation of one strain of Shiga toxin-producing Escherichia coli 091: H21 from a child with diarrhea in the city of Porto Alegre, RS, Brazil. Considering the pathogenic potential of STEC, these organisms should be looked for more carefully among our population.

Triagem para detecçäo de resistência de Streptococcus pneumoniae à penicilina: estudo usando discos de oxacilina de três diferentes fabricantes / Screening for penicillin resistance of Streptococcus pneumoniae: a study with oxacillin discks of three different manufacturers

Resistência de Streptococcus pneumoniae à penicilina é um problema emergente global. Triagem para resistência à penicilina com uso de discos de oxacilina em teste em difusäo em ágar permite reconheciemnto de isolados suscetíveis. Isolados resitentes por este teste säo encaminhados para determinaçäo da concentraçäo inibitória mínima. Estudamos o desempenho do teste de triagem com discos de oxacilina em 119 isolados de S. peneumoniae, determinando o valor preditivo do teste para definiçäo da resitência. Discos de oxacilina de três diferentes fontes comerciais foram empregados. O valor preditivo do teste de triagem variou de 52,6-65,5 por cento entre discos testados, näo havendo, no entando, diferença significativa entre os valores. O valor preditivo do teste de triagem foi compatível com os observados na literatura para populaçöes com prevalência de resistência semelhante

Agar diffusion tests with cefuroxime disks for predicting ceftriaxone susceptibility among isolates of "Streptococcus pneumoniae"

The performance of agar diffusion tests using disks of cefuroxine (30 micrograms) for predicting ceftriaxone susceptibility in 33 isolates of "Streptococcus pneumoniae"was studied. All 7 resistant isolates to ceftriaxone (MIC>= 1.0 microgram/ml) exhibited zones of inhibition < 28 mm. The procedure can be easily adapted to clinical laboratories.

Resistência aos níveis elevados de aminoglicosídeos entre amostras de enterococcus isoladas na Irmandade Santa Casa de Misericórdia do Porto Alegre / High-Level resitance of aminoglycosides among isolates of enterococcus from irmandade Snta Casa de Misericórdia de Porto Alegre (ISCMPA)

O Enterococcus tem emergido como um dos mais importantes patógenos nosocomiais no mundo inteiro. Cepas resistentes aos níveis elevados de aminoglicosídeos, principalmente à gentamicina e à estreptomicina, têm aparecido com certa freqüencia em ambientes hospitalares, o que tem sido causa de preocupaçäo, pois causa de preocupaçäo, pois estes antimicrobianos säo utilizados no tratamento de infecçöes sérias causadas por este microrganismo. Alguns pesquisadores do sudeste do país têm se preocupado com esses dados, mas, no Rio Grande do Sul, ainda näo há dados disponíveis em relaçäo a esta resistência. Trinta e seis amostras isoladas da Irmandade Santa Casa de Misericórdia de Porto Alegre (ISCMPA), 35 (97,2 por cento) de E. faecalis e uma (2,8 por cento) de E. faecium, foram analisadas. Resistência aos níveis elevados de gentamicina, estreptomicina e aos dois aminoglicosídeos foi abservada em 50 por cento, 8,33 por cento e 2,77 por cento das amostras, respectivamente. Nossos resultados, apesar de pequena amostragem, indicam que a resistência aos níveis elevados de gentamicina tem alta prevalência em nosso meio e enfatizam a necessidade de estabelecer técnicas in vitro precisas de detecçäo desta resistência

Streptococcus agalactiae näo-hemolítico: identificaçäo por automaçäo / Streptococcus agalactiae non-hemolytic: identification by automation

Säo relatados quatro isolamentos de Streptococcus agalactiae grupo B (GBS) näo-hemolítico em urina. Amostras näo-hemolíticas de GBS raramente causam doença e säo habitualmente detectadas em funçäo perinatal. A identificaçäo foi obtida usando procedimento de automaçäo com um sistema comercial. Os isolados foram provenientes de amostras com contagens superiores a 100.000UFC/ml. A natureza retrospectiva deste estudo näo permitiu utilizar testes convencionais para caracterizaçäo do S. agalactiae. É preconizada a utilizaçäo de, pelo menos, um teste convencional de características para confirmaçäo do diagnóstico

Estafilococos resistentes à Oxacilina säo encontrados entre portadores da comunidade? / Are there oxacilin resistent staphilococci among carriers in the communitty?

O objetivo deste trabalho é investigar a existência de estafilococos resistentes à oxacilina entre portadores da comunicade. Para tanto, estudamos amostras obtidas de 93 estudantes de medicina näo expostos ao ambiente hospitalar. Dois testes laboratoriais foram empregados para detecçäo de resistência à oxacilina: teste de difusäo em ágar e teste de triagem com meio salino. Isolou-se Staphylococcus aureus em 21 (22,6 por cento) dos estudantes, näo tendo sido observada resistência a oxacilina. Em três estudantes encontramos estafilococos coagulares negativos apresentando resistência a oxacilina. Os dois testes laboratoriais usados na detecçäo de resistência apresentaram boa correlaçäo. Os dados obtidos apoiam o uso de oxacilina para tratamento de estafilococcias adquiridas na comunidade

Resistência de "Haemophilus influenzae" à ampicilina: uma revisäo / Resistance of \"Haemophilus influenzae\" to ampicillin: a review

Resistência de "Haemophilus influenzae" à ampicilina é um tema pouco conhecido e discutido em nosso meio. Na presente revisäo säo abordados aspectos relativos a diferentes mecanismos de resistência do microrganismo à ampicilina e os efeitos que os mesmos tem na definiçäo de resistência. A prevalência da resistência à ampicilina em diferentes países é apresentada, observando-se considerável variaçäo regional. A controvérsia sobre as alternativas para documentaçäo laboratorial da resistência é explorada, sendo discutidas as alternativas mais viáveis para laboratórios médicos. É destacada a necessidade de mais estudos que documentem resistência de "Haemophilus influenzae" à ampicilina em nosso país