Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Caracterización molecular de cepas invasivas de Streptococcus dysgalactiae subsp. equisimilis: estudio multicéntrico Argentina 2011-2012 / Molecular characterization of invasive Streptococcus dysgalactiae subsp. equisimilis. Multicenter study: Argentina 2011-2012

Streptococcus dysgalactiae subsp. equisimilis (SDSE) has virulence factors similar to those of Streptococcus pyogenes. Therefore, it causes pharyngitis and severe infections indistinguishable from those caused by the classic pathogen. The objectives of this study were: to know the prevalence of SDSE invasive infections in Argentina, to study the genetic diversity, to determine the presence of virulence genes, to study antibiotic susceptibility and to detect antibiotic resistance genes. Conventional methods of identification were used. Antibiotic susceptibility was determined by the disk diffusion and the agar dilution methods and the E-test. Twenty eight centers from 16 Argentinean cities participated in the study. Twenty three isolates (16 group G and 7 group C) were obtained between July 1 2011 and June 30 2012. Two adult patients died (8.7%). Most of the isolates were recovered from blood (60.9%). All isolates carried speJ and ssa genes. stG62647, stG653 and stG840 were the most frequent emm types. Nineteen different PFGE patterns were detected. All isolates were susceptible to penicillin and levofloxacin, 6 (26.1%) showed resistance or reduced susceptibility to erythromycin --#91;1 mef(A), 3 erm(TR), 1 mef(A) + erm(TR) and 1 erm(TR) + erm(B)--#93; and 7 (30.4%) were resistant or exhibited reduced susceptibility to tetracycline --#91;2 tet(M), 5 tet(M) + tet(O)--#93;. The prevalence in Argentina was of at least 23 invasive infections by SDSE. A wide genetic diversity was observed. All isolates carried speJ and ssa genes. Similarly to other studies, macrolide resistance (26.1%) was mainly associated to the MLS B phenotype.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) posee factores de virulencia similares a Streptococcus pyogenes y, en consecuencia, produce faringitis e infecciones graves indistinguibles de las generadas por este patógeno clásico. Los objetivos del estudio fueron conocer la prevalencia de SDSE en infecciones invasivas en Argentina, estudiar su diversidad genética, determinar la presencia de genes de virulencia, ensayar su sensibilidad a los antibióticos y conocer los genes de resistencia. Se emplearon métodos convencionales de identificación. La sensibilidad se determinó por difusión, Etest y dilución en agar. Participaron 28 centros de 16 ciudades argentinas. Se obtuvieron 23 aislamientos (16 del grupo G y 7 del grupo C) desde el 1-7-2011 hasta el 30-6-2012. Se registraron 2 muertes en adultos (8,7%). La mayoría de los aislamientos fueron obtenidos de sangre (60,9%). Todos eran portadores de los genes speJ y ssa. Los genotipos más frecuentes fueron stG62647, stG653 y stG840. Se detectaron 19 pulsotipos distintos. Todos los aislamientos fueron sensibles a penicilina y levofloxacina, 6 (26,1%) presentaron resistencia o sensibilidad disminuida a eritromicina (1 mef--#91;A--#93;, 3 erm--#91;TR--#93;, 1 mef--#91;A--#93; + erm--#91;TR--#93; y 1 erm--#91;TR--#93; + erm--#91;B--#93;) y 7 (30,4%) fueron resistentes o tuvieron sensibilidad disminuida a tetraciclina (2 tet--#91;M--#93;, 5 tet--#91;M--#93; + tet--#91;O--#93;). La prevalencia anual en la Argentina fue de al menos 23 infecciones invasivas por SDSE y se observó una amplia diversidad genética. Todos los aislamientos presentaron los genes ssa y speJ. Como en otros estudios, la resistencia a macrólidos (26,1%) estuvo asociada, principalmente, al fenotipo MLS B.

Genotipificación y estudio de los genes pauA y sua de aislamientos de Streptococcus uberis de mastitis bovina / Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6 % concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8 % (134/137) and 94.9 % (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95 % and 100 % with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis.

Este estudio pretendió determinar la relación clonal entre 137 aislamientos de S. uberis obtenidos de leche de bovinos con mastitis clínica o subclínica en la Argentina, como así también la prevalencia y la conservación de los genes sua y PauA entre dichos aislamientos. Esta información es crítica para el diseño racional de una vacuna que prevenga la mastitis bovina por S. uberis. Los aislamientos se tipificaron molecularmente por amplificación al azar del ADN polimórfico (RAPD) y mediante electroforesis de campos pulsados (PFGE). Los 137 aislamientos mostraron 61 pulsotipos mediante PFGE y 25 tipos de RAPD diferentes. Los índices de Simpson calculados fueron 0,983 por PFGE y 0,941 por RAPD; esto evidencia el elevado poder discriminatorio de ambas técnicas. El análisis de la relación entre pares de aislamientos mostró un 92,6 % de concordancia entre ambas técnicas, lo que indica que cualquier par de aislamientos que fue distinguido por un método tendió a ser distinguido por el otro. La prevalencia de los genes sua y puaA fue del 97,8 % (134/137) y 94,9 % (130/137), respectivamente. Las secuencias de nucleótidos y de aminoácidos codificados por los genes sua y pauA de los 20 aislamientos de S. uberis seleccionados sobre la base de su tipo de PFGE y RAPD y origen geográfico tuvieron un porcentaje de identidad de entre 95 % y 100 % con respecto a todas las secuencias de referencia registradas en GenBank. Estos resultados demuestran que, a pesar de la diversidad clonal de S. uberis, los genes sua y pauA son prevalentes y están altamente conservados y deberían ser incluidos en futuros estudios de vacunas para prevenir mastitis bovina causada por S. uberis.

The First Case of Concurrent Infective Endocarditis and Spondylitis Caused by Streptococcus tigurinus

No abstract available.

Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

Resistencia a los antibacterianos en América Latina: consecuencias para la infectología / Antibacterial drug resistance in Latin America: consequences for infectiousdisease control

Antibacterial drug resistance is a particularly significant issue in Latin America. This article explores antimicrobial resistance in three classes of clinically important bacteria: gram-positive bacteria, enterobacteria, and nonfermenting gram-negativebacilli. The gram-positive bacteria frequently responsible for infections in humans are for the most part cocci: staphylococci, streptococci (including pneumococci), and enterococci,in both community and hospital settings. This situation is no different in theRegion of the Americas. Among the gram-positive bacteria, the causative agents of bacteremia are most commonly strains of coagulase-negative Staphylococcus, followed by enterococci. This report explores the resistance of these species to different antimicrobial drugs, resistance mechanisms in community and hospital strains, and new drugs for treating infections caused by these bacteria. In Latin America, antimicrobialresistance in Enterococcus strains is still a minor problem compared to the situation in the United States. The strains of the genus Streptococcus isolated from respiratory infections are still sensitive to penicillin. Furthermore, the resistance of enterobacteriais extremely important in the Region, particularly because of the broad dissemination of CTX-M extended-spectrum beta-lactamases (ESBL), some of which originated in Latin America. This article analyzes the resistance of Streptococcus pneumoniae, betahemolytic streptococci, and viridans group streptococci. Among the nonfermentinggram-negative bacilli, while Pseudomonas aeruginosa strains remain the leading cause of bacteremia, infections caused by strains of Acinetobacter spp. have proliferatedextensively in some areas. With regard to antibiotics, several options are available for treating gram-positive bacterial infections...

La resistencia a los fármacos antibacterianos tiene particular importancia en América Latina. En este artículo se analiza la resistencia a los antimicrobianos de tres clases de bacterias de importancia clínica: bacterias grampositivas, enterobacterias y bacilos gramnegativos no fermentadores.Las bacterias grampositivas que producen infecciones humanas frecuentes son, en su mayoría, cocos: estafilococos, estreptococos (incluidos neumococos) y enterococos, tanto en elmedio comunitario como en el nosocomial. Esta situación no es diferente en la Región de las Américas. Entre las bacterias grampositivas, las que causan bacteriemia con mayor frecuencia corresponden a cepas de estafilococos coagulasa negativos, seguidas de las de enterococos. Eneste informe se analiza la resistencia de estas especies a distintos antimicrobianos, los mecanismosde resistencia para las cepas de origen hospitalario y comunitario y los nuevos medicamentos para tratar las infecciones por estas bacterias. La resistencia a los antimicrobianos delas cepas de Enterococcus en América Latina todavía es un problema menor en relación con la situación en los Estados Unidos de América. Las cepas del género Streptococcus aisladasde infecciones respiratorias aún son sensibles a penicilina. Por otra parte, la resistencia de las enterobacterias es de gran importancia en la Región, particularmente por la gran difusión debetalactamasas de espectro extendido (BLEE) de tipo CTX-M, algunas de las cuales se originaron en América Latina. En el presente artículo se analizan la situación de la resistencia de las cepas de Streptococcus pneumoniae, y de los estreptococos betahemolítico y del grupo viridans. Entre los bacilos gramnegativos no fermentadores, si bien las cepas de Pseudomonasaeruginosa siguen siendo la causa principal de bacteriemias, la proliferación de infecciones por cepas de Acinetobacter spp. tiene en algunas partes gran magnitud...

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM) cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml) collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF), hyaluronidase (HYA), capsule (CAP) and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE). S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively), but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.

Caracterización fenotípica y genotípica de Streptococcus uberis aislados de mastitis bovina subclínica en tambos de Argentina. El objetivo de este estudio fue investigar las características fenotípicas y genotípicas de Streptococcus uberis aislados de casos de mastitis subclínica (MSC) y examinar la posible asociación entre ambas características. Un total de 32 cepas de S. uberis fueron aisladas de 772 muestras de leche de cuartos mamarios (MSC > 25 0000 células/ml) colectadas de 195 vacas seleccionadas al azar pertenecientes a 18 tambos lecheros localizados en Argentina. Las cepas de S. uberis fueron caracterizadas fenotípicamente sobre la base de la presencia de factores de virulencia tales como el factor activador del plasminógeno (FAP), la hialuronidasa (HIA), la cápsula (CAP) y el factor CAMP. Además, fueron caracterizadas genotípicamente por electroforesis de campos pulsados (PFGE). Las cepas de S. uberis expresaron el factor activador del plasminógeno, la hialuronidasa o la cápsula (65,5 %, 56,3 % y 59,4 %, respectivamente), pero solo el 25 % fueron CAMP positivas. Sobre la base de la combinación de los factores de virulencia se identificaron 13 perfiles de virulencia diferentes. Asimismo, se identificaron 18 patrones de PFGE con un 90 % de similitud entre las 32 cepas de S. uberis. Se presentó una gran diversidad de perfiles de virulencia y patrones de PFGE entre los tambos. Cepas con el mismo patrón de PFGE presentaron perfiles de virulencia diferentes. Las cepas de S. uberis causantes de MSC en bovinos incluidas en el presente estudio mostraron heterogeneidad con respecto a sus características fenotípicas y genotípicas, y los patrones de PFGE no estuvieron asociados con los perfiles de virulencia.

Detection of Streptococcus dysgalactiae subsp. equisimilis in equine nasopharyngeal swabs by PCR

Streptococcus (S.) dysgalactiae subsp. equisimilis is responsible for severe diseases in humans, including primary bacteraemia, pneumonia, endocarditis, and toxic shock syndrome. Infection in some animal species can also occur, although a few studies have looked into cross-species infectivity. In horses, S. equisimilis is generally considered infrequent or opportunistic, but has recently been isolated from cases of strangles-like disease. Rapid and sensitive diagnostic techniques could enable epidemiological studies and effective investigation of outbreaks involving these bacteria. In this study, PCR protocols previously described in cattle and in humans to detect the species S. dysgalactiae and the subspecies equisimilis were evaluated to detect specific sequences in equine samples. For this purpose, 99 monolateral nasal swabs were collected from horses from stud farms with a history of S. equisimilis infection and were tested blindly by bacteriological isolation and by single and duplex PCR. DNA for PCR was extracted both from the colonies grown on agar media and from enrichment broth aliquots after incubation with nasal swab samples. S. equisimilis was identified by bacteriological isolation in 23 out of 99 swab samples, and PCR assays on these colonies were fully concordant with bacteriological identification (kappa statistic = 1.00). In addition, PCR of the enrichment broth aliquots confirmed the bacteriological results and detected S. equisimilis in 6 samples more than the bacteriological examination (kappa statistic = 0.84). The PCR protocols appeared to be reliable for the rapid identification of S. equisimilis in equine nasal swab samples, and could be useful for microbiological diagnosis.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations / Vírus influenza e bactéria proteolítica co-infectantes em trato respiratório de indivíduos com manifestações respiratórias

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09 percent) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64 percent) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13 percent) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51 percent) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 ºC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

O papel da bactéria proteolítica na exacerbação do vírus influenza tem sido demonstrado em hospedeiros naturais como porcos e humanos. Foram coletadas 407 amostras do trato respiratório de indivíduos apresentando manifestações clínicas, durante a estação da influenza (2003-2005) na cidade de São Paulo. Este trabalho teve como objetivo avaliar a incidência de determinadas bactérias que junto com vírus co-infectarem o trato respiratório humano. Testes bacteriológicos, e virológicos como imunofluorescência (IF), RT/PCR e hemaglutinação (HA) foram usados nas investigações viral e bacteriana. Pelo teste de IF foram selecionadas trinta e sete (9,09 por cento) amostras positivas para o vírus influenza. A presença do vírus influenza foi confirmada pela técnica de RT/PCR. Pelos testes bacteriológicos e do agar caseina, verificou-se que 18 (48,64 por cento) dos indivíduos foram infectados com bactérias proteolíticas tais como Staphylococcus spp., Streptococcus spp. e Pseudomonas spp. Destas amostras, 13 (35,13 por cento) foram co-infectadas com vírus influenza tipo A, e 5 (13,51 por cento) com influenza tipo B. No experimento in vitro com influenza e S. aureus, detectou-se aumento do título hemaglutinante deste vírus, após contacto de 30 min a 25 ºC. Os resultados obtidos revelaram a ocorrência de co-infecção com bactéria proteolítica e vírus influenza nos indivíduos avaliados. Estes achados corroboram com a investigação do sinergismo, entre bactéria e vírus, que poderia ser capaz de potencializar infecção respiratória, aumentando os riscos aos hospedeiros.

Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island

Streptococcus sp. is gram-positive coccus that causes streptococcal infections in fish due to intensification of aquaculture and caused significant economic losses in fish farm industry. A streptococcal infection occurred from cultured diseased olive flounder (Paralichthys olivaceus) in May, 2005 at a fish farm in Jeju Island, Korea. The diseased flounder exhibited bilateral exophthalmic eyes and rotten gills; water temperature was 16~18oC when samples were collected. Of the 22 fish samples collected, 3 samples were identified as Lactococcus garvieae and 18 samples were identified as Streptococcus parauberis by culture-based, biochemical test. Serological methods such as slide agglutination, hemolysis and antimicrobial susceptibility test were also used as well as multiplex PCRbased method to simultaneously detect and confirm the pathogens involved in the infection. S. parauberis and L. garvieae have a target region of 700 and 1100 bp., respectively. One fish sample was not identified because of the difference in the different biochemical and serological tests and was negative in PCR assay. In the present study, it showed that S. parauberis was the dominant species that caused streptococcosis in the cultured diseased flounder.

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae and Streptococcus sp. by polymerase chain reaction for the diagnosis of bacterial meningits

Avaliamos o desempenho da reação em cadeia da polimerase (PCR) para detecção simultânea da Neisseria meningitidis, Haemophilus influenzae e Streptococcus sp. no diagnóstico das meningites bacterianas e sua aplicabilidade na rotina diagnóstica. Foi realizado um estudo de coorte com 182 crianças apresentando suspeita de meningite bacteriana. Em 84, havia alterações clínicas e laboratoriais sugestivas de meningite bacteriana. Destas, 65 tiveram o agente etiológico identificado pelos métodos laboratoriais de rotina e 19 ficaram sem diagnóstico etiológico. Em 98 pacientes foi excluído o diagnóstico de meningite bacteriana. Analisando o desempenho da PCR encontramos sensibilidade de 88,1%, especificidade de 99,0% e valores preditivos positivo e negativo de 98,7% e 90,1% respectivamente. Nos 19 pacientes com meningite bacteriana mas sem diagnóstico etiológico a PCR detectou microrganismos em 14, sendo 12 N. meningitidis, um H. influenzae e um Streptococcus sp. A PCR possui o potencial de poder aumentar os índices de identificação das técnicas tradicionais, principalmente nas situações onde a microscopia direta, cultura ou identificação antigênica são negativos ou inconclusivos.

Rapid discrimination between strains of beta haemolytic streptococci by intact cell mass spectrometry.

BACKGROUND & OBJECTIVES: Beta haemolytic streptococci belonging to Lancefield group A, B, C and G cause a wide spectrum of clinical diseases. Hence there is a need for rapid and accurate typing of these strains. The present study was undertaken to evaluate the use of intact cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for rapid discrimination between strains of beta haemolytic streptococci. METHODS: Colonies of beta haemolytic streptococci were emulsified with chemical matrix on the sample slide, dried and analyzed by MALDI-TOF-MS. RESULTS: The reproducibility of results for all groups of beta haemolytic streptococci was good and spectra obtained for Lancefield group A, C and G streptococci showed discrimination between the groups on visual comparison. A finer difference in spectrum was observed among group A streptococci isolated from different locations at different periods of time. INTERPRETATION & CONCLUSION: MALDI-TOF-MS may be a potential tool in discriminating between strains of beta haemolytic streptococci, and also in the characterisation of untypable strains of group A streptococci.

Genetic analysis of Streptococcus uberis plasminogen activators.

BACKGROUND & OBJECTIVES: Streptococci produce a diverse range of secreted plasminogen activators capable of converting mammalian plasminogen to plasmin in a species-specific manner. In all examples to date, the host animal's plasminogen and that of a number of additional species have been shown to interact with these molecules leading to the conclusion that the pathogenesis of streptococci is in some way dependent upon activation of host plasminogen. PauA was the first plasminogen activator described from Streptococcus uberis, a pathogen frequently isolated from cases of bovine mastitis. Recently, a second S. uberis plasminogen activator (PauB) was identified from a Danish mastitis isolate. Interestingly, the pauB open reading frame occupied the locus normally filled by pauA. In the present study a genetic screen of streptococcal and field isolates frequently associated with mastitis was undertaken to assess the distribution, chromosomal location and sequence variation of these putative virulence factors. METHODS: Southern analysis of a diverse panel of streptococci and additional bacterial isolates frequently associated with bovine mastitis was performed using pauA and pauB probes. Sequence variation of PauA was assessed at the protein level following nucleotide sequence analysis of pauA alleles amplified from isolates picked from different geographical locations. RESULTS: We observed plasminogen activators to be universally distributed amongst S. uberis. A pauA allele was identified in all but one strain of S. uberis. This strain had a pauB allele substituted for pauA at the same locus. The remarkably low level of sequence variation demonstrated by PauA was further restricted to a limited number of residues within the molecule. INTERPRETATION & CONCLUSION: The high prevalence of PauA alleles in field isolates of S.uberis supported the observation that plasminogen activators are likely to confer an advantage with respect to colonization and growth. The findings of the present study support the theory that PauA plays a critical role in the pathogenesis of S. uberis.

Comparative genomics of streptococcal species.

Microbial genome sequencing has produced an unprecedented amount of new information and insights into an organism's metabolic activities, virulence properties, and evolution. The complete genome sequence has been reported for four different species of streptococci, including Streptococcus pyogenes, S. agalactiae, S. pneumoniae and S. mutans. Comparative genome analysis among organisms of the same species not only shows a high degree of similarity in gene content and organization, but also a high degree of sequence heterogeneity as evidenced by the large number of single nucleotide polymorphisms present. Considerable differences were also observed in the number of mobile genetic elements found in each organism, including complete and partial bacteriophage genomes, IS elements, transposons, and plasmids. S. pyogenes was the only species to contain complete bacteriophage genomes in its genome, while only S. pneumoniae and S. mutans contained the full complement of competence genes essential for natural transformation. Comparative genome analysis between the species showed that S. pyogenes was more closely related to S. agalactiae than with S. pneumoniae or S. mutans.

Genetic basis for mutacin N and of its relationship to mutacin I.

BACKGROUND & OBJECTIVES: The mutans streptococci (MS) are a group of 7 species of dental cariesassociated bacteria of which Streptococcus mutans and Streptococcus sobrinus are the most important in humans. Many MS produce bacteriocin-like inhibitory substances (BLIS), some of which have been characterised as small peptides capable of inhibiting the growth of closely-related species. These peptides have most commonly been referred to as mutacins. S. mutans strains N and UA140 appear to have closely similar BLIS activities. Both produce mutacins that seem to target the same species of bacteria. On closer analysis however, these two strains have been shown to produce distinctly different mutacins, known as mutacin N and mutacin I respectively. In the present study the mutacin N structural gene (mutN) was cloned and compared with the mutacin I structural gene (mutA). METHODS: Cloning and sequencing of S. mutans N was done. The distribution of mutN using DNA from 216 streptococcal strains was determined by dot blotting. RESULTS: Mut N was cloned and sequenced from an 1800 bp Bam HI/Eco RI fragment. PCR with the mutN primers mutNF and mutNR on the four mutN-positive strains identified identical bands to S. mutans N. The location of mutN differs significantly from that of mutA in that it is directly upstream of comC, a gene encoding a putative competence stimulating factor. INTERPRETATION & CONCLUSION: The close upstream proximity of mutN to comC suggests a link between mutacin N production and competence development. Further studies need to be done to detect competence-related genes in S. mutans strain N.

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmatic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0 percent ammonium sulfate, when a linear grandient was applied. Theree major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 muM and kcat = 0.82 s(-1), when assayed with a chromogen-coupled subtrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.