PCR-based analytics of gene therapies using adeno-associated virus vectors: Considerations for cGMP method development.

The field of gene therapy has evolved and improved so that today the treatment of thousands of genetic diseases is now possible. An integral aspect of the drug development process is generating analytical methods to be used throughout clinical and commercial manufacturing. Enumeration and identification assays using genetic testing are critical to ensure the safety, efficacy, and stability of many active pharmaceutical ingredients. While nucleic acid-based methods are already reliable and rapid, there are unique biological, technological, and regulatory aspects in gene therapies that must be considered. This review surveys aspects of method development and validation using nucleic acid-based testing of gene therapies by focusing on adeno-associated virus (AAV) vectors and their co-transfection factors. Key differences between quantitative PCR and droplet digital technologies are discussed to show how improvements can be made while still adhering to regulatory guidance. Example validation parameters for AAV genome titers are described to demonstrate the scope of analytical development. Finally, several areas for improving analytical testing are presented to inspire future innovation, including next-generation sequencing and artificial intelligence. Reviewing the broad characteristics of gene therapy assessment serves as an introduction for new researchers, while clarifying processes for professionals already involved in pharmaceutical manufacturing.

The Effect of Bifidobacterium animalis subsp. lactis Bl-04 on Influenza A Virus Infection in Mice.

Influenza A virus infection is a major global disease requiring annual vaccination. Clinical studies indicate that certain probiotics may support immune function against influenza and other respiratory viruses, but direct molecular evidence is scarce. Here, mice were treated with a placebo or Bifidobacterium animalis subsp. lactis Bl-04 (Bl-04) orally via food (cereal) and also by gavage and exposed to Influenza A virus H1N1 (H1N1). The symptoms of the infection were observed, and tissues and digesta were collected for viral load RT-qPCR, transcriptomics, and microbiomics. The treatment decreased the viral load by 48% at day 3 post-infection in lungs and symptoms of infection at day 4 compared to placebo. Tissue transcriptomics showed differences between the Bl-04 and placebo groups in the genes in the Influenza A pathway in the intestine, blood, and lungs prior to and post-infection, but the results were inconclusive. Moreover, 16S rRNA gene profiling and qPCR showed the presence of Bl-04 in the intestine, but without major shifts in the microbiome. In conclusion, Bl-04 treatment may influence the host response against H1N1 in a murine challenge model; however, further studies are required to elucidate the mechanism of action.

An in vitro model of the chicken gastrointestinal tract with special emphasis to the cecal microbiota.

An in vitro model of the upper gastrointestinal tract as well as the chicken cecum was developed to have a predictive tool for estimating the production performance of animals by analyzing the feeding value of a certain diet. The upper gastrointestinal tract consists of a batch type model, whereas the cecal model is comprised of 4 semi-continuous connected vessels inoculated with cecal or fecal microbes. The upper gastrointestinal tract and cecal simulations were both run with a corn- and a wheat-based diet to simulate 2 typical feed types. Samples were collected after the 5-h cecal simulations and aliquots were frozen to assess inoculum stability. The microbiota was analyzed by 16S rRNA gene sequencing, whereas short chain fatty acids as microbial metabolites were analyzed by using gas chromatography. As expected, some significant differences in microbial abundance after simulation between the cecal and fecal slurry samples (P = 0.001) were detected, as well between the fresh and frozen status (P = 0.001), hence simulations inoculated with cecal and fresh samples being more diverse. For the measured metabolites, almost all of them increased (P < 0.05) significantly when comparing fresh and frozen inoculum. The present chicken intestinal in vitro model represents a rapid systematic screening system for studying dietary related microbial changes and reducing the need of animal sacrifice for experimentation.

Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium.

Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR-based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics.

Transcriptomics reveal different metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in select Levilactobacillus brevis strains.

BACKGROUND: Of the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental health outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health. RESULTS: Three strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108™ (Lbr-6108), formerly known as L. brevis DPC 6108, and Levilactobacillus brevis Lbr-35 ™ (Lbr-35) had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early within the growth phase and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14869 after the stationary phase. The global gene expression during GABA production at several timepoints was determined by RNA sequencing. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only 6 h of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG. CONCLUSIONS: This study is the first to evaluate whole transcriptome changes in Levilactobacillus brevis during GABA production in different growth phases. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain's potential as a therapeutic and the overall value of screening microbes for effector molecule output.

Epigenetics: A New Frontier in Probiotic Research.

Research into the benefits of probiotics has progressed beyond interventional studies to identifying the underlying molecular mechanisms. Health-promoting effector molecules produced by probiotics are well documented and have been linked to specific genes and even individual nucleotides. However, the factors controlling the expression of these molecules are poorly understood and we argue that epigenetic influences likely play an important role in mediating the health-promoting attributes of probiotics. Here, we review established epigenetic regulation of important microbial genetic systems involved in health promotion, safety, and industrialization to provide evidence that the same regulation occurs in probiotic organisms. We advocate for studies combining genomic and meta-epigenomic data to better understand the mode of action of probiotics, their associated microbiomes, and their effects on consumers.

Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™.

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values > 0.99), and 87, 84, and 86% (R square values > 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2'-fucosyllactose (2'-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5-0.7) and late-log phases (1.0-2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2'-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2'-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2'-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2'-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Droplet Digital PCR Is an Improved Alternative Method for High-Quality Enumeration of Viable Probiotic Strains.

Traditional microbiological enumeration methods have long been employed as the standard evaluation procedure for probiotic microorganisms. These methods are labor intensive, have long-time to results and inherently have a high degree of variability - up to 35%. As clinical probiotic and microbiome science continues to grow and develop, it is increasingly important that researchers thoroughly define and deliver the targeted probiotic dose. Furthermore, to establish high quality commercial products, the same dosage level must be administered to consumers. An ISO method for the use of flow cytometry has been established which does speed up the time to results and reduce variability, but the method has not yet gained widespread adoption across the probiotic industry. This is possibly due to expertise needed to implement and maintain a new testing platform in an established quality system. In this study we compare enumeration using plate counts and flow cytometry to the use of droplet digital PCR (ddPCR), which in addition to giving faster time to results than plate count and less variability than both plate count and flow cytometry, has additional benefits such as strain-specific counts. Use of ddPCR gives the ability to design primers to target deletions and single base pair differences which will allow for strain profiling in microbiome analyses. We demonstrate that ddPCR probiotic enumeration results are positively correlated to both plate count and flow cytometry results and should be considered a viable, next generation enumeration method for the evaluation of probiotics.

Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline.

Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.

Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04 ® via Chip-Based Digital PCR.

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.

Metabolic Fate of 13C-Labeled Polydextrose and Impact on the Gut Microbiome: A Triple-Phase Study in a Colon Simulator.

The present study introduces a novel triple-phase (liquids, solids, and gases) approach, which employed uniformly labeled [U-13C] polydextrose (PDX) for the selective profiling of metabolites generated from dietary fiber fermentation in an in vitro colon simulator using human fecal inocula. Employing 13C NMR spectroscopy, [U-13C] PDX metabolism was observed from colonic digest samples. The major 13C-labeled metabolites generated were acetate, butyrate, propionate, and valerate. In addition to these short-chain fatty acids (SCFAs), 13C-labeled lactate, formate, succinate, and ethanol were detected in the colon simulator samples. Metabolite formation and PDX substrate degradation were examined comprehensively over time (24 and 48 h). Correlation analysis between 13C NMR spectra and gas production confirmed the anaerobic fermentation of PDX to SCFAs. In addition, 16S rRNA gene analysis showed that the level of Erysipelotrichaceae was influenced by PDX supplementation and Erysipelotrichaceae level was statistically correlated with SCFA formation. Overall, our study demonstrates a novel approach to link substrate fermentation and microbial function directly in a simulated colonic environment.

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics.

Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation "well defined strains," encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species using plate counting and targeted genotyping. Strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS) of the 16S rRNA sequence using paired-end 2 × 250 bp Illumina MiSeq technology. Using these methods, we tested the hypothesis that products do contain the quantitative and qualitative list of labeled microbial species. We found that 17 samples (33%) were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58%) of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species, and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to determine the correct taxonomy and quantify the relative amounts of mixed microbial populations in commercial probiotic products.

CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus.

UNLABELLED: Many bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (Streptococcus thermophilus-phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR. Mutation rates in phage genomes highly exceed those of the host. The presence of multiple phages increased phage persistence by enabling recombination-based formation of chimeric phage genomes in which sequences heavily targeted by CRISPR were replaced. Collectively, our results establish CRISPR-Cas adaptive immunity as a key driver of phage genome evolution under the conditions studied and highlight the importance of multiple coexisting phages for persistence in natural systems. IMPORTANCE: Phages remain an enigmatic part of the biosphere. As predators, they challenge the survival of host bacteria and archaea and set off an "arms race" involving host immunization countered by phage mutation. The CRISPR-Cas system is adaptive: by capturing fragments of a phage genome upon exposure, the host is positioned to counteract future infections. To investigate this process, we initiated massive deep-sequencing experiments with a host and infective phage and tracked the coevolution of both populations over hundreds of days. In the present study, we found that CRISPR immunity drives the accumulation of phage genome rearrangements (which enable longer phage survival) and escape mutations, establishing CRISPR as one of the fundamental drivers of phage evolution.

Strong bias in the bacterial CRISPR elements that confer immunity to phage.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness.

Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation.

BACKGROUND: The broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources. RESULTS: Differences in L. casei genome inventory revealed an open pan-genome comprised of 1,715 core and 4,220 accessory genes. Extrapolation of pan-genome data indicates L. casei has a supragenome approximately 3.2 times larger than the average genome of individual strains. Evidence suggests horizontal gene transfer from other bacterial species, particularly lactobacilli, has been important in adaptation of L. casei to new habitats and lifestyles, but evolution of dairy niche specialists also appears to involve gene decay. CONCLUSIONS: Genome diversity in L. casei has evolved through gene acquisition and decay. Acquisition of foreign genomic islands likely confers a fitness benefit in specific habitats, notably plant-associated niches. Loss of unnecessary ancestral traits in strains collected from bacterial-ripened cheeses supports the hypothesis that gene decay contributes to enhanced fitness in that niche. This study gives the first evidence for a L. casei supragenome and provides valuable insights into mechanisms for genome evolution and lifestyle adaptation of this ecologically flexible and industrially important lactic acid bacterium. Additionally, our data confirm the Distributed Genome Hypothesis extends to non-pathogenic, ecologically flexible species like L. casei.