The Experts below are selected from a list of 3087 Experts worldwide ranked by ideXlab platform
Todd R Klaenhammer - One of the best experts on this subject based on the ideXlab platform.
-
Deletion-based escape of CRISPR-Cas9 targeting in Lactobacillus gasseri.
Microbiology (Reading England), 2018Co-Authors: Emily Stout, Todd R Klaenhammer, Yong Jun Goh, Rosemary Sanozky-dawes, Alexandra B. Crawley, Rodolphe BarrangouAbstract:Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52–70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1–15 spacers, and systematically included the leader-proximal targeting spacer. This study shows that deletions of spacers within CRISPR arrays constitute a key escape mechanism to evade CRISPR targeting, while preserving the functionality of the CRISPR-Cas system. This mechanism enables cells to maintain an active immune system, but allows the uptake of potentially beneficial plasmids. Our study revealed the co-occurrence of other genomic mutations associated with various phenotypes, showing how this selection process uncovers population diversification.
-
Development of an integration mutagenesis system in Lactobacillus gasseri
Gut microbes, 2014Co-Authors: Kurt Selle, Yong Jun Goh, Sarah O'flaherty, Todd R KlaenhammerAbstract:Lactobacillus gasseri ATCC 33323 is a member of the acidophilus-complex group, microbes of human origin with significant potential for impacting human health based on niche-specific traits. In order to facilitate functional analysis of this important species, a upp-based counterselective chromosomal integration system was established and employed for targeting the lipoteichoic acid (LTA) synthesis gene, ltaS, in L. gasseri ATCC 33323. The ltaS gene encodes a phosphoglycerol transferase responsible for building the glycerol chain of LTA. No isogenic mutant bearing the deletion genotype was recovered, but an integration knockout mutant was generated with insertion inactivation at the ltaS locus. The ltaS deficient derivative exhibited an altered cellular morphology and significantly reduced ability to adhere to Caco-2 intestinal cell monolayers, relative to the wild-type parent strain.
-
Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health
FEMS microbiology reviews, 2013Co-Authors: Kurt Selle, Todd R KlaenhammerAbstract:Certain lactic acid bacteria (LAB) have the capacity to occupy mucosal niches of humans, including the oral cavity, gastrointestinal tract, and vagina. Among commensal, LAB are species of the acidophilus complex, which have proven to be a substantial reservoir for microorganisms with probiotic attributes. Specifically, Lactobacillus gasseri is an autochthonous microorganism which has been evaluated for probiotic activity based on the availability of genome sequence and species-specific adaptation to the human mucosa. Niche-related characteristics of L. gasseri contributing to indigenous colonization include tolerance of low pH environments, resistance to bile salts, and adhesion to the host epithelium. In humans, L. gasseri elicits various health benefits through its antimicrobial activity, bacteriocin production, and immunomodulation of the innate and adaptive systems. The genomic and empirical evidence supporting use of L. gasseri in probiotic applications is substantiated by clinical trial data displaying maintenance of vaginal homeostasis, mitigation of Helicobacter pylori infection, and amelioration of diarrhea.
-
Characterization of Lactobacillus gasseri isolates from a breast-fed infant.
Gut microbes, 2012Co-Authors: Luciana Rodrigues Da Cunha, Yong Jun Goh, Célia Lucia De Luces Fortes Ferreira, Evelyn Durmaz, Rosemary Sanozky-dawes, Todd R KlaenhammerAbstract:The potential health benefits of probiotic bacteria have led to the isolation of new microbial strains for incorporation into food products. However, newly isolated candidate probiotic organisms do not automatically share the “generally recognized as safe” (GRAS) status of traditional lactic acid bacteria (LAB). Before their introduction into food products, the safety of new isolates has to be evaluated. The objective of this study was to characterize LAB isolates from the stool of a newborn infant, and evaluate their safety and probiotic potential, in vitro. Thirty colonies were identified as Lactobacillus gasseri through sequencing of 16S rDNA. Pulsed Field Gel Electrophoresis using restriction enzymes SmaI and Apa I revealed that 29 of the L. gasseri were nearly identical, however one isolate exhibited a distinctive DNA fingerprint. All 30 L. gasseri were evaluated for resistance to antibiotics, bile tolerance, hemolytic activity and antagonism toward selected pathogens. All 30 strains harbored three p...
-
targeted expression of anthrax protective antigen by Lactobacillus gasseri as an anthrax vaccine
Future Microbiology, 2010Co-Authors: Mansour Mohamadzadeh, Evelyn Durmaz, Mojgan Zadeh, Krishna Chaitanya Pakanati, Matthew Gramarossa, Valeria C Cohran, Todd R KlaenhammerAbstract:Aim: Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice. Materials & methods: Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection. Results: Oral application of L. gasseri expressing the PA–DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expressio...
Hanan R Shehata - One of the best experts on this subject based on the ideXlab platform.
-
real time pcr assays for the specific identification of probiotic strains Lactobacillus gasseri bnr17 and Lactobacillus reuteri lrc ncimb 30242
Probiotics and Antimicrobial Proteins, 2020Co-Authors: Hanan R Shehata, Subramanyam Ragupathy, Shara Allen, Gregory Leyer, Steven G NewmasterAbstract:The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.
-
Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)
Probiotics and Antimicrobial Proteins, 2020Co-Authors: Hanan R Shehata, Subramanyam Ragupathy, Shara Allen, Gregory Leyer, Steven G NewmasterAbstract:The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was
Steven G Newmaster - One of the best experts on this subject based on the ideXlab platform.
-
real time pcr assays for the specific identification of probiotic strains Lactobacillus gasseri bnr17 and Lactobacillus reuteri lrc ncimb 30242
Probiotics and Antimicrobial Proteins, 2020Co-Authors: Hanan R Shehata, Subramanyam Ragupathy, Shara Allen, Gregory Leyer, Steven G NewmasterAbstract:The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.
-
Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)
Probiotics and Antimicrobial Proteins, 2020Co-Authors: Hanan R Shehata, Subramanyam Ragupathy, Shara Allen, Gregory Leyer, Steven G NewmasterAbstract:The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was
B Di Luccia - One of the best experts on this subject based on the ideXlab platform.
-
characterisation of efv12 a bio active small peptide produced by the human intestinal isolate Lactobacillus gasseri sf1109
Beneficial Microbes, 2020Co-Authors: M Di Napoli, B Di Luccia, Giuseppe Vitiello, Gerardino Derrico, Andrea Carpentieri, Alessandro Pezzella, Elio Pizzo, Eugenio Notomista, Mario Varcamonti, Anna ZanfardinoAbstract:EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties ...
-
Characterisation of EFV12 a bio-active small peptide produced by the human intestinal isolate Lactobacillus gasseri SF1109.
Beneficial microbes, 2020Co-Authors: M Di Napoli, B Di Luccia, Giuseppe Vitiello, Andrea Carpentieri, Alessandro Pezzella, Elio Pizzo, Eugenio Notomista, Mario Varcamonti, Gerardino D'errico, Anna ZanfardinoAbstract:EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties are characterised. In particular, we propose a possible mechanism of action for EFV12 involving bacterial membranes targeting. Moreover, we show that this small peptide is able to bind lipopolysaccharides (LPS) and to counteract its inflammatory insult preventing LPS action on Toll-like receptor 4, thus interfering with extracellular signal-regulated kinase, p38 and Jun N-terminal kinase, mitogen-activated protein kinases signalling pathways. Altogether these observations suggest that the bioactive peptide EFV12 is a good candidate to promote L. gasseri induced gut homeostasis and counteracting intestinal pathogens.
-
Lactobacillus gasseri SF1183 protects the intestinal epithelium and prevents colitis symptoms in vivo
Journal of Functional Foods, 2018Co-Authors: B Di Luccia, Arianna Mazzoli, Rosa Cancelliere, Raffaella Crescenzo, I. Ferrandino, A. Monaco, Antonio Bucci, Gino Naclerio, Susanna Iossa, Ezio RiccaAbstract:Abstract Lactobacillus gasseri SF1183 belongs to a subpopulation of bacteria tightly associated to the human ileal epithelium. Cells of SF1183 survive and grow in simulated intestinal and gastric conditions, have a strong antimicrobial activity against Gram-positives and Gram-negatives and secrete molecule(s) sensed by human intestinal cells. We report that the oral administration of SF1183 cells had a protective effect in a murine model of DSS (Dextran-Sulfate-Sodium)-induced colitis. The analysis of the intestinal microbial composition indicated that several bacterial genera were differently represented in the intestine of DSS-treated animals. An overall similar alteration was observed in the microbiota of DSS-treated animals that received SF1183, suggesting that the beneficial role of the probiotic was not played through a reshuffling of the intestinal flora. Based on our in vivo data we propose the SF1183 strain of L. gasseri as a new anti-inflammatory probiotic, potentially useful as a therapeutic agent for the treatment of IBDs.
-
Identification of a new small bioactive peptide from Lactobacillus gasseri supernatant.
Beneficial microbes, 2016Co-Authors: Anna Zanfardino, B Di Luccia, Andrea Carpentieri, Alessandro Pezzella, Elio Pizzo, Eugenio Notomista, G. Criscuolo, M.l. Ciavatta, Mario VarcamontiAbstract:Ten lactic acid bacteria (LAB) strains, previously isolated from human ileal biopsy of healthy volunteers, were screened for production and secretion of molecules having anti-bacterial and anti-biofilm activities. Because many recent reports indicate that LAB secreted molecules may exert immune-modulatory action, we also tested the effect on human intestinal HCT116 cells challenged with bacterial lipopolysaccharides. One of the Lactobacillus gasseri strains, SF1109, strongly inhibited: (1) Pseudomonas aeruginosa growth; (2) Escherichia coli biofilm production; (3) LPS induction of P-ERK1/2 in HCT116 cells, and was selected for further characterisation of the secreted active molecule. Cell-free supernatant of the L. gasseri SF1109 was analysed and one 1.3 kDa peptide has been characterised. Eight out twelve amino acids of this peptide were identified allowing the synthesis of an octa-peptide which still presented the mentioned activities.
Subramanyam Ragupathy - One of the best experts on this subject based on the ideXlab platform.
-
real time pcr assays for the specific identification of probiotic strains Lactobacillus gasseri bnr17 and Lactobacillus reuteri lrc ncimb 30242
Probiotics and Antimicrobial Proteins, 2020Co-Authors: Hanan R Shehata, Subramanyam Ragupathy, Shara Allen, Gregory Leyer, Steven G NewmasterAbstract:The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.
-
Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)
Probiotics and Antimicrobial Proteins, 2020Co-Authors: Hanan R Shehata, Subramanyam Ragupathy, Shara Allen, Gregory Leyer, Steven G NewmasterAbstract:The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was
