Cheese whey: a cost-effective alternative for hyaluronic acid production by Streptococcus zooepidemicus

This study focuses on the optimization of cheese whey formulated media for the production of hyaluronic acid HA by Streptococcus zooepidemicus. Culture media containing whey (W; 2.1 g/L) or whey hydrolysate (WH; 2.4 g/L) gave the highest HA productions. Both W and WH produced high yields on protein consumed, suggesting cheese whey is a good nitrogen source for S. zooepidemicus production of HA. Polysaccharide concentrations of 4.0 g/L and 3.2 g/L were produced in W and WH in a further scale-up to 5 L bioreactors, confirming the suitability of the low-cost nitrogen source. Cheese whey culture media provided high molecular weight (> 3000 kDa) HA products. This study revealed replacing the commercial peptone by the low-cost alternative could reduce HA production costs by up to a 70%compared to synthetic media.

Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase

A metabolic flux model was developed for Streptococcus zooepidemicus to compare the metabolism of glucose and maltose during aerobic batch cultivation. Lactic acid was the main product of glucose metabolism whereas acetic acid was the main product of maltose metabolism. This difference was chiefly attributed to the two-fold higher flux through NADH oxidase in maltose-grown cells that enabled the ATP generation rate to remain high despite a slower maltose consumption rate. The two-fold higher flux was matched by a two-fold increase in NADH oxidase activity, 2.53 +/- 0.1 mumol NADH min(-1) mg(-1) protein on maltose versus 1.07 +/- 0.04 Rmol NADH min(-1) mg(-1) protein on glucose, indicating that NADH oxidase activity is regulated by the energy status of the cell. Surprisingly, the energy status of the cell had little impact on hyaluronic acid (HA) yield and molecular weight. (C) 2003 Elsevier Science B.V. All rights reserved.

Stable production of hyaluronic acid in Streptococcus zooppidemicus chemostats operated at high dilution rate

Hyaluronic acid is routinely produced through fermentation of both Group A and C streptococci. Despite significant production costs associated with short fermentations and removal of contaminating proteins released during entry into stationary phase, hyaluronic acid is typically produced in batch rather than continuous culture. The main reason is that hyaluronic acid synthesis has been found to be unstable in continuous culture except at very low dilution rates. Here, we investigated the mechanisms underlying this instability and developed a stable, high dilution rate (0.4 h(-1)) chemostat process for both chemically defined and complex media operating for more than 150 h of production. In chemically defined medium, the product yield was 25% higher in chemostat cultures than in conventional batch culture when arginine or glucose was the limiting substrate. In contrast, glutamine limitation resulted in higher ATP requirements and a yield similar to that observed in batch culture. In complex, glucose-limited medium, ATP requirements were greatly reduced but biomass synthesis was favored over hyaluronic acid and no improvement in hyaluronic acid yield was observed. The successful establishment of continuous culture at high dilution rate enables both commercial production at reduced cost and a more rational characterization and optimization of hyaluronic acid production in streptococci. (c) 2005 Wiley Periodicals, Inc.

Estudo da microbiota vaginal de éguas com ênfase na pesquisa de lactobacilos

Pós-graduação em Medicina Veterinária - FMVZ

Perfil fenotípico e susceptibilidade antimicrobiana de Streptococcus equi isolados de equinos da região Sul do Brasil

As características fenotípicas [morfológicas, bioquímicas, susceptibilidade aos antimicrobianos, índice de resistência múltipla aos antimicrobianos (IRMA), concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) da benzilpenicilina] de 38 isolados de Streptococcus equi oriundos de amostras clínicas de animais com adenite equina foram alvo deste estudo. A fenotipia demonstrou três padrões de colônias, três biotipos de fermentação de carboidratos e variação de 0 a 0,4 no IRMA. Todos os isolados de S. equi demonstraram sensibilidade à penicilina, tanto pelo método de disco difusão quanto pelo método de microdiluição. A CIM e CBM média de benzilpenicilina foi de 0,0095μg/mL e 0,0267μg/mL para S. equi subesp. equi e de 0,0128μg/mL e 0,0380μg/mL para S. equi subesp. zooepidemicus. Os valores de CIM e CBM diferiram entre as subespécies (p<0,05). O diâmetro do halo de inibição de penicilina demonstrou relação com a CIM (ì=0,03638 - 0,00072x) para S. equi subesp. equi. Também foi demonstrada relação entre o diâmetro do halo de inibição de penicilina com a CBM para S. equi subesp. equi (ì=0,10931- 0,00223x). Entretanto para as amostras de S. equi subesp. zooepidemicus esta relação somente foi verificada para a CBM (ì=0,1322 - 0,00271x). A CIM de benzilpenicilina frente às amostras isoladas da região Central, Planalto e Sul do estado do Rio Grande do Sul foram estatisticamente semelhantes, mas diferiram do isolado do estado do Paraná, sugerindo o caráter atípico desta cepa. Todos os isolados de S. equi são sensíveis à penicilina e sulfazotrim, confirmando a eleição destes antimicrobianos para o tratamento das infecções por este agente na clínica veterinária. Os resultados obtidos não dispensam a utilização prudente dos antimicrobianos.

Streptococcus equi subsp ruminatorum subsp nov., isolated from mastitis in small ruminants

Six isolates of an unknown Gram-positive, catalase-negative, chain-forming, coccus-shaped organism isolated from ovine and caprine mastitis were characterized by phenotypic and molecular taxonomic methods. On the basis of cellular morphology and the results of biochemical tests, the organism was tentatively identified as a streptococcal species. Comparative 16S rRNA gene sequencing studies confirmed that the organism is a member of the genus Streptococcus, with Streptococcus equi as its closest phylogenetic relative (98(.)8% similarity). DNA-DNA pairing studies showed that the unidentified organism displayed more than 70% relatedness to the type strains of S. equi subsp. equi and subsp. zooepidemicus. Despite the relatively high DNA-DNA reassociation values, biotyping and ribotyping allowed clear differentiation of the unknown bacterium from the two recognized subspecies of S. equi. On the basis of phenotypic and molecular genetic evidence, it is proposed that the unknown Streptococcus isolates from ovine and caprine mastitis be classified as a novel subspecies, Streptococcus equi subsp. ruminatorum subsp. nov. The type strain is CECT 5772(T) (=CCUG 47520(T) = Mt 167(T)).

Two-component System Vicrk Regulates Functions Associated With Establishment Of Streptococcus Sanguinis In Biofilms.

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H2O2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRKSs regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.

Adesão de linhagem selvagem de Streptococcus thermophilus em superfície de aço inoxidável e efeitos da higienização na sua remoção

A wild strain of Streptococcus thermophilus isolated from pasteurized milk was evaluated using an experimental model with respect to its adhesion onto stainless steel surfaces and its behaviour when submitted to cleansing and sanification. In milk, the adhesion of the microorganism on to stainless steel surfaces was studied after 6 hours of contact at 45°C with agitation, and after a cleansing process involving cleaning stages with alkaline and acid detergents followed by sanification, in order to evaluate the resistance of the adhered cells. The microorganism adhered to stainless steel surfaces producing a cell load of 10(4) CFU/cm². After alkaline cleansing, no adhered cells were detected but 6 CFU/cm² were still detected on the surfaces after acid cleansing. Cleansing, followed by sanification with sodium hypochlorite, was sufficient to reduce the load of wild S. thermophilus on the stainless steel surfaces to non-detectable levels. The experimental model proved adequate for the study indicating that the wild microorganism S. thermophilus produces biofilms on stainless steel surfaces. Alkaline cleansing remove more that 99.9% of the adhered cells. The few cells adhered on the surface are removed by acid cleansing demonstrating the need to use different steps and types of detergent for efficient cleansing. The best results for the removal of these biofilms are obtained by using alkaline cleansing followed by acid cleaning, this procedure being more efficient when complemented by sanification with sodium hypochlorite.

In Vitro Adhesion of Streptococcus sanguinis to Dentine Root Surface After Treatment with Er:Yag Laser, Ultrasonic System, or Manual Curette

Objective: The purpose of this in vitro study was to evaluate the dentine root surface roughness and the adherence of Streptococcus sanguinis (ATCC 10556) after treatment with an ultrasonic system, Er:YAG laser, or manual curette. Background Data: Bacterial adhesion and formation of dental biofilm after scaling and root planing may be a challenge to the long-term stability of periodontal therapy. Materials and Methods: Forty flattened bovine roots were randomly assigned to one of the following groups: ultrasonic system (n = 10); Er:YAG laser (n = 10); manual curette (n = 10); or control untreated roots (n = 10). The mean surface roughness (Ra, mu m) of the specimens before and after exposure to each treatment was determined using a surface profilometer. In addition, S. sanguinis was grown on the treated and untreated specimens and the amounts of retained bacteria on the surfaces were measured by culture method. Results: All treatments increased the Ra; however, the roughest surface was produced by the curettes. In addition, the specimens treated with curettes showed the highest S. sanguinis adhesion. There was a significant positive correlation between roughness values and bacterial cells counts. Conclusion: S. sanguinis adhesion was the highest on the curette-treated dentine root surfaces, which also presented the greatest surface roughness.

Acute Streptococcus agalactiae endocarditis: Outcomes of early surgical treatment

The incidence of Streptococcus agalactiae is widely recognized as increasing in nonpregnant adults. This report describes a retrospective case series of S. agalactiae endocarditis at the Heart Institute of Sao Paulo, Brazil, between 2000 and 2006. Early diagnosis and valve surgery seem to have contributed to improved patient outcomes. (Heart Lung (R) 2010;39:331-334.)

Antimicrobial activity of Rheedia brasiliensis and 7-epiclusianone against Streptococcus mutans

This in vitro study evaluated the antimicrobial activity of extracts obtained from Rheedia brasiliensis fruit (bacupari) and its bioactive compound against Streptococcus mutans. Hexane, ethyl-acetate and ethanolic extracts obtained (concentrations ranging from 6.25 to 800 mu g/ml) were tested against S. mutans UA159 through MIC/MBC assays. S. mutans 5-days-old biofilms were treated with the active extracts (100 x MIC) for 0, 1, 2, 3 and 4 h (time-kill) and plated for colony counting (CFU/ml). Active extracts were submitted to exploratory chemical analyses so as to isolate and identify the bioactive compound using spectroscopic methods. The bioactive compound (concentrations ranging from 0.625 to 80 mu g/ml) was then tested through MIC/MBC assays. Peel and seed hexane extracts showed antimicrobial activity against planktonic cells at low concentrations and were thus selected for the time kill test. These hexane extracts reduced S. mutans biofilm viability after 4 h, certifying of the bioactive compound presence. The bioactive compound identified was the polyprenylated benzophenone 7-epiclusianone, which showed a good antimicrobial activity at low concentrations (MIC: 1.25-2.5 mu g/ml; MBC: 10-20 mu g/ml). The results indicated that 7-epiclusianone may be used as a new agent to control S. mutans biofilms; however, more studies are needed to further elucidate the mechanisms of action and the anticariogenic potential of such compound found in R. brasiliensis. (C) 2008 Elsevier GmbH. All rights reserved.

Effects of 7-Epiclusianone on Streptococcus mutans and Caries Development in Rats

The aim of this study was to evaluate the effects of 7-epiclusianone (7-epi) on specific virulence attributes of Streptococcus mutans in vitro and on development of dental caries in vivo. 7-Epi was obtained and purified from fruits of Rheedia brasiliensis. We investigated its influence on surface-adsorbed glucosyltransferase (Gtf) B activity, acid production, and viability of S. mutans in biofilms, as well as on caries development using a rodent model. 7-Epi (100 mu g/mL) significantly reduced the activity of surface-adsorbed GtfB (up to 48.0 +/- 1.8 of inhibition at 100 mu g/mL) and glyco-lytic pH-drop by S. mutans in biofilms (125 and 250 mu g/mL) (vs. vehicle control, p < 0.05). In contrast, the test compound did not significantly affect the bacterial viability when compared to vehicle control (15% ethanol, p > 0.05). Wistar rats treated topically with 7-epi (twice daily, 60-s exposure) showed significantly smaller number of and less severe smooth-and sulcal-surface carious lesions (p < 0.05), without reducing the S. mutans viable population from the animals` dental biofilms. In conclusion, the natural compound 7-epiclusianone may be a potentially novel pharmacological agent to prevent and control dental caries disease.

Growth and acidification performance of probiotics in pure culture and co-culture with Streptococcus thermophilus: The effect of inulin

Inulin was used as a prebiotic to improve the quality and consistency of skim milk fermented by co-cultures and pure Cultures of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Bifidobacterium lactis with Streptococcus thermophilus. We compared, either in the presence or absence of 4 g inulin/100 g, the results of the main kinetic parameters, specifically the generation time (t(g)), the maximum acidification rate (V(max)). and the times to reach V(max) (t(max)), to attain pH 5.0 (t(pH5.0)) and to complete the fermentation (t(pH4.5)). Post-acidification, lactic acid formation and cell counts were also determined and compared, either 1 day after the fermentation was complete or after 7 day storage at 4 degrees C. In general, inulin addition to the milk increased in co-cultures V(max), decreased t(max), t(g) and t(pH4.5), favored post-acidification, exerted a bifidogenic effect, and preserved almost intact cell viability during storage. In addition, S. thermophilus was shown to stimulate the metabolism of the other lactic bacteria. Contrary to co-cultures, most of the effects in pure Cultures were not statistically significant. The most important aspect of this paper is the use of the generation time as a toot to investigate the microbial response to inulin addition. (c) 2009 Elsevier Ltd. All rights reserved.

Effect of inulin on growth and acidification performance of different probiotic bacteria in co-cultures and mixed culture with Streptococcus thermophilus

Inulin was used as a prebiotic to improve the quality and consistency of skim milk fermented by Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Lactobacillus bulgaricus (Lb) and Bifidobacterium lactis (BI) with Streptococcus thermophilus (St), either in binary co-cultures or in cocktail containing all microorganisms. We compared, either in the presence of 40 mg inulin g(-1) or not, the results of the maximum acidification rate (V(max)) and the times to reach it (t(max)), to reach pH 5.0 (t(PH5.0)) and to complete the fermentation (t(f)). Post-acidification, lactic acid formation and cell counts were also compared after either 1 day (D1) or 7 days of storage at 4 degrees C (N). In co-culture, inulin addition to the milk increased V(max), decreased t(max) and t(f), favored post-acidification and exerted a bifidogenic effect. S. thermophilus proved to stimulate the metabolism of the other lactic bacteria and enhanced the product features. After D7, a significant prebiotic effect of inulin was observed in all co-cultures. Either after D1 or D7, the enumerations of Lr and BI in mixed culture markedly decreased compared to their respective co-cultures because of greater competition for the same substrates. (C) 2008 Elsevier Ltd. All rights reserved.

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: nonselective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L acidophilus La05, L casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 degrees C or 42 degrees C, anaerobiosis; L acidophilus: MRS agar with bile salts, 37 degrees C or 42 degrees C, aerobiosis; L casei: MRS agar with lithium chloride and sodium propionate, 37 degrees C or 42 degrees C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 degrees C or 42 degrees C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 degrees C, anaerobiosis, L acidophilus: LC medium, 42 degrees C, aerobiosis or anaerobiosis and L casei: LP-MRS, 42 degrees C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P <= 0.05). (C) 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.