Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

We developed a PCR-based method to detect and quantify viable Bifidobacterium bifidum BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 10 10 to 10 6 cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4=,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 10 5.3 to 10 10.3 cells/g feces (wet weight) (r > 0.99, P < 0.001). After 12 healthy subjects ingested 10 10.3 to 10 11.0 CFU of BF-1 in a fermented milk product daily for 28 days, 10 4.5 ؎ 1.5 (mean ؎ standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 10 6.2 ؎ 0.4 viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 10 7.6 ؎ 0.7 BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (P < 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces.

Section: Fujimoto and Watanabesupporting

confidence: 64%

Quantitative Detection of Viable Bifidobacterium bifidum BF-1 Cells in Human Feces by Using Propidium Monoazide and Strain-Specific Primers

Fujimoto

Watanabe

2013

“…LGG_00154 encodes a hypothetical membrane associated protein, designated map, located outside LGGISL1,2 (locus 168611 to 168105). Moreover, LGG_00154 is located on an amplicon previously selected for specific identification of L. rhamnosus GG (12).…”

Section: Methodsmentioning

confidence: 99%

Genome Instability in Lactobacillus rhamnosus GG

Sybesma¹,

Molenaar²,

IJcken

et al. 2013

We describe here a comparative genome analysis of three dairy product isolates of Lactobacillus rhamnosus GG (LGG) and the ATCC 53103 reference strain to the published genome sequence of L. rhamnosus GG. The analysis showed that in two of three isolates, major DNA segments were missing from the genomic islands LGGISL1,2. The deleted DNA segments consist of 34 genes in one isolate and 84 genes in the other and are flanked by identical insertion elements. Among the missing genes are the spaCBA genes, which encode pilin subunits involved in adhesion to mucus and persistence of the strains in the human intestinal tract. Subsequent quantitative PCR analyses of six commercial probiotic products confirmed that two more products contain a heterogeneous population of L. rhamnosus GG variants, including genotypes with or without spaC. These results underline the relevance for quality assurance and control measures targeting genome stability in probiotic strains and justify research assessing the effect of genetic rearrangements in probiotics on the outcome of in vitro and in vivo efficacy studies. R ecently, the concept of "generic probiotics" was introduced, as a practical solution to create access to probiotics for people in the developing world (1). This concept refers to the free use of probiotic bacteria introduced under a novel brand name, after intellectual property rights have expired. In analogy to generic drugs, also patent-expired probiotics are free to be used by others, and health and safety claims from the expired patents can be linked to the generic probiotic strains, provided that the genotype of the original strain is identical to that of the generic strain. This raises questions about the extent of genome stability in probiotic bacterial strains and its impact on probiotic functionality.Today, only a few examples exist about active components of probiotics, which could be targeted to confirm the presence of the associated functionality (2). Lebeer et al. (3) report on the classification of several genes and molecules that contribute to probiotic and health-promoting actions of Lactobacillus: (i) adaptation factors, including determinants of stress resistance, metabolism in the host, and adherence to the gut mucosa, and (ii) probiotic factors directly mediating health effects including antipathogenic, epithelium barrier-preserving, and immunomodulatory molecules. In another recent study, a collection of lactic acid bacteria isolated from fermented foods has been screened for more than 30 probiotic functionality related genes. The authors defined genes as "probiotic" that are involved in survival in the gastrointestinal tract (resistance to low pH and bile salt), starch metabolism, and folate and riboflavin synthesis (4).Many of the probiotic strains marketed today originate from intestinal or plant isolates, and the shift from their complex and highly variable niche to the relatively constant and nutrient-rich industrial production environment is likely to be linked with selection for metabolic simplifica...

“…Three technical replicates from three biological samples were performed for each time point. Two sets of primers were employed, the pair srtC1-for (5=-AGTGCGACTATTAGCTTTA-3=) and srtC1-rev (5=-GGATCTTGT GACCTTAATG-3=) and a GG-specific primer pair (29).…”

Section: Methodsmentioning

confidence: 99%

Polymorphisms, Chromosomal Rearrangements, and Mutator Phenotype Development during Experimental Evolution of Lactobacillus rhamnosus GG

Douillard

Ribbera

Xiao

et al. 2016

Lactobacillus rhamnosus GG is a lactic acid bacterium widely marketed by the food industry. Its genomic analysis led to the identification of a gene cluster encoding mucus-binding SpaCBA pili, which is located in a genomic island enriched in insertion sequence (IS) elements. In the present study, we analyzed by genome-wide resequencing the genomic integrity of L. rhamnosus GG in four distinct evolutionary experiments conducted for approximately 1,000 generations under conditions of no stress or salt, bile, and repetitive-shearing stress. Under both stress-free and salt-induced stress conditions, the GG population (excluding the mutator lineage in the stress-free series [see below]) accumulated only a few single nucleotide polymorphisms (SNPs) and no frequent chromosomal rearrangements. In contrast, in the presence of bile salts or repetitive shearing stress, some IS elements were found to be activated, resulting in the deletion of large chromosomal segments that include the spaCBA-srtC1 pilus gene cluster. Remarkably, a high number of SNPs were found in three strains obtained after 900 generations of stress-free growth. Detailed analysis showed that these three strains derived from a founder mutant with an altered DNA polymerase subunit that resulted in a mutator phenotype. The present work confirms the stability of the pilus production phenotype in L. rhamnosus GG under stress-free conditions, highlights the possible evolutionary scenarios that may occur when this probiotic strain is extensively cultured, and identifies external factors that affect the chromosomal integrity of GG. The results provide mechanistic insights into the stability of GG in regard to its extensive use in probiotic and other functional food products. IMPORTANCELactobacillus rhamnosus GG is a widely marketed probiotic strain that has been used in numerous clinical studies to assess its health-promoting properties. Hence, the stability of the probiotic functions of L. rhamnosus GG is of importance, and here we studied the impact of external stresses on the genomic integrity of L. rhamnosus GG. We studied three different stresses that are relevant for understanding its robustness and integrity under both ex vivo conditions, i.e., industrial manufacturing conditions, and in vivo conditions, i.e., intestinal tract-associated stress. Overall, our findings contribute to predicting the genomic stability of L. rhamnosus GG and its ecological performance. Lactobacillus rhamnosus GG is a human intestinal isolate (1) that has been extensively studied over the last 2 decades for its probiotic properties and impact on human health (2, 3). In numerous clinical trials, L. rhamnosus was shown to display beneficial properties in humans, such as the reduction of diarrhea (4-7), atopic eczema (2), and respiratory infections (8, 9). Its implementation in a large variety of applications, food products, and clinical trials was possible, thanks to the remarkably high adaptability and resilience of L. rhamnosus GG to adverse culturing, handling, or stora...