Predictive Power of the "Trigger Tool" for the detection of adverse events in general surgery: a multicenter observational validation study

Background In spite of the global implementation of standardized surgical safety checklists and evidence-based practices, general surgery remains associated with a high residual risk of preventable perioperative complications and adverse events. This study was designed to validate the hypothesis that a new “Trigger Tool” represents a sensitive predictor of adverse events in general surgery. Methods An observational multicenter validation study was performed among 31 hospitals in Spain. The previously described “Trigger Tool” based on 40 specific triggers was applied to validate the predictive power of predicting adverse events in the perioperative care of surgical patients. A prediction model was used by means of a binary logistic regression analysis. Results The prevalence of adverse events among a total of 1,132 surgical cases included in this study was 31.53%. The “Trigger Tool” had a sensitivity and specificity of 86.27% and 79.55% respectively for predicting these adverse events. A total of 12 selected triggers of overall 40 triggers were identified for optimizing the predictive power of the “Trigger Tool”. Conclusions The “Trigger Tool” has a high predictive capacity for predicting adverse events in surgical procedures. We recommend a revision of the original 40 triggers to 12 selected triggers to optimize the predictive power of this tool, which will have to be validated in future studies

Effect of lactic acid probiotic cultures utilization on the vaginal microbiota of women diagnosed with bacterial and fungal infections

A microbiota vaginal saudável é constituída, majoritariamente, por espécies de lactobacilos que representam uma barreira natural contra microrganismos causadores de doenças como a candidíase vulvovaginal (CVV), vaginose bacteriana (VB) e infecções do trato urinário (ITU), que juntas acometem cerca de um bilhão de mulheres no mundo anualmente. Um melhor entendimento da ecologia microbiana vaginal pode ser útil na otimização de tratamentos existentes para as infecções urogenitais, os quais podem destruir parcialmente a microbiota autóctone, predispor a novas infecções, contribuir para a seleção de microrganismos resistentes e causar efeitos colaterais indesejáveis. A utilização de microrganismos certificadamente probióticos, Lactobacillus rhamnosus GR-1 e Lactobacillus reuteri RC-14, representa uma alternativa terapêutica promissora na abordagem de VB e CVV, uma vez que são capazes de colonizar o trato vaginal, apresentam atividade inibitória frente a diversos patógenos do trato urogenital, exibem risco mínimo para a seleção de microrganismos resistentes e podem auxiliar na restauração da microbiota vaginal. Os objetivos deste trabalho foram: (i) avaliar a prevalência de espécies de lactobacilos na microbiota vaginal de mulheres saudáveis e diagnosticadas com infecções vaginais (CVV e VB) da cidade de Ribeirão Preto (São Paulo, Brasil), (ii) avaliar a capacidade de isolados de lactobacilos produzirem peróxido de hidrogênio (H2O2) e (iii) determinar a eficácia da utilização de L. rhamnosus GR-1 e L. reuteri RC-14 no tratamento de CVV e VB, quando co-administrados com medicamentos antimicrobianos tradicionais. Participaram deste estudo, 196 pacientes voluntárias, atendidas por médicos ginecologistas de centros de saúde ligados à Universidade de São Paulo, campus de Ribeirão Preto (64 saudáveis, 68 diagnosticadas com CVV e 64 diagnosticadas com VB) e duas amostras vaginais de cada paciente foram coletadas com o auxílio de zaragatoas esterilizadas. Uma zaragatoa foi utilizada para semeadura em ágar MRS (de Man, Rogosa & Sharpe), os isolados de bactérias láticas obtidos foram analisados através da técnica de PCR-ARDRA (Reação em cadeia da polimerase Análise de restrição do DNA ribossomal amplificado) e a habilidade de Lactobacillus spp. produzir H2O2 foi determinada semi-quantitativamente. A outra zaragatoa foi utilizada para a análise das espécies de lactobacilos da microbiota vaginal pela técnica independente de cultivo PCR-DGGE (Reação em cadeia da polimerase Eletroforese em gel de gradiente de desnaturação). As leveduras do gênero Candida foram obtidas através da semeadura das amostras vaginais provenientes de pacientes saudáveis e com CVV no meio Chromagar® Candida e identificadas através de provas bioquímicas de referência. As pacientes diagnosticadas com CVV participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de fluconazol (150mg) e suplementação diária durante 28 dias com (i) duas cápsulas contendo os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 (Urex-Cap-5®) ou (ii) duas cápsulas de placebo. As pacientes com VB também participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de tinidazol (2g) e suplementação diária com cápsulas do probiótico (Urex-Cap-5®) ou placebo, conforme descrito acima. Todas as pacientes foram reavaliadas ao final do tratamento, no 28º dia. Foram realizados experimentos para averiguar o possível efeito de L. rhamnosus GR-1 e L. reuteri RC-14 na modulação in vitro da infecção por Candida albicans em culturas de células epiteliais vaginais humanas (VK2/E6E7) Os resultados mostraram que, pela técnica de PCR-ADRA, L. crispatus foi a espécie mais prevalente nos grupos de pacientes saudáveis (37,0%) e com CVV (35,9%), enquanto que L. gasseri foi predominante no grupo de pacientes com VB (34,6%). De acordo com o método de PCR-DGGE, L. iners foi o microrganismo mais prevalente nos três grupos de pacientes avaliados: saudáveis, com CVV e VB (48,7%, 44,7% e 65,0%, respectivamente). A maioria dos isolados de Lactobacillus spp. obtidos nos grupos de pacientes saudáveis (98,6%) e diagnosticadas com CVV (97,4%) foram capazes de produzir H2O2 (1 a 100mg/L), em comparação a apenas 68,2%, determinado no grupo de pacientes com VB (p<0,05). L. crispatus e L. johnsonii produziram as maiores quantidades médias de H2O2 (30mg/L). A taxa de colonização por leveduras do gênero Candida foi de 26,6% no grupo de pacientes saudáveis (C. albicans correspondeu a 52,4% de todos os isolados), enquanto que no grupo de pacientes com CVV, 89,2% dos isolados leveduriformes foram identificados como C. albicans. Para as análises estatísticas dos dados obtidos com os testes clínicos, foram consideradas 55 pacientes diagnosticadas com CVV (pela presença de sinais e sintomas da infecção e cultura positiva para Candida sp.) e foi observado que a utilização de dose única de fluconazol e suplementação diária com o probiótico, resultou em taxa de cura mais elevada para a infecção (89,7%) em comparação com aquela verificada no grupo placebo (65,4%) (p<0,05). A utilização de tinidazol em associação com a ingestão diária de Urex-Cap-5® no tratamento de pacientes com VB também resultou em taxa de cura mais elevada para a condição (87,5%), em comparação àquela observada no grupo placebo (50,0%) (p<0,05). A atividade anti-Candida de L. rhamnosus GR-1 e L. reuteri RC-14 foi observada através do modelo in vitro para infecção vaginal. Em conclusão, quando as técnicas de PCR-ARDRA e PCR-DGGE foram comparadas entre si, foi observado que ambas apresentaram limitações, evidenciando a importância do emprego de diferentes metodologias para avaliação adequada das espécies de lactobacilos presentes na microbiota vaginal. As espécies de lactobacilos vaginais determinadas nas mulheres saudáveis do presente trabalho foram semelhantes àquelas verificadas em estudos prévios descritos na literatura com pacientes com dieta e localização geográfica notadamente distintas. Os dados do presente trabalho sugerem que a presença de lactobacilos produtores de H2O2, isoladamente, não confere proteção contra CVV, enquanto que a ausência desses microrganismos pode ser um fator contribuinte para VB. Além disso, foi demonstrado que a utilização de medicamentos antimicrobianos tradicionais e suplementação com os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 foi mais eficiente no tratamento de CVV e VB em comparação com medicamentos clássicos e placebo. Estes resultados podem contribuir para prolongar a vida útil de medicamentos cuja eficácia pode ser comprometida devido à seleção de microrganismos resistentes e também reduzir o tempo de tratamento para pacientes que necessitam de terapias clássicas por períodos prolongados.The vaginal microbiota is mainly constituted by lactobacilli species, which represent a natural barrier against microorganisms that cause vulvovaginal candidiasis (VVC), bacterial vaginosis (BV), and urinary tract infections (UTI). Together, these conditions afflict each year an estimated one billion women worldwide. A better understanding of the vaginal microbial ecology may be useful to improve the current available treatments for urogenital infections, which can partially destroy the autochthonous microbiota, predispose to other infections, contribute for the selection of resistant microorganisms and cause undesirable collateral effects. The use of microorganisms with demonstrated probiotic properties, Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, represents a promising therapeutic alternative for BV and VVC, since they are able to colonize the vaginal tract, present inhibitory against several urogenital pathogens, pose minimal risk for the selection of resistant microorganisms and can help to restore the vaginal microbiota. The objectives of this work were: (i) to evaluate the prevalence of lactobacilli species in the vaginal microbiota of healthy women and those diagnosed with vaginal infections (VVC and BV) in the city of Ribeirão Preto (São Paulo, Brazil), (ii) to evaluate the ability of the lactobacilli isolates to produce hydrogen peroxide (H2O2) and (iii) to determine the efficacy of the use of L. rhamnosus GR-1 and L. reuteri RC-14 in the treatment of VVC and BV, in co-administration with traditional antimicrobials. 196 voluntary subjects were examined by the gynecologists team from health centers affiliated with Universidade de São Paulo, campus at Ribeirão Preto (64 healthy, 68 diagnosed with VVC and 64 diagnosed with BV) and two vaginal samples from each patient were collected by the use of two sterile swabs. One swab was cultured in MRS (de Man, Rogosa & Sharpe) agar, the isolates of lactic acid bacteria obtained were analyzed by PCR-ARDRA (Polymerase chain reaction - Amplified ribosomal DNA restriction analysis) and the ability of Lactobacillus spp. to produce hydrogen peroxide was determined semi-quantitatively. The other swab was used for the analysis of lactobacilli species from the vaginal microbiota using the culture-independent PCR-DGGE (Polymerase chain reaction Denaturating gradient gel electrophoresis). The yeasts belonging to Candida genus were obtained also by culturing the vaginal material from healthy and VVC patients in Chromagar® Candida and were identified by standard biochemical tests. VVC patients were enrolled in a randomized, double-blind, placebo-controlled trial and treated with a single dose fluconazole (150mg) and daily supplementation for 28 days with (i) two capsules containing the probiotic microorganisms L. rhamnosus GR-1 and L. reuteri RC-14 (Urex-Cap-5®) or (ii) two placebo capsules. BV patients were also enrolled in a randomized, double-blind, placebo controlled trial and treated with a single dose of tinidazole (2g) and supplementation with probiotic capsules (Urex-Cap-5®) or placebo, as described above. All patients were re-evaluated at the end of treatment, on the 28th day. Experiments were also conducted to assess the possible effect of L. rhamnosus GR-1 and L. reuteri RC-14 in the in vitro modulation of vaginal infection by Candida albicans on cultures of human vaginal epithelial cells (VK2/E6E7). The results revealed that according to PCR-ADRA, L.crispatus was the most prevalent species in the groups of healthy women (37.0%) and those with VVC (35.9%), while L. gasseri was dominant in BV patients (34.6%). By PCR-DGGE method, L. iners was the most prevalent Lactobacillus species in all the three groups evaluated: healthy, VVC and BV (48.7%, 44.7% and 65.0%, respectively). The majority of the isolates of Lactobacillus spp. from healthy women (98.6%) and those with VVC (97.4%) were able to produce H2O2 (1 to 100mg/L) in comparison with only 68.2% assessed for the BV group (p<0.05). L. crispatus and L. johnsonii produced the highest average levels of H2O2 (30mg/L). Colonization rate by yeasts belonging to Candida genus was 26.6% in the group of healthy patients (C. albicans represented 52.4% of all isolates), whereas in the VVC group, 89.2% of yeast isolates were identified as C. albicans. For the performance of statistical analysis of the results obtained with the clinical trials, 55 patients diagnosed with VVC (by the presence of symptoms and signals of the infection and positive culture for Candida sp.) were taken into consideration and it was observed that the use of a single dose of fluconazole and daily supplementation with probiotics, yielded a higher cure rate (89.7%), in comparison with the placebo group (65.4%) (p<0.05). The use of tinidazole plus probiotic also resulted in higher cure rate of the infection (87.5%), compared to placebo group (50.0%) (p<0.05). An anti-Candida activity of L. rhamnosus GR-1 and L. reuteri RC-14 was observed in the in vitro model of vaginal infection. In conclusion, when PCR-ARDRA and PCR-DGGE were compared, it was verified that both presented limitations, which evidences the need of using different techniques for a better knowledge of lactobacilli species present in the vaginal microbiota. The lactobacilli species found in healthy women in this work were similar to those reported in previous studies described in the literature for patients with distinctly different diet and geographic localization. The data of the present work indicate that solely the presence of H2O2-producing isolates does not render protection against VVC, whereas the absence of those microorganisms may be a contributing factor for BV. Moreover, it was demonstrated that the use of classical medicines supplemented with the probiotics L. rhamnosus GR-1 and L. reuteri RC-14 was more efficient to treat VVC and BV in comparison with classical medicines plus placebo. These results may contribute to extend the longevity of drugs whose efficacy is compromised due to the selection of resistant microorganisms and also to shorten the length of treatment courses for patients that require long regimens with standard therapy

Assessment Of The Inhibitory Effect Of Free And Encapsulated Commercial Nisin (nisaplin (r)), Tested Alone And In Combination, On Listeria Monocytogenes And Bacillus Cereus In Refrigerated Milk

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The antimicrobial effect of free and encapsulated (carrier agent: gum Arabic) commercial nisin (Nisaplin (R)) against Listeria monocytogenes ATCC 7644 (Lm) and Bacillus cereus IAL 55 (Bc) in refrigerated (6 +/- 1 degrees C) milk was determined throughout 21 days (d). Skim and whole milk samples containing free and encapsulated commercial nisin (0.25-1.0 mg/L, alone and combined) were contaminated, individually, with Lm or Bc (vegetative cells and spores) and the microorganisms' counts assessed every 3 d. Encapsulated commercial nisin presented characteristic traits of spray-dried products and stable antimicrobial activity under refrigeration (90 days). In both skimmed and whole milk, free and encapsulated Nisaplin (R) combined (0.5 mg/L each) exhibited the strongest antilisterial effect (d21 - d0; P < 0.05), although Lm resistant cells were observed. Free and encapsulated commercial nisin (0.25 mg/L) were highly effective against Bc spores germination and for the pathogen outgrowth inhibition (d21 - d0; P < 0.05) in both types of milk, improving the food product microbiological safety. (C) 2015 Elsevier Ltd. All rights reserved.686775CNPq ("Conselho Nacional de Desenvolvimento Cientifico e Tecnologico") [151287/2014-7, 150464/2015-0, 162748/2015-9]CNPq [302763/2014-7]CAPES ("Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior")Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Assessment of the inhibitory effect of free and encapsulated commercial nisin (nisaplin (r)), tested alone and in combination, on listeria monocytogenes and bacillus cereus in refrigerated milk

The antimicrobial effect of free and encapsulated (carrier agent: gum Arabic) commercial nisin (Nisaplin®) against Listeria monocytogenes ATCC 7644 (Lm) and Bacillus cereus IAL 55 (Bc) in refrigerated (6 ± 1 °C) milk was determined throughout 21 days (d). Skim and whole milk samples containing free and encapsulated commercial nisin (0.25–1.0 mg/L, alone and combined) were contaminated, individually, with Lm or Bc (vegetative cells and spores) and the microorganisms' counts assessed every 3 d. Encapsulated commercial nisin presented characteristic traits of spray-dried products and stable antimicrobial activity under refrigeration (90 days). In both skimmed and whole milk, free and encapsulated Nisaplin® combined (0.5 mg/L each) exhibited the strongest antilisterial effect (d21 – d0; P < 0.05), although Lm resistant cells were observed. Free and encapsulated commercial nisin (0.25 mg/L) were highly effective against Bc spores germination and for the pathogen outgrowth inhibition (d21 – d0; P < 0.05) in both types of milk, improving the food product microbiological safety686775CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES#151287/2014-7; 150464/2015-0; 162748/2015-9; #302763/2014-7Sem informaçã

Elongated cells of Listeria monocytogenes in biofilms in the presence of sucrose and bacteriocin-producing Leuconostoc mesenteroides A11

Listeria monocytogenes is a foodborne pathogen which may survive in biofilms and persist in food processing plants. In this study, the ability of Leuconostoc mesenteroides (bac+ and bac-) to inhibit biofilm formation by L. monocytogenes ATCC 19115 was studied with stainless steel coupons immersed in BHI broth and BHI broth plus sucrose in combination with the Lactic Acid Bacteria (LAB). Adhered cells were collected with swabs and enumerated on selective agars (Oxford for listeria and MRS for leuconostoc). Leuconostoc mesenteroides bac+ in co-culture with L. monocytogenes was effective to inhibit biofilm formation by listeria for up to 3 hours of incubation, but at 24 hours, biofilm was present in all conditions tested, as confirmed by observations of stainless steel coupons under Scanning Electron Microscopy (SEM). It was also observed that in the presence of L. mesenteroides bac+ in BHI plus sucrose, a high number of elongated cells of L. monocytogenes was present, which may indicate an adaptation response of the pathogen to stress conditions with important implications for food safety

Targeting bacteriocin genes in lactic acid bacteria: what we need to know

In the last decade, the aim of several research papers has focused on the determination of bacteriocin genes in genomic or plasmid DNA of lactic acid bacteria (LAB). These reports can be divided in two major groups; the first of which contains articles reporting the detection of these genes and giving a scientific proof of their expression, usually through RT-PCR, or by the amino-acid sequencing of the produced bacteriocin. In the second group, the presence of known bacteriocin genes in genomic or plasmid DNA of newly isolated LAB is reported, but authors do not provide any significant evidence that the detected genes are or might be expressed. These latter reports need to be received with high criticism and skepticism. Combination of the bio-molecular and bio-chemical approaches is critical in the better understanding of the expression of genes encoding the bacteriocins from LAB.In this review we summarize these two groups of research: those targeting the bacteriocin genes with a solid proof of expression of the bacteriocins and those that only report on the presence of the bacteriocins genes in the genomic and plasmid DNA.Fil: Todorov, Svetoslav Dimitrov. Universidade de Sao Paulo; BrasilFil: Kruger, Monika Francisca. Universidade de Sao Paulo; BrasilFil: Chacon Ruiz Martinez, Rafael. Universidade de Sao Paulo; BrasilFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Gombossy de Melo Franco, Bernadette Dora. Universidade de Sao Paulo; Brasi

Biochemical, antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.Fil: Chacon Ruiz Martinez, Rafael. Universidade de Sao Paulo; BrasilFil: Wachsman, Mónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Torres, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Todorov, Svetoslav Dimitrov. Universidade de Sao Paulo; BrasilFil: Melo Franco, Bernadette Dora Gombossy de. Universidade de Sao Paulo; Brasi

Populations of <i>B. animalis</i> Bb-12, <i>L. acidophilus</i> La-5, and <i>L. sakei</i> 2a assessed in the probiotic <i>petit-suisse</i> cheese (F2) throughout storage (1, 14, and 28 days) at 4°C and during the <i>in vitro</i> assay simulating the gastrointestinal conditions [time-point zero, and after 2 h (gastric phase), 4 h (enteric phase I), and 6 h (enteric phase II)].

<p>Populations of <i>B. animalis</i> Bb-12, <i>L. acidophilus</i> La-5, and <i>L. sakei</i> 2a assessed in the probiotic <i>petit-suisse</i> cheese (F2) throughout storage (1, 14, and 28 days) at 4°C and during the <i>in vitro</i> assay simulating the gastrointestinal conditions [time-point zero, and after 2 h (gastric phase), 4 h (enteric phase I), and 6 h (enteric phase II)]. The samples were analyzed using three methods: plate counts, Real-time PCR (qPCR) and Real-time PCR combined with propidium monoazide (PMA-qPCR).</p><p><b>Footnote:</b></p><p>Values are expressed as mean log CFU/g ± standard deviation (SD) obtained by plate count method and as log CFU equivalents/g as calculated from Ct values for qPCR and PMA-qPCR;</p>A,B,C<p>Different superscript capital letters in a row denote significant differences between methods (<i>P</i><0.05).</p>a,b,c<p>Different superscript lowercase letters in the same column for each phase denote significant differences between storage days (<i>P</i><0.05).</p

Morphological changes in <i>S. thermophilus</i>, La-5, Bb-12, and <i>L. sakei</i> 2a during simulated digestive stress.

<p>Morphological changes, observed through scanning electron microscopy, in <i>S. thermophilus</i>, <i>L. acidophilus</i> La-5, <i>B. animalis</i> Bb-12, and <i>L. sakei</i> 2a throughout the different phases of the in <i>vitro</i> assay simulating the gastrointestinal conditions. Over the entire experiment, some representative photographs were obtained at time-point zero (untreated cells) (<b>A</b>); after 2 h, gastric phase (pH 2.3–2.6 in the presence of pepsin and lipase, 2 h) (<b>B</b>); after 4 h, enteric phase I (pH 5.4–5.7 in the presence of pancreatin and bile, 2 h) (<b>C</b>), and after 6 h, enteric phase II (pH 6.8–7.2 in the presence of pancreatin and bile (<b>D</b>) are shown. (<b>1</b>) <i>L. sakei</i> 2a; (<b>2</b>) <i>L. acidophilus</i> La-5; (<b>3</b>) <i>B. animalis</i> Bb-12, and (<b>4</b>) <i>S. thermophilus</i>.</p