Streptococcus agalactiae - Distribuição sorotípica e relação com fatores de virulência e resistência antimicrobiana / Streptococcus agalactiae - Serotype distriburion and correlation with virulence factors and antibiotic resistance

Streptococcus agalactiae, ou Estreptococo do Grupo B, é um microrganismo que encontrado na microbiota intestinal, vaginal e/ou geniturinária de 10-30% de mulheres saudáveis. A principal infecção causada por S. agalactiae é a sepse neonatal. O bebê pode adquirir o microrganismo durante o parto ao passar pelo canal vaginal, ou até mesmo durante a gestação, caso haja ascensão de S. agalactiae para o útero. Existem diversos fatores associados à infecção do feto por S. agalactiae quando a mãe é colonizada, tais como fator CAMP, cápsula de polissacarídeos, hialuronidase, ß-citolisina/hemolisina e pili. Não existe consenso ou recomendação técnica sobre o tema no Brasil. Segundo o Caderno de Atenção Básica ao Pré-Natal, não existem estudos que levem à recomendação da antibioticoterapia intraparto. É necessário elucidar as características genotípicas de cepas de S. agalactiae isoladas no Brasil para alinhar as práticas clínicas às características fenotípicas do microrganismo. Desta forma, os objetivos deste projeto são: i) classificar cepas de S. agalactiae isoladas de gestantes e não gestantes quanto ao sorotipo capsular, por PCR Multiplex, ii) avaliar a presença e distribuição de fatores de virulência, por PCR e iii) avaliar o perfil de resistência antimicrobiana, pelo método de disco difusão e teste D. Os achados de virulência e resistência a antimicrobianos foram comparados com os sorotipos, gestação, localização geográfica e sítio de isolamento. Foram analisadas 292 cepas isoladas de gestantes e não gestantes em São Paulo, São José dos Campos e Rio de Janeiro. O sorotipo Ia foi o mais prevalente entre as cepas. Na cidade de São José dos Campos não houve diferença significativa entre a prevalência dos sorotipos Ia e V, sendo que o sorotipo V foi mais abundante do que nas cidades de São Paulo e Rio de Janeiro. O sorotipo II foi mais abundante em mulheres não gestantes do que gestantes. Não foram encontradas cepas resistentes à Penicilina e vancomicina; contudo a resistência a Cefepima, Eritromicina e Clindamicina ficou em torno de 22%. Foram encontradas diferenças entre os sorotipos quanto à resistência, genes de virulência e sítio de isolamento das cepas. Portanto essas diferenças podem se refletir no perfil epidemiológico da infecção por S. agalactiae quanto à localização geográfica também quanto à gestação. A incidência de sepse causada por S. agalactiae diminuiu muito nas últimas décadas, contudo o monitoramento constante é necessário para alinhar as práticas clínicas às características fenotípicas do microrganismo

Streptococcus agalactiae, or Group B Streptococcus, is a microorganism found in intestinal, vaginal and/or genitourinary microbiota from about 10-30% of all healthy women. The main infection caused by S. agalactiae is neonatal sepsis. The baby can contract the infection during labor when passing through the vaginal canal, or even during pregnancy, if S. agalactiae ascends from the vaginal canal to the uterus. There are several factors associated to the infection of the fetus by S. agalactiae when the mother is colonized, such as the CAMP factor, polysaccharide capsule, hyaluronidase, ß-cytolysin/hemolysin and pili. There is no consensus or technical recommendation regarding this theme in Brazil. According to the Brazilian guidelines to prenatal care, there is no research that justifies the implementation of intrapartum antibiotic therapy. There is a need to clarify genotype characteristics of S. agalactiae strains isolated in Brazil in order to align clinical practices to phenotypical characteristics of this microorganism. This way, the goals of this project are: i) to classify S. agalactiae strains isolated from pregnant and nonpregnant women according to their capsular serotype, using PCR Multiplex, ii) to evaluate the presence and distribution of virulence factors, using PCR and iii) to evaluate their antibiotic resistance profile, using disk-diffusion and D-zone tests. The findings regarding virulence and resistance were compared to serotypes, pregnancy, geographic localization and the site where the sample was isolated. A total of 292 strains from pregnant and nonpregnant women from the cities of São Paulo, São José dos Campos and Rio de Janeiro were analyzed. Serotype Ia was the most prevalent among the strains. In São José dos Campos there was no significate difference in the prevalence of serotypes Ia and V. Serotype V was the most abundant in São Paulo and Rio de Janeiro. Serotype II was most prevalent in nonpregnant women when compared to pregnant women. No resistance to Penicillin nor Vancomycin was found. However, resistance to Cefepime, Erythromycin or Clindamycin was found in around 22% of strains. There were differences among serotypes regarding resistance, virulence genes and site where the strain was isolated. Therefore these differences can reflect into the epidemiologic profile of S. agalactiae infection in regards to geographic localization and pregnancy. The incidence of sepsis caused by S. agalactiae has decrease in the last few decades, however constant monitoring is necessary in order to align clinical practice to the microorganisms phenotypical characteristics

Detection of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in Brazilian mastitic milk goats by multiplex-PCR / Detecção de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em leite caprino mastítico no Brasil, por multiplex-PCR

This study evalueted the prevalence of Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli in milk samples from 257 goats (513 half-udders) and ten bulk tanks, from ten dairy goat farms of São Paulo State, Brazil, by multiplex-PCR. The samples were screened by microbiological culture (gold-standard), and tested by different multiplex-PCR protocols for the detection of each bacterium. A total of 178 half-udders resulted positive by microbiological culture, with coagulase-negative staphylococci (70%), S. aureus (13.5%), S. intermedius (7.9%), and Enterobacteriaceae (4%) the prevalent pathogens. In other way, multiplex-PCR detected 173 pathogens in 151/523 (28.9%; CI95% 25.2-32.9%) milk samples 144/513 (28.1%) half-udders and 7/10 (70%) bulk tanks, with E. coli (86/162, 51.9%) and S. aureus (50/162, 30.9%) the prevalent ones in half-udders, and S. aureus (6/10, 60%) and E. coli (4/5, 36.4%) in bulk tanks. Multiplex-PCR showed a high performance for the detection of three bacteria at a time in mastitic goat milk direct from half-udders or bulk tanks. Thus, this multiplex-PCR protocol proved to be an adequate tool for the identification of the most common mastitis pathogens, independent of their phenotypic characteristics in the diagnosis of clinical mastitis in goats, allowing a continuous and better vigilance and monitoring the herd, being included in quality programs.(AU)

Este estudo avaliou por multiplex-PCR a prevalência de Staphylococcus aureus, Streptococcus agalactiae e Escherichia coli em amostras de leite de 257 caprinos (513 tetos) e dez tanques de expansão, em dez fazendas leiteiras do estado de São Paulo, Brasil. As amostras foram triadas por cultura microbiológica (padrão-uro) e testadas por diferentes protocolos multiplex-PCR para a detecção de cada bactéria. Um total de 178 amostras de leite foram positivos na cultura microbiológica, com estafilococos coagulase-negativos (70%), S. aureus (13,5%), S. intermedius (7,9%) e Enterobacteriaceae (4%) como patógenos prevalentes. Por outro lado, a PCR multiplex detectou 173 patógenos em 151/523 (28,9%, IC95% 25,2-32,9%) amostras de leite, 144/513 (28,1%) amostras de tetos e 7/10 (70%) em tanques de expansão, E. coli (86/162, 51,9%) e S. aureus (50/162, 30,9%) foram identificados nas amostras de tetos e S. aureus (6/10, 60%) e E. coli (4/5, 36,4%) em tanques expansão. Multiplex-PCR mostrou um alto desempenho para a detecção das três bactérias em leite de cabra com mastite ou em tanques de expansão. Dessa forma, este protocolo multiplex-PCR provou ser uma ferramenta adequada para a identificação dos patógenos mais comuns da mastite, independentemente de suas características fenotípicas no diagnóstico de mastite clínica em caprinos, permitindo uma vigilância contínua e melhor acompanhamento do rebanho, sendo incluído em programas de qualidade.(AU)

Polissacarídeo capsular do Streptococcus agalactiae como antígeno vacinal: desenvolvimento de um modelo vacinal para mucosas com Nanopartícula de quitosana / Capsular polysaccharide of Streptococcus agalactiae as vaccine antigen: development of a mucosal vaccine model with chitosan nanoparticle

A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal

Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen

Molecular epidemiology of Streptococcus agalactiae isolated from mastitis in Brazilian dairy herds

Abstract Streptococcus agalactiae is one of the most common pathogens leading to mastitis in dairy herds worldwide; consequently, the pathogen causes major economic losses for affected farmers. In this study, multilocus sequence typing (MLST), genotypic capsular typing by multiplex polymerase chain reaction (PCR), and virulence gene detection were performed to address the molecular epidemiology of 59 bovine (mastitis) S. agalactiae isolates from 36 dairy farms located in the largest milk-producing mesoregions in Brazil (Minas Gerais, São Paulo, Paraná, and Pernambuco). We screened for the virulence genes bac, bca, bibA, cfb, hylB, fbsA, fbsB, PI-1, PI-2a, and PI-2b, which are associated with adhesion, invasion, tissue damage, and/or immune evasion. Furthermore, five capsular types were identified (Ia, Ib, II, III, and IV), and a few isolates were classified as non-typeable (NT). MLST revealed the following eight sequence types (STs): ST-61, ST-67, ST-103, ST-146, ST-226, ST-314, and ST-570, which were clustered in five clonal complexes (CC64, CC67, CC103, CC17, and CC314), and one singleton, ST-91. Among the virulence genes screened in this study, PI-2b, fbsB, cfb, and hylB appear to be the most important during mastitis development in cattle. Collectively, these results establish the molecular epidemiology of S. agalactiae isolated from cows in Brazilian herds. We believe that the data presented here provide a foundation for future research aimed at developing and implementing new preventative and treatment options for mastitis caused by S. agalactiae.

Prevalence of Group B Streptococcus serotypes III and V in pregnant women of Rio de Janeiro, Brazil

GBS serotypes III and V were the most prevalent in pregnant women and exhibited resistance to tetracycline, clindamycin and sulfamethoxazole/trimethoprim. Serotype III showed high sialic acid content and PFGE analysis discerned 33 heterogeneous profiles. Phenotypic and genotypic characterization could be relevant to control GBS infections unaffected by intra-partum chemoprophylaxis.

Taxa de infecção e sorotipos de Streptococcus agalactiae em amostras de recém-nascidos infectados na cidade de Campinas (SP), Brasil / Infection rate and Streptococcus agalactiae serotypes in samples of infected neonates in the city of Campinas (São Paulo), Brazil

OBJETIVO: Descrever os casos e o perfil microbiológico dos sorotipos de Streptococcus agalactiae provenientes de recém-nascidos em um Centro de Referência da Saúde da Mulher na cidade de Campinas, São Paulo, Brasil. MÉTODOS: Estudo transversal clínico-laboratorial realizado de janeiro de 2007 a dezembro de 2011. As cepas suspeitas, isoladas em amostras de sangue e líquor, foram identificadas a partir da hemólise em ágar sangue, coloração de Gram, provas de catalase, teste de CAMP, hidrólise do hipurato ou por automação microbiológica Vitek 2 BioMerieux®. A seguir, estas cepas foram tipadas por PCR utilizando sucessivamente primers específicos para espécie e para nove sorotipos de S. agalactiae. RESULTADOS: Foram isoladas sete amostras de sangue, uma de líquor e uma de secreção ocular provenientes de nove recém-nascidos com infecções causadas pelo S. agalactiae, sendo sete casos de infecção de início precoce e duas de início tardio. Apenas um destes casos foi positivo para amostras pareadas mãe-filho. Para um total de 13.749 partos no período, os 7 casos correspondem a 0,5 caso de infecção precoce por Streptococcus do Grupo B a cada 1 mil nascidos vivos (ou 0,6 casos por 1 mil, incluindo os 2 de infecção tardia), tendo ocorrido 1, 3, 2, nenhum e 3 casos (um precoce e dois tardios), respectivamente, nos anos de 2007 a 2011. Foi possível realizar o PCR para sete amostras, sendo duas de cada um dos sorotipos Ia e V e o sorotipo III em três amostras, uma delas em um recém-nascido e outras duas em amostra pareada mãe-filho. CONCLUSÕES: Embora com casuística limitada, os sorotipos encontrados coincidem com os mais prevalentes na literatura mundial, mas diferem dos estudos brasileiros, exceto para o sorotipo Ia.

PURPOSE: To describe the epidemiological cases and microbiological profile of Streptococcus agalactiae serotypes isolated from infected newborns of a Women's Health Reference Centre of Campinas, São Paulo, Brazil. METHODS: Cross-sectional laboratory survey conducted from January 2007 to December 2011. The newborns' strains, isolated from blood and cerebrospinal fluid samples, were screened by hemolysis on blood ágar plates, Gram stain, catalase test, CAMP test, hippurate hydrolysis or by microbiological automation: Vitek 2 BioMerieux®. They were typed by PCR, successively using specific primers for species and nine serotypes of S. agalactiae. RESULTS: Seven blood samples, one cerebrospinal fluid sample and an ocular sample, were isolated from nine newborns with infections caused by S. agalactiae, including seven cases of early onset and two of late onset. Only one of these cases was positive for paired mother-child samples. Considering that 13,749 deliveries were performed during the study period, the incidence was 0.5 cases of GBS infections of early onset per 1 thousand live births (or 0.6 per 1 thousand, including two cases of late onset) with 1, 3, 2, zero and 3 cases (one early and two late onset cases), respectively, for the years from 2007 to 2011. It was possible to apply PCR to seven of nine samples, two each of serotypes Ia and V and three of serotype III, one from a newborn and the other two from a paired mother-child sample. CONCLUSIONS: Although the sample was limited, the serotypes found are the most prevalent in the literature, but different from the other few Brazilian studies available, except for type Ia.

Caracterización molecular en aislados chilenos de Streptococcus agalactiae / Molecular characterization of Chilean isolates of streptococcus agalactiae

Streptococcus agalactiae is the main causing organism of invasive infections such as sepsis and meningitis in the newborn. Aim: To perform a genotype characterization of Streptococcus agalactiae strains coming form invasive infections of newborns and colonized pregnant women. Material and methods: A group of 58 strains not related epidemiologically isolated from colonized pregnant women and invasive infections in newborns, were studied. Pulsed field electrophoresis (PFGE) and polymerase chain reaction amplification of hylB and IS 1548 genes, as possible virulence markers, were performed. Results: Among the studied strains, 37 genetic subtypes were observed. There were nine groups of identical PFGE patterns. Three corresponded to serotype la and six to serotype III. An erythromycin and clindamycin resistant clone was identified in three colonized women and a newborn with sepsis, which were not epidemiologically related. The hylB gene was equally present in cases of neonatal meningitis or colonized pregnant women. Conclusions: There was a great degree of polymorphism among the studied strains. The ample presence of hylB gene and the absence of the insertion element IS1548 in the hylB gene in invasive and colonizing strains, indicates that both groups of strains are potentially pathogenic.

Phenotypical characteristics of group B streptococcus in parturients

Colonization by Group B Streptococcus (GBS) is highly prevalent among pregnant women, with prevalence rates ranging between 4 percent and 30 percent. The infection may be transmitted vertically and may result in serious neonatal consequences. In the period from November 2003 to May 2004, a cross-sectional study was carried out among 316 parturients at the Jundiaí Teaching Hospital to establish the prevalence of genital GBS colonization, to identify the factors associated with colonization and the characteristic phenotypes of these streptococci. Samples from rectal and vaginal areas were collected for selective culture in Todd-Hewitt broth. Susceptibility to 7 antimicrobial agents was tested using the antibiotic diffusion disk technique, and the isolated strains were classified using specific antisera. The prevalence of GBS colonization was 14.6 percent. No strain was resistant to penicillin, ampicillin, erythromycin or nitrofurantoin. The majority of strains were sensitive to cephalothin. Greatest resistance was to gentamicin (76.1 percent), followed by clindamycin (17.4 percent). The most frequent serotype was Ib (23.9 percent), followed by serotypes II and Ia (19.6 percent and 17.4 percent, respectively). There was no correlation between serotype and greater antimicrobial resistance. In conclusion, the prevalence of GBS in parturients was high and penicillin continues to be the drug of choice for intrapartum prophylaxis. The most frequent serotype (Ib) found in this study differs from those found in the majority of studies carried out in other countries, revealing the need to identify prevalent serotypes in each region so that specific vaccines can be designed.

"Serotyping and antibiotic susceptibility of Group B streptococcal isolates from obstetric patients"

A total of 156 recent distinct isolates of Group B streptococcus [GBS] were recovered from clinical specimens of female patients at Madinah Maternity and Children Hospital and King Khalid University Hospital, Saudi Arabia. Serotyping results revealed that 96 percent of the strains were serotypable and the most frequent serotypes were III, II, and Ia which accounted for 35.26, and 15 percent strains respectively. Whereas serotypes Ib, IV, and V constituted 9, 7, and 4 percent respectively. The minimum inhibitory concentrations [MIC] of antibiotics against GBS isolates were determined by an agar dilution method. All GBS strains were susceptible to 0.125 mg/L of either penicillin, ampicillin, erythromycin, or clindamycin. Whereas at 0.06 mg/L the inhibitory effect of these antibiotics were 80, 69, 45, and 86 percent respectively. Cefoxtin, gentamicin, and tetracycline, however, showed remarkable variation in their activities. The majority of GBS strains showed intermediate susceptibility towards cefoxitin, whereas 35 and 77 percent of the isolates were resistant to gentamicin [GM] and tetracycline [Tc] respectively. The genetic basis of Tc-resistance was investigated was investigated. No plasmids were detected among the randomly selected different GBS sterotypes resistant to Tc. However, in all cases EcoRI- digested chromosomal DNA from resistant strains hybridized with the used tet-gene probe [ATCC 37234] of Kpnl/Hpal 0.39 [Kb] fragment. No differences were detected between GBS strains of various origin. No resistance to penicillin or ampicillin was found, nor was penicillinase production demonstrated. Yet tolerace to penicillin was found among GBS strains as tested by penicillinase-disk diffusion, penicillin-gradient replica plating and quantitative killing curves methods. Data of the latter method showed that 21 strains [14 percent] were tolerant to penicillin. Results suggest that penicillin tolerance is not rare among strains of GBS isolated in our hospitals and its routine testing may be warranted

Correlation of Serotypes and Genotypes of Macrolide-Resistant Streptococcus agalactiae

Despite the necessity for studies of group B streptococci (GBS), due to the increase in serious adult infections, the emergence of new serotypes, and the increased resistance to macrolide antibiotics, such studies have been limited in Korea. The primary purpose of the present study was to determine the frequency trends of GBS serotypes, including serotypes VI, VII, and VIII. The final objective was to elucidate the relationship between the genotypes and serotypes of macrolide-resistant GBS isolates from a Korean population. Among 446 isolates of Streptococcus agalactiae, isolated between January 1990 and December 2002 in Korea, the frequency of serotypes were III (36.5%), Ib (22.0%), V (21.1%), Ia (9.6%), VI (4.3%), II (1.8%), VIII (1.3%), IV (1.1%), and VII (0.9%). The resistance rates to erythromycin, by serotype, were 85% (V), 23% (III), 21% (VI), 3% (Ib), and 2% (Ia). Of 135 erythromycin- resistant S. agalactiae, ermB was detected in 105 isolates, mefA in 20 isolates, and ermTR in seven isolates; most type V isolates harbored the ermB gene, Ib type isolates had an equal distribution of resistance genes, type III isolates accounted for 70% of all isolates carrying mefA genes, and one fourth of type VI isolates had mefA genes.

Distribución de tipo capsular y sensibilidad antimicrobiana de Streptococcusagalactiae productores de infecciones en Argentina / Distribution of capsular types and antimicrobial susceptibility of Streptococcus agalactiae causing infections in Argentina

Streptococcus agalactiae es una bacteria colonizante que ha emergido en los últimos años como causante de infecciones neonatales, perinatales y en pacientes con compromiso inmunológico. La caracterización del polisacárido capsular, de las proteínas de superficie (c, X, R), así como el análisis de marcadores moleculares, permiten su clasificación en serotipos y genotipos. Esto resulta de utilidad para fines epidemiológicos y para estudios de virulencia de la bacteria. El objetivo de este trabajo fue conocer los serotipos prevalentes y la sensibilidad antimicrobiana de aislamientos provenientes de procesos infecciosos en pacientes de distintas zonas de Argentina. En la muestra analizada se obtuvo predominio de los serotipos Ia y III, seguido de II y IV. Todas las cepas resultaron sensibles a penicilina. Se observó 6% de resistencia a eritromicina y 4,5% a clindamicina. En 3 de las cepas se detectó fenotipo MLS (resistencia a macrólidos, lincosaminas y estreptograminas) constitutiva y en una cepa, resistencia MLS inducible. Los resultados logrados en este estudio destacan la importancia de efectuar un relevamiento de los serotipos más frecuentes en nuestro país en vistas a la prevención de esta infección con una vacuna que realmente sea eficaz, como así también el conocimiento de la sensibilidad antimicrobiana para lograr éxito terapéutico en los tratamientos.

Streptococcus agalactiae is an endogenous bacterium that has emerged in the last 20 years as an etiological agent in both neonatal and perinatal infections, and in immunocompromised patients. The differentiation of the capsular polysaccharide, the presence of surface proteins c, X, R, and molecular methods allow classification in serotypes and genotypes. This identification is a useful tool for epidemiological purposes and virulence studies in this bacterium. The objective of this work was to study the serotypes and the antimicrobial susceptibility of isolates recovered from invasive diseases in different areas of Argentina. In the analyzed sample a fair predominance of Ia and III serotypes was recovered, followed by II and IV serotypes. All the isolates were found to be sensitive to penicillin. A 6% of resistance to erythromycin and a 4.5% to clindamycin were detected. In three of the isolates, constitutive MLS phenotype (resistance to macrolides, lincosamins and streptogramins) was founded, while in the remaining one, inducible MLS phenotype was detected. These results stress the importance of conducting a surveillance of the prevalent serotypes in our country with the goal of future prevention of this disease with an effective vaccine. The knowledge of the antimicrobial susceptibility profile will be also important to obtain therapeutic success in the treatment.

Distribution of PFGE types of invasive Norwegian group B streptococci in relation to serotypes.

BACKGROUND & OBJECTIVES: Pulsed-field gel electrophoresis (PFGE) has been used for characterisation of group B streptococci, non-typeable by serotyping. We wanted to compare PFGE with serotyping in order to see the how well the method discriminates between epidemiological unrelated strains. METHODS: A total of 78 epidemiological unrelated invasive GBS strains were examined by PFGE using SmaI digested chromosomal DNA. Of these, 11 were nontypeable (NT) with regard to capsular polysaccharide (CPS) serotype. PFGE patterns were analyzed and classified in a dendrogram. RESULTS: 75 strains were typeable by PFGE, and a total of 62 restriction profiles were identified. At an 85 per cent similarity level, 53 different PFGE patterns were identified. Within each serotype, PFGE patterns differed considerably, the largest degree of heterogeneity observed among type IV, Ia, and II strains. Serotype Ib, III, and V strains were more homogeneous. Strains with identical macrorestriction profiles belonged to the same CPS type, but varied with regard to serosubtypes. Any strain among the ones investigated showing a greater than 88 per cent similarity to a restriction profile in the database, could correctly be ascribed to a particular CPS type. Of the 11 NT strains 10 restriction profiles were found, two of which were identical to the PFGE profile for a cluster of type V strains, and one profile were identical to the profile showed by a cluster of 5 type Ib strains. INTERPRETATION & CONCLUSION: PFGE is a useful technique for classifying strains that are non-typeable by conventional serotyping.

Vaginal & rectal carriage of Streptococcus agalactiae in the Czech Republic: incidence, serotypes distribution & susceptibility to antibiotics.

BACKGROUND & OBJECTIVES: Streptococcus agalactiae (group B streptococcus, GBS) is the predominant bacterial agent responsible for invasive perinatal infection. To obtain reliable data on vaginal and rectal carriage of S. agalactiae in pregnant women in Czech Republic, and to formulate a prevention programme of neonatal GBS disease for the Czech Republic, women at childbirth were screened for vaginal and anorectal carriage of GBS. The isolates were serotyped and tested for susceptibility to antimicrobials including those recommended for intrapartum prophylaxis. METHODS: A total of 586 women at childbirth were screened for GBS carriage in vaginal and anorectal regions using the non-enrichment and selective culture media. The isolates were serotyped by precipitation with antisera raised against various serotypes and antigenic extracts prepared according to Lancefield's modification. Mueller Hinton agar with 5 per cent defibrinated sheep blood was used for antimicrobial susceptibility testing. MIC values were evaluated according to the NCCLS criteria. RESULTS: Using selective media, GBS was detected in 172 (29.3%) of 586 women screened, vaginal and anorectal colonization was found in 21.7 and 24.4 per cent of them, respectively, concomitant vaginal and anorectal colonization was recorded in 16.5 per cent of the women studied. Serotypes III (33.2%), Ia (22.0%) and V (13.9%) prevailed among 172 isolates tested. All isolates were susceptible to penicillin, ampicillin and cefotaxime. The rates of GBS resistance to tetracycline, erythromycin and clindamycin were 83.9, 3.8 and 3.2 per cent, respectively. INTERPRETATION & CONCLUSION: GBS carriage in pregnant women in the Czech Republic is rather high as compared with that reported in literature. The most frequent serotypes III, Ia and V, identified in GBS-colonized pregnant women in the Czech Republic, were among those predominant in the USA and Western Europe. Our findings confirm uniform susceptibility of GBS isolates from pregnant women to penicillin and other beta-lactam antibiotics tested. Resistance to erythromycin remains low in the Czech Republic.

Characterization of vaginal & rectal colonization with multiple serotypes of group B streptococci using multiple colony picks.

BACKGROUND & OBJECTIVES: There is paucity of information on vaginal and rectal colonization with multiple serotypes of group B streptococci (GBS). As part of an ongoing cohort study evaluating the natural history of vaginal and rectal colonization by GBS, the colonization with multiple serotypes was studied in 102 non-pregnant women aged 18-30 yr. METHODS: Up to ten separate colony picks of beta-haemolytic streptococci (total 1515 isolates) were selected from vaginal and rectal primary culture plates. The colonies were identified as GBS, and their capsular polysaccharides (CPS) serotypes determined using monospecific rabbit antisera for types Ia-VIII by double immunodiffusion in agarose (DID). A colony dot immunoblot (DB) assay, using monospecific rabbit antisera to purified type polysaccharides conjugated to tetanus toxoid, was developed to serotype efficiently the multiple colony picks of GBS. RESULTS: The CPS serotype distribution, examining only the 177 "a" or first colony picks from the 102 patients, was 30.5 per cent for Ia; 28.2 per cent for type III; 15.3 per cent for type II; and 13.6 per cent for type V. Only 2.8 per cent were nontypeable. Eighty of the 102 patients (78.4%) were colonized with only one serotype; 20 (19.6%) had two serotypes and two patients (2%) had three serotypes in their vaginal and/or rectal paired cultures. Overall, 91.9 per cent of the culture sites colonized with one to three CPS types (from the total number of colonies picked) were identified with a minimum of three colony picks. In 75 patients with vaginal/rectal pairs the GBS serotype concordance of only the "a" colony was 89.3 per cent and concordance decreased to 80 per cent when the serotype concordance of the total colony picks was analyzed. INTERPRETATION & CONCLUSION: In conclusion, there was a relatively high prevalence of serotype nonconcordance in this population, and 21.6 per cent of patients had multiple GBS serotypes.

Bacteremia due to beta haemolytic streptococci.

The Beta haemloytic streptococci (BHS) are well recognised human pathogens causing a variety of infections, including septicemia. It is important to ensure their isolation from clinical specimens by using optimum media. Moreover, since the different groups have different pathogenic potential, it is equally important to routinely serogroup them; this is emphasized here. Since, BHS are uniformly will greatly decrease morbidity and mortality due to BHS infection.

Changing patterns of group B streptococcal serotypes associated with human infections.

One hundred strains of group B streptococci (GBS) isolated from diverse clinical specimens of patients seen in our hospital were subjected to serotyping by an indigenously prepared coagglutination system. Serotype NT/c was the most predominant (24%), followed by Ia (23%), II/c (12%) and Ib (11%). Type Ia was the most predominant in all specimens except genital samples where NT/c was the most predominant. Comparison of the distribution of serotypes identified during 1975-78 with that of the present study showed a great increase in the prevalence of types NT/c, II/c and Ia and a dramatic decline of types III, Ia/c and Ib. Despite the inclusion of reagents for newer serotypes, IV and V two strains were nontypable indicating the prevalence of hitherto unidentified GBS serotypes in our community.

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides

Epidemioiogicai studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemioiogically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemioiogically-unrelated isolates (ciassified into 5 different serotypes) resulted in ll distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.

Trend of isolation and serotypes of group B streptococci in Korea

Group B streptococci (GBS) neonatal infection, a prevalent disease in western countries, is considered rare in Korea. GBS neonatal infection is known to be often due to serotype III organisms, but the serotypes in Korea have not been reported. In this study, GBS were frequently isolated from specimens of genitalia, urine and various pus. Among the 186 isolates 14 (7.5%) were from neonates, two with concomitant bacteremia and meningitis and one with pneumonia. Frequently isolated GBS serotypes were Ib (9.2%), Ib/c (26.6%) and III/R (23.9%). Change of frequently isolated serotypes during the study was noted, but JM9 which became increasingly isolated in Japan was not found. It is concluded that less prevalence of severe neonatal GBS infection in Korea is not due to the absence of serotype III, but possibly due to low genital carriage rate of GBS by pregnant women.

Spot CAMP test for the prompt presumptive identification of group B streptococci.

Spot and conventional CAMP tests done on 200 strains (120 human and 80 bovine) of group B streptococci showed 96 per cent of human and 90 per cent of bovine strains positive by both tests, indicating 100 per cent agreement. None of the 20 non-group B streptococci strains was positive for CAMP test for group B streptococci. The advantage of spot CAMP test is that it can be performed on a single isolated colony, easy to perform, inexpensive and can presumptively identify group-B streptococci within 30 min on primary sheep blood agar plate.