Streptococcus thermophilus Phages in Whey Derivatives: From Problem to Application in the Dairy Industry.

Fifteen samples of whey protein concentrate (WPC) were tested against 37 commercial Streptococcus thermophilus strains to detect infective bacteriophages. Seventy-three diverse phages were isolated from 12 samples, characterized by using DNA restriction patterns and host range analyses. Sixty-two of them were classified as cos, two as pac, and nine as 5093, according to PCR multiplex assays. Phage concentration was greater than 104 PFU/g for 25.3% of isolated phages. Seven phages showed an unusual wide host range, being able to infect a high number of the tested strains. Regarding thermal resistance, pac phages were the most sensitive, followed by cos phages, those classified as 5093 being the most resistant. Treatments at 85 °C for 5 min in TMG buffer were necessary to completely inactivate all phages. Results demonstrated that the use, without control, of these whey derivatives as additives in dairy fermentations could be a threat because of the potential phage infection of starter strains. In this sense, these phages constitute a pool of new isolates used to improve the phage resistance of starter cultures applied today in the fermentative industry.

Atividade antimicrobiana da fibrina rica em plaquetas frente a cepas bacterianas orais / Antimicrobial activity of platelet-rich fibrin against oral bacteria

Objetivo: avaliar a atividade antimicrobiana da fibrina rica em plaquetas (PRF) frente a bactérias da cavidade oral. Material e métodos: foram coletadas amostras sanguíneas de seis pacientes que fizeram uso de PRF para fins de tratamento odontológico. Após a coleta, o sangue foi centrifugado seguindo duas metodologias distintas, uma a 1.800 rpm durante dez minutos e outra a 3.000 rpm durante dez minutos. A PRF obtida pelo processo foi separada e imediatamente utilizada para análise de sua atividade antimicrobiana. As cepas Streptococccus mutans, Streptococcus oralis, Streptococcus salivarius, Streptococcus parasanguinis, Streptococcus mitis, Lactobacillus acidophilus e Lactobacillus rhamnosus foram cultivadas em meio Brain Heart Infusion (BHI) suplementado com 1% de sacarose, sob atmosfera de microaerofilia a 37oC durante 48 horas. O potencial bactericida da PRF foi verificado através do teste de resistência ou sensibilidade bacteriana por difusão em ágar. Resultados: após a obtenção dos resultados, pôde-se observar que algumas bactérias, como a S. mutans, S. oralis, S. sanguise e S. parasanguinis, foram suscetíveis à PRF, segundo medição do halo de inibição. Ainda, houve diferenças quanto à atividade da PRF dentre as duas metodologias empregadas. Conclusão: a PRF possui atividade antimicrobiana considerável frente a determinadas espécies bacterianas orais, fato este que a torna um excelente produto para uso em Odontologia. Contudo, outros estudos devem ser realizados visando o mecanismo de ação antimicrobiana deste recurso. (AU)

Objective: to evaluate the antimicrobial activity of platelet-rich fibrin (PRF) against oral bacteria. Material and methods: blood samples were collected from 6 patients who used PRF for dental treatment. After collection, the blood was centrifuged following two different methodologies, one at 1,800 rpm for 10 minutes and the other at 3,000 rpm for 10 minutes. The PRF obtained by the process was separated and immediately used to analyze its antimicrobial activity. Streptococcus mutans, Streptococcus oralis, Streptococcus salivarius, Streptococcus parasanguinis, Streptococcus sanguis, Lactobacillus acidophilus and Lactobacillus rhamnosus were cultured in Brain Heart Infusion (BHI) supplemented with 1% sucrose under a microaerophilic atmosphere at 37oC for 48 hours. The bactericidal potential of the PRF was verified through the bacterial resistance test by diffusion in agar. Results: after obtaining the results, it was observed that some bacteria such as S. mutans, S. oralis, S. sanguis and S. parasanguinis were susceptible to PRF according to the measurement of the inhibition halo. It was also observed that there were differences in PRF activity among the two methodologies used. Conclusion: it was possible to perceive that PRF has considerable antimicrobial activity against certain oral bacterial species, a fact that makes it an excellent product for use in dentistry. However, other studies still needed to be performed aiming at the mechanisms of antimicrobial action of this resource. (AU)

Phage-host interactions in Streptococcus thermophilus: Genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array.

Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5'-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array.

Genome analysis of two virulent Streptococcus thermophilus phages isolated in Argentina.

Two Streptococcus thermophilus phages (ALQ13.2 and phiAbc2) were previously isolated from breakdowns of cheese manufacture in Argentina. Complete nucleotide sequence analysis indicated that both phages contained linear double-stranded DNA: 35,525 bp in length for the pac-type phage ALQ13.2 and 34,882 bp for the cos-type phage phiAbc2. Forty-four and 48 open reading frames (ORF) were identified for ALQ13.2 and phiAbc2, respectively. Comparative genomic analysis showed that these isolates shared many similarities with the eight previously studied cos- and pac-phages infecting different S. thermophilus strains. In particular, part of the phiAbc2 genome was highly similar to a region of phage 7201, which was thought to be unique to this latter phage. Protein analysis of the pac-phage ALQ13.2 using SDS polyacrylamide gel electrophoresis (SDS-PAGE) identified three major proteins and seven minor proteins. Parallel structural proteome analysis of phiAbc2 revealed seven protein bands, two of which were related to major structural proteins, as expected for a cos-type phage. Similarities to other S. thermophilus phages suggest that the streptococcal phage diversity is not extensive in worldwide dairy factories possibly because related high-performing bacterial strains are used in starter cultures.

IDENTIFICACIÓN MICROBIOLÓGICA, MOLECULAR; SEROLÓGICA DE Streptococcus pneumoniae, Y DETERMINACIÓN DE LA SUSCEPTIBILIDAD A PENICILINA Y ERITROMICINA EN LA CIUDAD DE COCHABAMBA / Microbiological molecular identification and serologic of Streptococcus pneumoniae and determination of the suceptibility to penicilin and eritromicin in the city of Cochabamba

Streptococcus pneumoniae es uno de los principales patógenos causantes de infecciones respiratorias e invasivas, por lo que es importante identificar rápidamente este microorganismo y determinar la susceptibilidad a los antibióticos frecuentemente utilizados, para la realización de terapias adecuadas. Con este fin se realizó la identificación de 32 aislamientos de Streptococcus pneumoniae, a partir de pacientes con infecciones respiratorias e invasivas; para su identificación se utilizaron métodos microbiológicos convencionales y como pruebas confirmatorias, métodos moleculares por reacción en cadena de la polimerasa (PCR), amplificando regiones de los genes lyt A y ply y métodos serológicos para la determinación del antígeno capsular. Se realizaron antibiogramas para determinar la susceptibilidad a Penicilina y Eritromicina. Los resultados revelaron, un mayor número de aislamientos en menores de 10 años y en mayores de 49 años y en relación a épocas estacionales, de aislamientos un mayor número durante los meses de invierno. En las pruebas de susceptibilidad a los antibióticos se encontró una sensibilidad disminuida a Penicilina (SDP) de 46.88 %; resistencia a eritromicina de 6.25 % y resistencia intermedia de 15,63%. Realizando un análisis estadístico de los tres métodos como pruebas de reconfirmación, se observó una correlación baja, por lo que la prueba de la optoquina con fines de reconfirmación para Streptococcus pneumoniae, es inferior a las pruebas serológica y molecular.

Streptococcus pneumoniae is one of the main pathogens that cause breathing and invasive infections, that is why is important a quick identification of this microorganism and determine the sensitivity of the frequently used antibiotics, to execute appropriate therapies. With this purpose the identification of 32 isolates of Streptococcus pneumoniae from patients with breathing and invasive infections were carried out, conventional microbiological tests were applied for identification and for confirmation were used molecular techniques based on the chain reaction of polymerase (PCR), amplifying the regions of lyt A and ply genes and serological methods for the determination of the capsular antigen. Antibiograms were carried out in order to determinate the sensitivity to penicillin and erythromycin. The results showed a higher amount of isolates in children under 10 and adults over 49, and according to seasons, more isolates on winter months. Regarding sensitivity to antibiotics, we found a drop of 46.88% to penicillin, a drop of 6.25 to erythromycin and an intermediate drop of 15.63%. The statistical analysis among the three techniques used for reconfirmation revealed there is a low correlation among them. Therefore, optochin, used as a re-confirmation method, is lower than serological and molecular tests.

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption.

AIMS: To determine whether the presence and type of exopolysaccharides (EPS), slime-EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. METHODS AND RESULTS: Phage-host interactions between eight EPS-producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (phiYsca, phi3, phi5, phi6, phi8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular-producing strains were more sensitive to phage attacks than the noncapsular-producing strains. Streptococcus thermophilus CRL1190 (capsular-producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular-negative, EPS low-producing mutant of strain CRL1190 was reduced, especially for phiYcsa and phi8. CONCLUSIONS: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Capsular-producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.

Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina.

Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.

Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries.

Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.

Characterization of phage receptors in Streptococcus thermophilus using purified cell walls obtained by a simple protocol.

A simple protocol was designed and applied to obtain Streptococcus thermophilus purified cell walls. To identify the structures involved in phage adsorption, the cell walls of two Strep. thermophilus strains were treated with sodium dodecyl sulphate and proteinase K. These treatments did not reduce the adsorption of phages CYM and 0BJ to the cell walls of Strep. thermophilus YSD10 and Strep. thermophilus BJ15, respectively. However, phage binding was reduced when the cell envelopes were treated with mutanolysin or trichloroacetic acid 5%, suggesting that the phage receptor component is part of the peptidoglycan or a polymer closely linked to it. The ability of several saccharides to inactivate both phages was also assayed. These phage inhibition experiments suggested that the phage CYM adsorbed to a component involving glucosamine and rhamnose, while glucosamine and ribose interfered with the adsorption of phage 0BJ.