Biology subjects

Marco, M. L.

Publications and source records attributed to Marco, M. L..

9 recordsLinked to original sources

bioRxiv2025

Variations in cow milk and teat skin microbiota across the lactation cycle with intra-mammary cephalosporin use at dry-off

Cephalosporins and other broad-spectrum antibiotics are frequently administered prophylactically into the udder when dairy cows end their lactation cycle, termed dry-off, to reduce mastitis risk. However, the use of antibiotics on cows that do not have signs of infection may result in the selection of microbes resistant to antibiotics and negatively alter the udder microbiome composition. In this study, the effects of intramammary cephalosporin therapy with either Cephapirin (CB) or Ceftiofur (CH) on milk and teat skin microbiota were examined for three dairies in California. Bacterial composition was measured for cows with low somatic cell counts (SCC,<100,000 cells /mL) and high, subclinical SCC (>200,000 cells/mL). Samples were collected at dry-off (before treatment), seven days later, and 55-75 Days in Milk (DIM) in the next lactation cycle. The milk and skin microbiota were largely separated based on dairy (milk: R2 = 6.22, skin: R2 = 7.56) and day of sampling (milk: R2 = 4.74 and skin: R2 = 3.77). CB or CH use was associated with a small but significant impact on the milk microbiota beta-diversity (Bray-Curtis, p =0.003, R2= 1.4%) but no effect was observed on the skin. At one dairy (Dairy 3), milk from cows receiving CB and CH had a reduction in proportions of Staphylococcaceae at 55-75DIM compared to untreated cows. Overall, antibiotic use did not result in large significant changes to bacterial diversity in milk or on the teat skin, and instead the microbiota at those sites mainly differed between the time and location of sampling. ImportanceThe use of antibiotics in agriculture is under increasing scrutiny due to the rising spread of antimicrobial resistant bacteria. Our study showed that common preventative antibiotic intramammary treatment of cows with cephalosporins at the end their lactation (dry-off) had minimal effects on the milk and teat skin microbiota on asymptomatic cows with high somatic cell counts.

microbiology↗

bioRxiv2024

The fermented cabbage metabolome and its protection against cytokine-induced intestinal barrier disruption of Caco-2 monolayers

Fermented vegetables, such as fermented cabbage (sauerkraut), have garnered growing interest for their associations with a myriad of health benefits. However, the mechanistic details underlying the outcomes of consuming these foods require further investigation. This study examined the capacity of soluble metabolites in laboratory-scale and commercial fermented cabbage to protect against disruption of polarized Caco-2 monolayers by IFN-{gamma} and TNF-. Laboratory-scale ferments (LSF) were prepared with and without the addition of Lactiplantibacillus plantarum NCIMB8826R (LP8826R) and sampled after 7- and 14-days of incubation. Trans-epithelial electrical resistance (TER) and paracellular permeability to fluorescein isothiocyanate-dextran (FITC) revealed that fermented cabbage, but not raw cabbage or brine, protected against cytokine-induced damage to the Caco-2 monolayers. Barrier-protective effects occurred despite increased IL-8 production following cytokine exposure. Metabolomic analyses performed using gas and liquid chromatography resulted in the identification of 149 and 333 metabolites, respectively. Significant differences were found between raw and fermented cabbage. LSF metabolomes changed over time and the profiles of LSF with LP8826R best resembled the commercial product. Overall, fermentation resulted in lower carbohydrate and increased lactic acid, lipid, amino acid derivative (including D-phenyl-lactate (D-PLA), indole-3-lactate (ILA), and {gamma}-aminobutyric acid (GABA)), and phenolic compound concentrations. Lactate, D-PLA, and ILA tested individually and combined only partially protected against cytokine-induced TER reductions and increases in paracellular permeability of Caco-2 monolayers. The findings show that intestinal barrier-protective compounds are consistently enriched during cabbage fermentations, irrespective of scale or microbial additions, that may contribute to the health-promoting potential of these foods. ImportanceFermented vegetables are increasingly associated with health benefits. However, the importance of microbial transformations to foods during the fermentation process remain to be determined. We found that the metabolites in spontaneously fermented cabbage protected polarized intestinal epithelial cells against damage induced by proinflammatory cytokines. Cabbage fermentations resulted in consistent metabolome profiles enriched in bioactive compounds known to be made by beneficial members of the human gut microbiome, including D-phenyl-lactate (D-PLA) and indole-3-lactate (ILA). The metabolomes were distinct from raw cabbage and further differentiated based on sampling time, addition of an exogenous Lactiplantibacillus plantarum strain, and source. Because only partial protection against intestinal barrier disruption was found when individual metabolites (D-PLA, ILA, lactate) were applied, the findings indicate that the complex mixture of metabolites in a cabbage fermentation offer advantages over single metabolites to benefit intestinal health.

microbiology↗

bioRxiv2023

Lactiplantibacillus plantarum uses ecologically relevant, exogenous quinones for extracellular electron transfer

Extracellular electron transfer (EET) is a metabolic process that frequently uses quinones to couple intracellular redox reactions with extracellular electron acceptors. The physiological relevance of this metabolism for microorganisms that are capable of EET, but unable to synthesize their own quinones, remains to be determined. To address this question, we investigated quinone utilization by Lactiplantibacillus plantarum, a microorganism required for food fermentations, performs EET, and is also a quinone auxotroph. L. plantarum selectively used 1,4-dihydroxy-2-naphthoic acid (DHNA), 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), 1,4-naphthoquinone, and menadione for EET reduction of insoluble iron (ferrihydrite). However, those quinones used for EET also inhibited L. plantarum growth in non-aerated conditions. Transcriptomic analysis showed that DHNA induced oxidative stress in L. plantarum and this was alleviated by the inclusion of an electron acceptor, soluble ferric ammonium citrate (FeAC), in the laboratory culture medium. The presence of DHNA and FeAC during growth also induced L. plantarum EET metabolism, although activity was still dependent on the presence of exogenous electron shuttles. To determine whether quinone-producing bacteria frequently found together with L. plantarum in food fermentations could be a source of those electron shuttles, L. plantarum EET was measured after incubation with Lactococcus lactis and Leuconostoc mesenteroides. Quinone-producing L. lactis, but not a quinone-deficient L. lactis {Delta}menC mutant, increased L. plantarum ferrihydrite reduction and medium acidification through an EET-dependent mechanism. L. plantarum EET was also stimulated by L. mesenteroides, and this resulted in greater environmental acidification and transient increases in L. plantarum growth. Overall, our findings revealed that L. plantarum overcomes the toxic effects of exogenous quinones to use those compounds, including those made by related bacteria, for EET-conferred, ecological advantages during the early stages of food fermentations.

microbiology↗

bioRxiv2022

Calcium determines Lactiplantibacillus plantarum intraspecies competitive fitness

The importance of individual nutrients for microbial strain robustness and coexistence in habitats containing different members of the same species is not well understood. To address this for Lactiplantibacillus plantarum in food fermentations, we performed comparative genomics and examined the nutritive requirements and competitive fitness for L. plantarum strains B1.1 and B1.3 isolated from a single sample of teff injera fermentation batter. Compared to B1.1 and other L. plantarum strains, B1.3 has a smaller genome, limited biosynthetic capacities, and large mobilome. Despite these differences, B1.3 was equally competitive with B1.1 in a suspension of teff flour. In commercially-sourced, nutrient-replete MRS (cMRS) medium, strain B1.3 reached three-fold higher numbers than B1.1 within two days of passage. Because B1.3 growth and competitive fitness was poor in mMRS, a modified MRS lacking beef extract, we used mMRS to identify nutrients needed for robust B1.3 growth. No improvement was observed when mMRS was supplemented with nucleotides, amino acids, vitamins, or monovalent metals. Remarkably, the addition of divalent metal salts increased the growth rate and cell yields of B1.3 in mMRS. Metal requirements were confirmed by Inductively Coupled Plasma Mass Spectrometry, showing that total B1.3 intracellular metal concentrations were significantly (up to 2.7-fold) reduced compared to B1.1. Supplemental CaCl2 conferred the greatest effect, resulting in equal growth between B1.1 and B1.3 over successive five passages in mMRS. Moreover, calcium supplementation reversed a B1.3 strain-specific stationary phase, flocculation phenotype. These findings show how L. plantarum calcium requirements affect competitive fitness at the strain level. ImportanceEcological theory states that the struggle for existence is stronger between closely related species. Contrary to this assertion, fermented foods frequently sustain conspecific individuals, despite their high levels of phylogenetic relatedness. Therefore, we investigated two isolates of Lactiplantibacillus plantarum B1.1 and B1.3 randomly selected from a single batch of teff injera batter. These strains spanned the known genomic and phenotypic range of the L. plantarum species, and in nutrient-replete, laboratory culture medium, strain B1.3 exhibited poor growth and was outcompeted by the more robust strain B1.1. Despite those differences, B1.1 and B1.3 were equally competitive in teff flour. This result shows how these bacteria have adapted for co-existence in that environment. The capacity for the single macronutrient calcium to restore B1.3 competitive fitness in laboratory culture medium suggests that L. plantarum intraspecies diversity found in food systems is fine-tuned to nutrient requirements at the strain level.

microbiology↗

bioRxiv2022

Improved assessments of milk microbiota composition via sample preparation and DNA extraction methods

Although bacterial detection by 16S rRNA gene amplicon DNA sequencing is now a widely-applied technique, standardized methods for sample preparation and DNA extraction are needed to ensure accuracy, reproducibility, and scalability for automation. To develop these methods for bovine milk, we assembled a bacterial cell mock community (BCMC) containing bacterial species commonly found in milk. Variations to the following methods were examined: BCMC enumeration method (colony enumeration or microscopy), sample volume (200 l to 30 ml), sample storage condition (frozen in PBS or 25% glycerol or exposure to freeze-thaw cycles), cell lysis method (bead-beating, vortex, enzymatic), and DNA extraction procedure (MagMAX Total, MagMAX CORE, and MagMAX Ultra 2.0, with and without either Proteinase K or RNase A). Cell enumeration by microscopy was shown to be more accurate for quantification of the BCMC contents. We found that least 10 mL ([≥] 104 cells in high quality milk) is needed for reproducible bacterial detection by 16S rRNA gene amplicon DNA sequencing, whereas variations in storage conditions caused minor differences in the BCMC. For DNA extraction and purification, a mild lysis step (bead-beating for 10 s at 4 m/s or vortexing at 1800 rpm for 10 s) paired with the MagMAX Total kit and Proteinase K digestion provided the most accurate representation of the BCMC. These approaches were confirmed to provide similar results in commercial milk samples. Overall, variations in cell lysis methods conferred the greatest changes to milk microbiota composition and therefore should be optimized for sample type.

microbiology↗

bioRxiv2021

Extracellular electron transfer increases fermentation in lactic acid bacteria via a hybrid metabolism

Energy conservation in microorganisms is classically categorized into respiration and fermentation, however recent work shows some species can use mixed or alternative bioenergetic strategies. We explored the utility of a flavin-based extracellular electron transport (FLEET) system for energy conservation within diverse lactic acid bacteria (LAB), microorganisms that mainly rely on fermentative metabolism and are important in food fermentations. The LAB Lactiplantibacillus plantarum uses extracellular electron transfer to increase its NAD+/NADH ratio, generate more ATP through substrate-level phosphorylation and accumulate biomass more rapidly. This novel, hybrid metabolism was dependent on a type-II NADH dehydrogenase (Ndh2) and conditionally required a flavin-binding extracellular lipoprotein (PplA) in the FLEET system to confer increased fermentation yield, metabolic flux, and environmental acidification in both laboratory media and food fermentation. The discovery of a single pathway that blends features of fermentation and respiration expands our knowledge of energy conservation metabolism and provides immediate biotechnology applications.

microbiology↗

bioRxiv2021

A chemically-defined growth medium to support Lactobacillus-Acetobacter community analysis

Lactobacilli and acetobacters are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of Drosophila melanogaster fruit flies. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growths. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would greatly facilitate identification of the precise metabolic interactions between these two groups of bacteria. While numerous such media have been developed that support specific strains of lactobacilli and acetobacters, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous Lactobacillus CDM, by modifying the nutrient abundances to improve growth of both groups of bacteria. We further simplified the medium by substituting casamino acids for individual amino acids and the standard Wolfes vitamins and mineral stocks for individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. The new CDM and variations of it support robust growth of lactobacilli and acetobacters. We provide the composition and an example of its use to measure nutritional interactions.

microbiology↗

bioRxiv2021

Strain diversity of plant-associated Lactiplantibacillus plantarum

The intraspecific phenotypic and genetic diversity of Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) was examined for five strains isolated from fermented olives and eight strains from cactus fruit, fermented tomatoes, teff injera, wheat boza, and wheat sourdough starter sources. Carbohydrate utilization and stress tolerance characteristics showed that the olive isolates grew more robustly in galactose and raffinose, showed higher tolerance to 12% v/v EtOH, and exhibited a greater capacity to inhibit an olive spoilage strain of Saccharomyces cerevisiae than L. plantarum from the other plant sources. Certain traits were variable between fermented olive isolates such as the capacity for biofilm formation and survival at pH 2 or 50 {degrees}C. By comparison, all L. plantarum from fruit sources grew better at a pH of 3.5 than the strains from fermented grains. Multi-locus sequence typing and genome sequencing indicated that strains from the same source type tended to be genetically related. Comparative genomics was unable to resolve strain differences, with the exception of the most phenotypically impaired and robust isolates. The findings show that L. plantarum is adapted for growth on specific plants or plant food types, but that intraspecific variation may be important for ecological fitness of L. plantarum within individual habitats.

microbiology↗

bioRxiv2018

Impact of DNA sequencing and analysis methods on 16S rRNA gene bacterial community analysis in dairy products

DNA sequencing and analysis methods were compared for 16S rRNA V4 PCR amplicon and gDNA mock communities encompassing nine bacterial species commonly found in milk and dairy products. The communities were examined using Illumina MiSeq and Ion Torrent PGM DNA sequencing methods followed by the QIIME 1 (UCLUST) and Divisive Amplicon Denoising Algorithm 2 (DADA2) data analysis pipelines including taxonomic comparisons to the Greengenes and Ribosomal Database Project (RDP) databases. Examination of the PCR amplicon mock community with these methods resulted in Operation Taxonomy Units (OTUs) and Amplicon Sequence Variants (ASVs) that ranged from a low of 13 to high of 118 and were dependent on the DNA sequencing method and read assembly step. The elevated numbers of OTUs and ASVs included assignments to spurious taxa as well as sequence variants of the nine species included in the mock community. Comparisons between the gDNA and PCR amplicon mock communities showed that combining gDNA from the different strains prior to PCR resulted in up to 8.9-fold greater numbers of spurious OTUs and ASVs. However, the DNA sequencing method and initial data assembly steps conferred the largest effects on predictions of bacterial diversity, independent of the mock community type (PCR amplicon or gDNA; Bray-Curtis R2 = 0.88 and weighted Unifrac, R2 = 0.32). Overall, DNA sequencing performed with the Ion Torrent PGM and analyzed with DADA2 and the Greengenes database resulted in the most accurate predictions of the mock community phylogeny, taxonomy, and diversity.\n\nImportanceValidated methods are urgently needed to improve DNA-sequence based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk and dairy-associated environments.

microbiology↗