Flow cytometric assessment of the protectants for enhanced in vitro survival of probiotic lactic acid bacteria through simulated human gastro-intestinal stresses

Chen, Song; Cao, Yu; Ferguson, Lynnette R.; Qin, Shu; Garg, Sanjay

doi:10.1007/s00253-012-4030-3

Cited by 35 publications

(14 citation statements)

References 44 publications

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“…Further, the large number of bacteria analyzed by flow cytometry increases counting precision and accuracy; according to Nebe-von-Caron, variation between readings is less than 1% when more than 1000 events are used for viability measurements [27]. Flow cytometry has been used to monitor the inactivation of Escherichia coli on solid food, to control water quality after chlorination, to verify the vitality of probiotic lactic acid bacteria exposed to gastric acid and bile salts [28][29][30]. Previous work from our group also used FCM for measuring viability of several oral pathogens after photo-inactivation [9,14].…”

Section: Discussionmentioning

confidence: 99%

Repeated exposures to blue light-activated eosin Y enhance inactivation of E. faecalis biofilms, in vitro

Marinic

Manoil

Filieri

et al. 2015

Photodiagnosis and Photodynamic Therapy

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Background: In dentistry, antibacterial photodynamic therapy (a-PDT) has shown promising results for inactivating bacterial biofilms causing carious, endodontic and periodontal diseases. In the current study, we assessed the ability of eosin Y exposed to 3 irradiation protocols at inactivating Enterococcus faecalis biofilms, in vitro. Methods: E. faecalis biofilms formed on hydroxyapatite disks were incubated with eosin Y (10-80 M), then activated with blue light using different irradiation protocols. Biofilms exposed to continuous exposure were incubated for 40 min before being light-activated for 960 s. For the intermittent exposure, biofilms were exposed 4 times to the light/photosensitizer combination (960 s total) without renewing the photosensitizer. For repeated a-PDT, the same light dose was delivered in a series of 4 irradiation periods separated by dark periods; fresh photosensitizer was added between each light irradiation. After treatment, bacteria were immediately labeled with LIVE/DEAD BacLight Bacterial Viability kit and viability was assessed by flow cytometry (FCM). Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (˛= 0.05). * Corresponding author. Results:The viability of E. faecalis biofilms exposed to 10 M eosin Y, was significantly reduced compared to controls (light only-eosin Y only). After a second exposure to blue light-activated eosin Y, viability significantly decreased from 58% to 12% whereas 6.5% of the bacterial biofilm remained live after a third exposure (p < 0.05). Only 3.5% of the bacterial population survived after the fourth exposure. Conclusions:The results of this study indicate that blue light-activated eosin Y can photoinactivate E. faecalis biofilms grown on hydroxyapatite disks. Also, repeated exposures to blue light-activated eosin Y were shown to significantly improve efficacy. Further studies seem warranted to optimize the antibacterial activity of blue light-activated eosin Y on major oral pathogens.

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Section: Discussionmentioning

confidence: 99%

Repeated exposures to blue light-activated eosin Y enhance inactivation of E. faecalis biofilms, in vitro

Marinic

Manoil

Filieri

et al. 2015

Photodiagnosis and Photodynamic Therapy

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“…Flow cytometry allows the study of a large number of cells at a time, recording, for each event (bacteria) several parameters. Different fluorescent probes can be applied to examine the physiological characteristics of probiotic living cells, such cell membrane integrity, intracellular enzyme activity, cytoplasmic pH, and membrane potential [92]. Limitations and new challenges in probiotic enumeration and identification can be found in Davis [85].…”

Section: Enumeration Of Probiotic Strainsmentioning

confidence: 99%

Probiotic Lactic Acid Bacteria: A Review

Quinto¹,

Jiménez²,

Caro³

et al. 2014

FNS

120

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Lactic acid bacteria (LAB) play a critical role in food production and health maintenance. There is an increasing interest in these species to reveal the many possible health benefits associated with them. The actions of LAB are species and strain specific, and depend on the amount of bacteria available in the gastrointestinal tract. Consumers are very concerned of chemical preservatives and processed foods. However, products with or processed with LAB are accepted as a natural way to preserve food and promote health. This paper aimed to review the recent data in regard to the role of probiotic LAB in the preservation of foods, in the immunomodulation in the gastrointestinal tract, and in its health benefits.The hypothetical first niche of the ancestral LAB is considered soil and plants and, subsequently, the gut of herbivorous animals [6]. The mammalian intestine is colonized by 100 trillions of microorganisms (called "microbiota") that are essential for health [7] [8]. The transition from soil and plants to the animal gut has three areas of genomic adaptation [9]: resistance to host barriers, adhesion to intestinal cells, and fermentation of some substrates in the gut. The membrane lipid composition is affected by low pH and bile salts. Microarray analysis has shown the expression of a glycerophosphatase in Lactobacillus reuteri after an acid shock, and an increase in the sensitivity to acid in Lactobacillus acidophilus [10]. Extracelullar lipopolysaccharides (LPS) also play a role in the resistance, but it is unclear [6]. The adhesion of LAB to intestinal cells is associated with the peristaltic flow, a good adherence capacity and the presence of mucins to protect and lubricate the epithelial surfaces [11]. Resident intestinal bacteria are able to inhibit the adherence of pathogenic bacteria to intestinal epithelial cells as a result of their ability to increase the production of intestinal mucins [12]. Lactobacillus plantarum increases the levels of expression of the mRNA of some mucins, inhibiting the cell attachment of enteropathogenic Escherichia coli [13] [14]. LAB have access to simple sugars and complex carbohydrates, so bacteria with genes involved in its degradation are probably in better condition to multiply in the gut [6].The resident gastrointestinal microbiota provide a microbial barrier against microbial pathogens [12]. Lactobacillus and Bifidobacterium spp. of human intestinal origin produce antimicrobial substances that are active in vitro and in vivo against enteropathogenic microorganisms involved in diarrhea [15]; both genera have the capacity for interfering with or block the pathogenic process of enteric bacterial pathogens [12]. Strains of Lactobacillus acidophilus, L. johnsonii, L. rhamnosus, L. casei, L. acidophilus, and L. rhamnosus interfere with a wide range of pathogens, such as enteropathogenic Escherichia coli, enterohemorrhagic E. coli, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and S. flexneri [16]-[22]. Blocking the process of pathogenicity of enteric pat...

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“…; Chen et al . ), antibiotic susceptibility (Novo et al . ) and microbial communities study (Apajalahti et al .…”

Section: Introductionmentioning

confidence: 99%

“…The use of flow cytometry in combination with fluorescent staining has increased within the microbiology field over the last 20 years (Comas-Riu and Rius 2009;Davis 2014;Sohier et al 2014). Viability staining and flow cytometry (viable flow cytometry) has been extensively used to monitor bacteria physiological state (Joux and Lebaron 2000;Doherty et al 2010), survival upon physical or chemical stresses (Amor et al 2002;Rault et al 2007;Chen et al 2012), antibiotic susceptibility (Novo et al 2000) and microbial communities study (Apajalahti et al 2002;Muller and Nebe-von-Caron 2010;Koch et al 2014). Viable flow cytometry techniques have also been employed for LAB and probiotic quantification on various products such as lyophilized powders (Kramer et al 2009), fermented products (Doherty et al 2010), dairy (Gunasekera et al 2000), food (Raymond and Champagne 2015) and complex matrices, such as artificial gut microbiota (Grootaert et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products

2018

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Aims: Classical microbiology techniques are the gold standard for probiotic enumeration. However, these techniques are limited by parameters of time, specificity and incapacity to detect viable but nonculturable (VBNC) microorganisms and nonviable cells. The aim of the study was to evaluate flow cytometry as a novel method for the specific quantification of viable and nonviable probiotics in multistrain products. Methods and Results: Custom polyclonal antibodies were produced against five probiotic strains from different species (Bifidobacterium bifidum R0071, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium longum ssp. longum R0175, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011). Evaluation of specificity confirmed that all antibodies were specific at least at the subspecies level. A flow cytometry method combining specific antibodies and viability assessment with SYTO â 24 and propidium iodide was applied to quantify these strains in three commercial products. Analyses were conducted on two flow cytometry instruments by two operators and compared with classical microbiology using selective media. Results indicated that flow cytometry provides higher cell counts than classical microbiology (P < 0Á05) in 73% of cases highlighting the possible presence of VBNC. Equivalent performances (repeatability and reproducibility) were obtained for both methods. Conclusions: This study showed that flow cytometry methods can be applied to probiotic enumeration and viability assessment. Combination with polyclonal antibodies can achieve sufficient specificity to differentiate closely related strains. Significance and Impact of the Study: Flow cytometry provides absolute and specific quantification of viable and nonviable probiotic strains in a very short time (<2 h) compared with classical techniques (>48 h), bringing efficient tools for research and development and quality control.

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Flow cytometric assessment of the protectants for enhanced in vitro survival of probiotic lactic acid bacteria through simulated human gastro-intestinal stresses

Cited by 35 publications

References 44 publications

Repeated exposures to blue light-activated eosin Y enhance inactivation of E. faecalis biofilms, in vitro

Repeated exposures to blue light-activated eosin Y enhance inactivation of E. faecalis biofilms, in vitro

Probiotic Lactic Acid Bacteria: A Review

Flow cytometry: a versatile technology for specific quantification and viability assessment of micro-organisms in multistrain probiotic products

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