Sensibilidad a trimetoprima-sulfametoxazol de Streptococcus pyogenes aislados de infecciones invasivas / Susceptibility of Streptococcus pyogenes isolated from invasive infections to trimethoprim-sulfamethoxazole

Se cree erróneamente que los estreptococos del grupo A (EGA) son universalmente resistentes a trimetoprima-sulfametoxazol (TMS). Esto se debe a que la timidina presente en los medios habitualmente usados para determinar sensibilidad in vitro a antibióticos antagoniza el efecto antibiótico de TMS. El objetivo de este trabajo fue determinar la sensibilidad de EGA a TMS, en presencia y ausencia de timidina. A tal fin, fueron analizados 95 aislamientos clínicos obtenidos de tejidos normalmente estériles con infección invasiva por EGA. La pruebas de sensibilidad por difusión con discos de TMS fueron realizadas en agar Mueller Hinton adicionado ya sea con 5% de sangre de carnero (MH-SC) o con 5% de sangre equina lisada (MH-SEL). La sangre equina lisada contiene timidina fosforilasa, que degrada este nucleósido. Como método de referencia se utilizó la epsilometría (Etest). El control de calidad con la cepa Enterococcus faecalis ATCC 29212 fue satisfactorio para ambos medios. La sensibilidad a TMS por difusión fue 100% en MH-SEL; en agar MH-SC 6 (6.3%) aislamientos resultaron resistentes; por Etest todos fueron sensibles, excepto uno de esos seis que presentó sensibilidad intermedia (CIM = 1.5/28.5 μg/ml). En este aislamiento no se encontraron las mutaciones genéticas de EGA más frecuentemente asociadas a resistencia a TMS. Probablemente, si se establecieran mejores puntos de corte para difusión, específicos para EGA, podría optimizarse la correlación con métodos de dilución o con Etest, aun empleando MH-SC.

It is erroneously believed that group A streptococci (GAS) are universally resistant to trimethoprim-sulfamethoxazole (TMS). This is mainly because media commonly used for in vitro determination of susceptibility to antibiotics contain thymidine, a nucleoside that antagonizes the antibiotic effect of TMS. The objective of this work was to determine EGA sensitivity to TMS in the presence and absence of thymidine. To this aim, 95 GAS isolates obtained from clinical tissues with i nvasive infections were analyzed. Susceptibility tests were performed by diffusion with TMS discs in Mueller Hinton agar supplemented either with 5% sheep blood or with 5% lysed equine blood (MH-LEB). Lysed equine blood contains thymidine phosphorylase, which degrades this nucleoside. Epsilometry (Etest) was used as gold standard. Quality controls with Enterococcus faecalis strain ATCC 29212 were satisfactory with both media. A 100% sensitivity to TMS was found in MH-SEL whereas 6 isolates (6.3%) resulted resistant in MH-SC; only one of them was found to have intermediate susceptibility by Etest (MIC > 1.5/28 μg/ml). The genetic determinants most frequently associated to TMS resistant EGA were not found in this isolate. Probably, if more accurate GAS-specific cut-off points were established for diffusion, the correlation with dilution methods or with the Etest could be improved, even employing MH-SB.

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.

Experimentally induced anachoresis in the periapical region after root canal filling / Anacoresis inducida experimentalmente en la región periapical después de obturación del conducto radicular

Anachoresis is the phenomenon through which blood-borne bacteria, dyes, pigments and other materials are attracted and fixed to circumscribed areas of inflammation. This study evaluated the occurrence of anachoresis in the periapical region of dogs submitted to root canal fillings. One hundred and four roots from four dogs were endodontically treated and root canals were filled with zinc-oxide-eugenol cement. Fifty percent were filled up to the dentinocemental junction and the others were overfilled. At 120 days after root canal treatment, experimental bacteremia was induced by intravenous inoculation of 105 CFU Streptococcus pyogenes. The dogs were sacrificed 48 hours and 30 days after the bacteremia. Culture and DNA amplification by PCR revealed the presence of the inoculated bacteria just in periapical tissues of dogs sacrificed 48 hours after bacteremia and not in animals sacrificed after 30 days. AP-PCR fingerprints of recovered colonies of S. pyogenes and the presence of genetic markers of resistance to antimicrobials were similar to the inoculated strain. Endodontically treated periapices seemed to be prone to the occurrence of anachoresis and there was no relationship between the phenomenon and the level of root canal filling.

Anacoresis es el fenómeno por el cual las bacterias transmitidas por la sangre, colorantes, pigmentos y otros materiales se atraen y se fija a zonas circunscritas de la inflamación. Este estudio evaluó la incidencia de anacoresis en la región periapical de los perros presentados a raíz de los rellenos del canal. Un total de ciento cuatro raíces de cuatro perros fueron tratados con endodoncia y tratamientos de conducto se rellena con cemento de óxido de zinc-eugenol. El cincuenta por ciento estaban llenos hasta el cruce dentinocemental y los otros se llene en exceso. A los 120 días después del tratamiento de conducto radicular, bacteriemia experimental fue inducida por la inoculación intravenosa de 105 UFC por Streptococcus pyogenes. Los perros fueron sacrificados 48 horas y 30 días después de la bacteriemia. La cultura y la amplificación del ADN por PCR reveló la presencia de las bacterias inoculadas sólo en los tejidos periapicales de los perros sacrificados 48 horas después de la bacteriemia y no en los animales sacrificados después de 30 días. AP-PCR huellas dactilares de las colonias recuperadas de S. pyogenes y la presencia de marcadores genéticos de resistencia a los antimicrobianos fueron similares a la cepa inoculada. Periápices endodonciados parecía ser propensos a la ocurrencia de anacoresis y no había ninguna relación entre el fenómeno y el nivel de llenado del conducto radicular.

Análisis de los fenotipos y genotipos de resistencia a eritromicina y clindamicina en cepas de Streptococcus pyogenes aisladas en Chile en un período de 10 años / Resistance phenotypes and genotypes of Streptococcus pyogenes clinical isolates in Chile over a 10-year period

Background: Macrolide and lincosamide resistance in Streptococcus pyogenes is due to the acquisition of mef, ermB and ermA genes, which confer different resistance phenotypes, namely M, MLSBconstitutive and MLSBinducible respectively. The last report of resistance in Chile was done in the period 1990-1998, in which resistance to macrolides was 5.4 percent, with M phenotype as the predominant one. Aim: To characterize the evolution of erythromycin and clindamycin resistance and their associated genes in S. pyogenes strains isolated from patients with invasive and non-invasive infections in the period 1996 to 2005. Material and Methods: Resistance to erythromycin and clindamycin was determined in 1,282 clinical isolates using the disk diffusion test. Resistant isolates were analyzed by polymerase chain reaction (PCR) for the presence of the above mentioned resistance genes. Results: Global resistance to erythromycin and clindamycin was 3.5 and 0.7 percent respectively. Eighty percent of the resistant strains possessed the M. phenotype. Conclusions: Resistance levels of S. pyogenes have decreased in Chile in the last years. Most resistant strains have M phenotype in contrast to many countries in which the MLSB constitutive phenotype is the predominant one.

Group A Sstreptococcus virulence factors genes in north India & their association with emm type in pharyngitis.

Background & objectives: Group A streptococcal (GAS) pharyngitis, especially among children, leads to high prevalence of rheumatic fever (RF)/rheumatic heart disease (RHD) in India, as compared to the western world where invasive diseases are common. GAS encodes numerous virulence factors that cause diseases by exhibiting extraordinary biological diversity. Hence, we studied the virulence factors genes of GAS isolated from the throat of children with pharyngitis and also asymptomatic carriers. Methods: Fifty GAS isolates cultured from throats of north Indian children aged 5-15 yr with mild pharyngitis (20), severe pharyngitis (24) and asymptomatic pharyngeal carriers (6), during 2000-2003 along with reference M1 strain were emm typed and characterized for virulence factors genes by PCR. The presence of virulence factors was also checked for their association with emm type in pharyngitis. Results: Twenty emm types, six sequence types, and one non-typeable strain were found circulating in north India. The five most prevalent types were emm 74 (12%), 11 & StI129 (8% each) and emm 68 and NS292 (6% each). The spe B gene was found to be significantly higher (P=0.0007) in opacity factor (OF) negative isolates. emm 3, 11, 77, 86, 87, 109 and StI129 showed maximum virulence factors genes. Interpretation & conclusions: GAS isolates collected from throats of children from north India possess highly virulent antigens. This study also supports concept of isolate-associated virulence rather than type relatedness.

Interpersonal relationships and group A streptococcus spread in a Mexican day-care center

OBJECTIVE: To study the effect of different degrees of centrality on the carrying of identical group A streptococcus (GAS) clones in the nasopharynx of children from a Mexican public day-care center. MATERIAL AND METHODS: Nasopharyngeal cultures were performed in children from rooms B (RB) (n = 35) and C (RC) (n = 37). The Restriction Fragment Length Polymorphism (RFLP) patterns were compared among GAS isolates. A social networks questionnaire was filled out for each child and 10 classmates. Structure coefficients were compared among children with and without GAS. RESULTS: Four GAS clones were identified; clone I in five children from RC; clone II in two from RC and one from RB; clone III in one from RB and one from RC; and clone IV in one from RC. Social network structure: Density of RB and RC = 0.40 (± 0.87) and 0.35 (± 0.80), respectively. In RB, the homophily pattern of interaction was different in carriers (0.00), non-carriers (0.47) and both (0.47) p = 0.35. In RC, the homophily pattern was also different in carriers (0.46), non-carriers (0.68) and mixed (0.19), p = .001. In 4/5 with clone I, the values of degree, closeness and betweenness were above the group mean. In 3/3 with clone II, the values of degree and betweenness were also above the mean. In contrast, in those with clone III and IV, the values of degree, closeness and betweenness were below the group mean. CONCLUSION: The spread of specific GAS clones was associated with groups of children having a high proportion of ties and a high centrality level. This is evidence that spread of GAS strains among children attending day-care centers is not random but dependent on the degree of communication and physical contact between pairs.

OBJETIVO: Evaluar el efecto de grados diferentes de centralidad con la presencia de clonas idénticas de estreptococo del grupo A (EGA) en la nasofaringe de niños de una guardería pública de México. MATERIAL Y MÉTODOS: Se realizaron cultivos nasofaríngeos en niños de los salones B (SB) (n = 35) y C (SC) (n = 37). El patrón de polimorfismos de longitud de fragmento por restricción (Restriction Fragment Length Polymorphism, RFLP) fue comparado entre aislamientos de EGA. Un cuestionario de redes sociales fue llenado para cada niño y 10 compañeros. Los coeficientes de estructura fueron comparados entre niños con y sin EGA. RESULTADOS: Se identificaron cuatro clonas de EGA. Clona I en cinco niños del SC; clona II en dos del SC y en uno del SB; clona III en uno del SB y uno del SC; y clona IV en uno del SC. Estructura de redes sociales: Densidad SB y SC = 0.40 (± 0.87) y 0.35 (± 0.80), respectivamente. En SB, el patrón de homofilia de la interacción fue distinto en portadores (0.00), no portadores (0.47) y ambos (0.47) p = 0.35. En SC, el patrón de homofilia fue distinto en portadores (0.46), no portadores (0.68) y mixto (0.19), p = .001. En 4/5 con la clona I, los valores de grado, cercanía e intermediación estuvieron por arriba de la media grupal. En 3/3 con la clona II, los valores de grado e intermediación estuvieron por arriba de la media grupal. En contraste, en los niños con clonas III y IV, los valores de grado, cercanía e intermediación estuvieron por debajo de la media grupal. CONCLUSIONES: La diseminación de clonas específicas de EGA se asoció a grupos de niños con gran proporción de lazos entre ellos y un alto nivel de centralidad. Esto evidencia que la transmisión de EGA entre niños de guardería no ocurre al azar sino que depende del grado de comunicación y contacto físico entre éstos.

Genetic Diversity and Exotoxin A Production of Group A Streptococci Causing Sepsis

The M protein and streptococcus pyrogenic exotoxin (SPE A) are important virulence factors in group A streptococci (GAS) infections. The emm types of GAS strains isolated from patients with sepsis were determined by sequencing the 5' N-terminus of the emm gene, encoding the M protein, and clonality analysis using pulsed-field gel electrophoresis. The presence of speA and production of SPE A were also examined. There were no predominant GAS clones. The emm genotypes were variable, and the most common genotype was emm13 (17.9%). The production prevalence of SPE A was 21.4%. The low mortality rate (7.1%) of GAS sepsis might be attributable to the low incidence of virulent strains such as emm1 (10.7%) and emm3 (7.1%), as well as to low production rate of SPE A.

Emm types of invasive group A streptococcal isolates from Thai patients at King Chulalongkorn Memorial Hospital from 1995-1999.

Emm (M protein gene) typing was used to analyze group A streptococcal (GAS) clinical isolates in Thailand from in-patients at Chulalongkorn University Hospital (CUH) between January 1, 1995 and December 31, 1999. Forty GAS isolates were recovered from blood and other sterile sites from 40 patients presenting with different types of infections. A variety of emm sequences (24 types) have been reported in this study including the identification of 2 novel emm variants demonstrating the diverse population of invasive GAS isolates in Thailand. The common emm types include emm1 (5 isolates, 12.5%), emm22 (4 isolates, 10%), emm25 (3 isolates, 7.5%), emm61 (3 isolates, 7.5%), and STNS1033 (3 isolates, 7.5%). No particular emm types of GAS tend to be frequently associated with specific clinical presentation, complication, or anatomic site of infection. This report provides epidemiological information from Thailand where streptococcal infections and their sequelae are important public health problems. In addition, the results are useful for the development of a suitable M protein based vaccine in the future.

A half-century of streptococcal research: then & now.

Research on Group A streptococci (GAS) before 1950 paved the way for successful clinical trials to prevent acute rheumatic fever (ARF) by treating the prior streptococcal infection with penicillin. Prevention of ARF has led to almost complete disappearance of rheumatic heart disease in the industrialized world, but has yet to be accomplished in developing countries, where most of the world's populations reside. Twenty years of research beginning in 1918 by Lancefield and others delineated the modern classification of haemolytic streptococci and led to the recognition that only Group A is responsible for the pharyngitis that causes ARF. M-protein, identified as a major virulence factor, is a powerful inhibitor of phagocytosis, and antibodies to it promote type-specific phagocytosis and therefore type-specific immunity. Other virulent properties of GAS include a bulky capsule, as well as extracellular toxins such as streptolysins S and O and streptococcal proteinase. McCarty and others pursued the cell biology of GAS and identified the cellular localization of various antigenic components. The discovery of purified M-protein as a helical coiled-coiled fibrillar protein has sparked development of M-protein vaccine. US, UK, and Trinidad scientists described differences between streptococcal infections of the throat and skin and noted particularly that many of the GAS M-types that cause impetigo are less likely to cause pharyngitis. GAS impetigo may cause acute glomerulonephritis, but such infections do not result in ARF. The changing manifestations of disease over time and the evolution of microbes are common themes in medicine today. These themes are relevant to GAS pharyngitis and ARF, especially the decline in the incidence of severe ARF and the decrease in severity of GAS pharyngitis. Research on GAS bacteriophages led to the discovery of a relationship between lysogenic GAS and production of erythrogenic toxin and has broadened approaches to the molecular epidemiology of GAS virulence. The 21st century begins with determination of the complete genome sequence of M-1, M-18, and M-3 strains of GAS. These studies provide evidence for phage-encoded toxins, high-virulence phenotypes, and clone emergence. This research will reveal genetic processes at the molecular level that control the emergence and decline of streptococcal diseases in different places and times and the shifting patterns in clinical manifestations.

Detección de los genes de las exotoxinas pirogénicas SpeA, SpeB y SpeC en cepas chilenas de streptococcus pyogenes y su asociación con la clínica / Detection of pyrogenic exotoxin SpeA, SpeB and SpeC genes in chilean streptococci isolates and their association with clinical manifestations

Background: The virulence of Streptococcus pyogenes is determined by a variety of structural molecules, toxins and complex enzymes. Pyrogenic exotoxins cause fever, erythematous reactions, cytotoxic and immunological effects. Aim: To assess the frequency of speA, speB and speC genes in Chilean Streptococcus pyogenes strains and their association with the invasiveness of infections. Material and methods: The genes for pyrogenic exotoxins SpeA, SpeB and SpeC were determined by polymerase chain reactions in 114 strains of group A Streptococcus pyogenes isolated from Chilean patients with invasive or non invasive infections. Results: The gene for SpeA was present in 30.7 percent of isolates, the gene for SpeB was present in 69.3 percent and the gen for SpeC in 44.7 percent of isolates. The gene for SpeA was present in 20 of 33 invasive infections and in 15 of 81 non invasive infections (p <0.0001). On the contrary, the gene for SpeC was present in 11 of 33 invasive infections and in 41 of 81 non invasive infections (p <0.05). The frequency of speB was similar in invasive and non invasive infections. Conclusions: There is a clear relationship between the presence of SpeA genes and the severity of infections caused by Streptococcus pyogenes

Fatal group a streptococcal toxic shock-like syndrome in a child with varicella: report of the first well documented case with detection of the genetic sequences that code for exotoxins SPE A and B, in Sao Paulo, Brazil

Crianca de sete anos, previamente higida, foi admitida na unidade de terapia intensiva por quadro de toxemia associado a varicela. Evoluiu rapidamente para choque e insuficiencia de multiplos orgaos e sistemas e, apesar do tratamento intensivo, morreu no quarto dia apos a admissao. A cultura de secrecao colhida por puncao profunda de partes moles em regiao toracica foi positiva para Streptococcus pyogenes, proteina-M nao tipavel e carreador dos genes codificadores da producao de exotoxinas pirogenicas estreptococicas A e B, preenchendo os criterios para definicao de Sindrome do choque toxico estreptococico. Os autores discutem aspectos clinicos e fisiopatologicos desta sindrome, bem como alguns aspectos incomuns relacionados a este caso