Bacterial Population Dynamics Research Articles

Mathematical Models of Plasmid Population Dynamics.

With plasmid-mediated antibiotic resistance thriving and threatening to become a serious public health problem, it is paramount to increase our understanding of the forces that enable the spread and maintenance of drug resistance genes encoded in mobile genetic elements. The relevance of plasmids as vehicles for the dissemination of antibiotic resistance genes, in addition to the extensive use of plasmid-derived vectors for biotechnological and industrial purposes, has promoted the in-depth study of the molecular mechanisms controlling multiple aspects of a plasmids’ life cycle. This body of experimental work has been paralleled by the development of a wealth of mathematical models aimed at understanding the interplay between transmission, replication, and segregation, as well as their consequences in the ecological and evolutionary dynamics of plasmid-bearing bacterial populations. In this review, we discuss theoretical models of plasmid dynamics that span from the molecular mechanisms of plasmid partition and copy-number control occurring at a cellular level, to their consequences in the population dynamics of complex microbial communities. We conclude by discussing future directions for this exciting research topic.

Design principles of collateral sensitivity-based dosing strategies

Collateral sensitivity (CS)-based antibiotic treatments, where increased resistance to one antibiotic leads to increased sensitivity to a second antibiotic, may have the potential to limit the emergence of antimicrobial resistance. However, it remains unclear how to best design CS-based treatment schedules. To address this problem, we use mathematical modelling to study the effects of pathogen- and drug-specific characteristics for different treatment designs on bacterial population dynamics and resistance evolution. We confirm that simultaneous and one-day cycling treatments could supress resistance in the presence of CS. We show that the efficacy of CS-based cycling therapies depends critically on the order of drug administration. Finally, we find that reciprocal CS is not essential to suppress resistance, a result that significantly broadens treatment options given the ubiquity of one-way CS in pathogens. Overall, our analyses identify key design principles of CS-based treatment strategies and provide guidance to develop treatment schedules to suppress resistance.

Selenium Yeast Dietary Supplement Affects Rumen Bacterial Population Dynamics and Fermentation Parameters of Tibetan Sheep (Ovis aries) in Alpine Meadow.

Selenium (Se) deficiency is a widespread and seasonally chronic phenomenon observed in Tibetan sheep (Ovis aries) traditionally grazed on the Qinghai–Tibet Plateau (QTP). Effects of the dietary addition of Se-enriched yeast (SeY) on the bacterial community in sheep rumen and rumen fermentation were evaluated with the aim of gaining a better understanding of the rumen prokaryotic community. Twenty-four yearling Tibetan rams [initial average body weight (BW) of 31.0 ± 0.64 kg] were randomly divided into four treatment groups, namely, control (CK), low Se (L), medium Se (M), and high Se (H). Each group comprised six rams and was fed a basic diet of fresh forage cut from the alpine meadow, to which SeY was added at prescribed dose rates. This feed trial was conducted for over 35 days. On the final day, rumen fluid was collected using a transesophageal sampler for analyzing rumen pH, NH3-N content, volatile fatty acid (VFA) level, and the rumen microbial community. Our analyses showed that NH3-N, total VFA, and propionate concentrations in the M group were significantly higher than in the other groups (P < 0.05). Both the principal coordinates analysis (PCoA) and the analysis of similarities revealed that the bacterial population structure of rumen differed among the four groups. The predominant rumen bacterial phyla were found to be Bacteroidetes and Firmicutes, and the three dominant genera in all the samples across all treatments were Christensenellaceae R7 group, Rikenellaceae RC9 gut group, and Prevotella 1. The relative abundances of Prevotella 1, Rikenellaceae RC9 gut group, Ruminococcus 2, Lachnospiraceae XPB1014 group, Carnobacterium, and Hafnia-Obesumbacterium were found to differ significantly among the four treatment groups (P < 0.05). Moreover, Tax4fun metagenome estimation revealed that gene functions and metabolic pathways associated with carbohydrate and other amino acids were overexpressed in the rumen microbiota of SeY-supplemented sheep. To conclude, SeY significantly affects the abundance of rumen bacteria and ultimately affects the rumen microbial fermentation.

Changes in physico-chemical characteristics and viable bacterial communities during fermentation of alfalfa silages inoculated with Lactobacillus plantarum.

This study investigated the effect of inoculating Lactobacillus (L.) plantarum PS-8 in fermentation of alfalfa silages. We monitored the fermentation characteristics and bacterial population dynamics during the ensiling process. PacBio single molecule real time sequencing was combined with propidium monoazide (PMA) treatment to monitor the viable microbiota dynamics. We found that inoculating L. plantarum PS-8 may improve the silage quality by accelerating acidification, reducing the amounts of clostridia, coliform bacteria, molds and yeasts, elevating the protein and organic acid contents (except butyrate), and enhancing lactic acid bacteria (LAB) while suppressing harmful microorganisms. Some significant differential abundant taxa were found between the PMA-treated and non-treated microbiota. For example, the relative abundances of L. brevis, L. plantarum, and Pediococcus pentosaceus were significantly higher in the PMA-treated group than the non-PMA-treated group, suggesting obvious differences between the viable and non-viable microbiota. It would thus be necessary to distinguish between the viable and non-viable microbial communities to further understand their physiological contribution in silage fermentation. By tracking the dynamics of viable microbiota in relation with changes in the physico-chemical parameters, our study provided novel insights into the beneficial effects of inoculating L. plantarum PS-8 in silage fermentation and the physiological function of the viable bacterial communities.

Characteristics of planktonic and sediment bacterial communities in a heavily polluted urban river.

Urban rivers represent a unique ecosystem in which pollution occurs regularly, altering the biogeochemical characteristics of waterbodies and sediments. However, little is presently known about the spatiotemporal patterns of planktonic and sediment bacterial community diversities and compositions in urban rivers. Herein, Illumina MiSeq high-throughput sequencing was performed to reveal the spatiotemporal dynamics of bacterial populations in Liangtan River, a heavily polluted urban river in Chongqing City (China). The results showed the richness and diversity of sediment bacteria were significantly higher than those of planktonic bacteria, whereas a strong overlap (46.7%) in OTUs was identified between water and sediment samples. Bacterial community composition remarkably differed in waters and sediments. Planktonic bacterial communities were dominated by Proteobacteria, Bacteroidetes, Cyanobacteria and Actinobacteria, while sediment bacterial communities mainly included Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Additionally, several taxonomic groups of potential bacterial pathogens showed an increasing trend in water and sediment samples from residential and industrial areas (RI). Variation partition analysis (VPA) indicated that temperature and nutrient were identified as the main drivers determining the planktonic and sediment bacterial assemblages. These results highlight that bacterial communities in the polluted urban river exhibit spatiotemporal variation due to the combined influence of environmental factors associated with sewage discharge and hydropower dams.

Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of Pseudomonas aeruginosa Biofilms: An In Vitro Model.

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization.

SummaryBacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini‐Tn7‐lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro‐ and micro‐scale imaging techniques. We demonstrate that a dual bioluminescence‐fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro‐ and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.

Characterization of Bacterial Populations in Urban and Rural Houston Watershed Soil Samples Following a Flooding Event

Since Houston is prone to flooding events, bacterial population dynamics in Houston watershed soils pre- and post-Hurricane Harvey were evaluated. Unexpectedly, bayous closer to Houston’s urban core, including Buffalo, Halls, Mustang, and Horsepen Bayous, had significantly higher enteric bacterial loads during the winter than the summer, likely due to water flow rate changes or proximity to wastewater outflow. Following bacterial load determination, isolated colonies were identified using biochemical tests. Additionally, metagenomic sequencing of 16S rDNA allowed for identification of both culturable and unculturable organisms. The phyla Proteobacteria, Actinobacteria, Bacteriodetes and Firmicutes were found to be dominant in our metagenomic analysis and are human gut bacteria. Some opportunistic bacterial Proteobacteria pathogens identified in our metabolomic analysis were Serratia marcenscens, Pseudomonas mendocina, Pseudomonas fulva, and Pseudomonas putida. To our knowledge, this is the first study that compares Houston-area bacterial populations before and after a major flooding event. Taken together, Hurricane Harvey likely contributed to a redistribution of enteric bacteria, as there was a significant increase in the enteric population of Buffalo and Halls Bayous. Similarly, our 2018 winter data set followed the same trend, as significant increases were seen in the enteric populations of Horsepen, Mustang, and Cypress Creek watershed soils.

Bacterial Population Dynamics of Natural Fermentation of Sumbawa Mare's Milk Using Metagenomic Approach

This study aimed to assess the changing of bacterial density and the physicochemical aspects during natural fermentation of Sumbawa mare’s milk, and to evaluate the dynamics of bacterial population during the natural fermentation using metagenomic approach. Mare’s milk sample obtained from Regency of Dompu were fermented for 60 days. On the day 0, 7, 15, 30 and 60 mare milk sample were collected for further analysis, such as bacterial density enumeration, nutrition content, physical properties of the milk, and total DNA isolation. The total DNA samples obtained were analyzed using next generation sequencing. The density of lactic acid bacteria was decreased along with fermentation periods. Meanwhile, the density of aerobic bacteria on was relatively fluctuated. The physicochemical content of mare’s milk also changed during fermentation periods. Carbohydrate content and total sugar was decrease along with the decreasing of pH value. Moreover, the lipid content increase, and the protein content was fluctuated. The changing in physical properties such as whey color, acidity and gas was observed until the end of mare’s milk natural fermentation process. Using metagenomics analysis, the bacterial diversity from each sample periods categorized as low because of the dominance of Lactobacillus helveticus until the end of the fermentation. Lactobacillus helveticus as a member of LAB did not grow on isolation media on the late stage of fermentation periods (day-60). The presence of uncultivable bacteria can be detected with metagenomic approach, fulfilling the limited information on the bacterial composition of fermented Sumbawa mare’s milk products.

Polyclonality, Shared Strains, and Convergent Evolution in Chronic Cystic Fibrosis Staphylococcus aureus Airway Infection

Rationale: Staphylococcus aureus is the most common respiratory pathogen isolated from patients with cystic fibrosis (CF) in the United States. Although modes of acquisition and genetic adaptation have been described for Pseudomonas aeruginosa, resulting in improved diagnosis and treatment, these features remain more poorly defined for S. aureus.Objectives: To characterize the molecular epidemiology and genetic adaptation of S. aureus during chronic CF airway infection and in response to antibiotic therapy.Methods: We performed whole-genome sequencing of 1,382 S. aureus isolates collected longitudinally over a mean 2.2 years from 246 children with CF at five U.S. centers between 2008 and 2017. Results were integrated with clinical and demographic data to characterize bacterial population dynamics and identify common genetic targets of in vivo adaptation.Measurements and Main Results: Results showed that 45.5% of patients carried multiple, coexisting S. aureus lineages, often having different antibiotic susceptibility profiles. Adaptation during the course of infection commonly occurred in a set of genes related to persistence and antimicrobial resistance. Individual sequence types demonstrated wide geographic distribution, and we identified limited strain-sharing among children linked by common household or clinical exposures. Unlike P. aeruginosa, S. aureus genetic diversity was unconstrained, with an ongoing flow of new genetic elements into the population of isolates from children with CF.Conclusions: CF airways are frequently coinfected by multiple, genetically distinct S. aureus lineages, indicating that current clinical procedures for sampling isolates and selecting antibiotics are likely inadequate. Strains can be shared by patients in close domestic or clinical contact and can undergo convergent evolution in key persistence and antimicrobial-resistance genes, suggesting novel diagnostic and therapeutic approaches for future study.

Diversity and dynamics of bacterial and fungal communities in cider for distillation

Bacterial and fungal population dynamics in cider for distillation have so far been explored by culture-dependant methods. Cider for distillation can be produced by the spontaneous fermentation of apples that do not undergo any intervention during the process. In this study, cider microbiomes extracted from six tanks containing ciders for distillation from four producers in Normandy were characterized at three main stages of the fermentation process: fermentation Initiation (I), end of the alcoholic Fermentation (F) and end of the Maturation period (M). Cider samples were subjected to Illumina MiSeq sequencing (rRNA 16S V1-V3 and ITS1 region targeting) to determine bacterial and fungal communities. Yeasts (YGC), Zymomonas (mZPP) and lactic acid bacteria selective media (mMRS, mMLO, mPSM) were also used to collect 807 isolates. Alcoholic levels, glycerol, sugar content (glucose, fructose and sucrose), pH, total and volatile acidity, nitrogen, malic and lactic acid contents were determined at all sampling points. Alpha diversity indexes show significant differences (p < 0.05) in microbial populations between I, F and M. Fungal communities were characterized by microorganisms from the environment and phytopathogens at I followed by the association of yearsts with alcoholic fermentation like Saccharomyces and non-Saccharomyces yeasts (Hanseniaspora, Candida). A maturation period for cider leads to an increase of the Dekkera/Brettanomyces population, which is responsible for off-flavors in cider for all producers. Among bacterial communities, the genera community associated to malolactic fermentation (Lactobacillus sp., Leuconostoc sp., Oenococcus sp.) was the most abundant at F and M. Acetic acid bacteria such as Acetobacter sp., Komagataeibacter sp. and Gluconobacter sp. were also detected during the process. Significant differences (p < 0.05) were found in fungal and bacterial populations between the four producers and during the fermentation process. The development of microorganisms associated with cider spoilage such as Zymomonas mobilis, Lactobacillus collinoides or Brettanomyces/Dekkera sp. was anticipated by a metagenomic approach. The monitoring of microbial diversity via high throughput sequencing combined with physical-chemical analysis is an interesting approach to improve the fermentation performance of cider for distillation and therefore, the quality of Calvados.

Cultivable bacterial communities associated with the salivary gland of Aedes aegypti

Rapid spread of vector borne diseases, especially through mosquitoes is in alarming rise worldwide. Among mosquitoes, Aedes aegypti is a major vector transmitting arboviruses such as dengue and zika to human population. The ineffectiveness of control measures against these arboviruses has necessitated alternate strategies to curb disease transmission. Recent studies show that symbiotic bacteria from insects hinder pathogen transmission by altering vector competence. Therefore, analyzing the bacterial communities of mosquitoes is essential to identify potential bacteria that play a critical role in vector competence. Salivary gland, a key organ in viral transmission however, is minimally explored towards their microbiota. In this study, we have screened 28 bacterial isolates from salivary glands of non-blood fed (NBF) and blood fed (BF) female Ae. aegypti, collected from the field population of Coimbatore, India. Among them, 11 bacterial species from distinct genera belonging to three major phyla; Proteobacteria, Firmicutes and Actinobacteria were identified through 16S rRNA gene sequencing. Interestingly, the differences in bacterial species associated with salivary glands were clearly discernible. This suggests that the dynamics of bacterial population after blood feeding is not restricted to the mosquito gut, but envisioned in the salivary glands as well. Overall, the results shed more light into the understating on salivary glands microbiota of dreadful vector Ae. aegypti.

Characterization of the marine aquaculture microbiome: A seasonal survey in a seabass farm

Vibriosis is a recurrent disease in marine aquaculture environments. Monitoring the dynamics of bacterial populations in several compartments of the aquaculture infrastructure is of paramount importance to understand the occurrence of the disease. In the present study, the fish farm, located in the north of France, is a flow-through system where the tanks are supplied by two water inlets. Water from breeding tanks, and inlets and the biofilms in the tanks were sampled weekly in a seabass farm for 7 months. DNA was extracted from 477 samples to sequence the variable V4-V5 region of the 16S rRNA gene and analyze the sequences using the QIIME2 platform. Proteobacteria (54.52%) and Bacteroidetes (26.85%) composed more than 80% of the phylum diversity. Alpha- and beta-diversity indicators showed that bacterial communities were more diverse in samples from biofilms and tanks than in samples from water inlets. Some families were specific to one compartment, e.g. ammonium-oxidizing bacteria in biofilms, whereas Vibrionaceae family isolates were mostly found in tank water samples. Taxa were determined up to the species level for the Vibrionaceae. Besides, all six determined genera of Vibrionaceae were identified, with mostly Vibrio and Photobacterium isolates detected in almost all samples. Vibrionaceae persisted over time in the biofilms. Our results suggest that biofilms may be a potential reservoir in case of dysbiosis. This study helps better understand microbial interactions, especially with Vibrionaceae in aquaculture environments.

Stochastic bacterial population dynamics restrict the establishment of antibiotic resistance from single cells

A better understanding of how antibiotic exposure impacts the evolution of resistance in bacterial populations is crucial for designing more sustainable treatment strategies. The conventional approach to this question is to measure the range of concentrations over which resistant strain(s) are selectively favored over a sensitive strain. Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying resistance plasmids, we show that single resistant cells have <5% probability of detectable outgrowth at antibiotic concentrations as low as one-eighth of the resistant strain's minimum inhibitory concentration (MIC). This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, well below the MIC of resistant strains, may effectively restrict de novo emergence of resistance even though they cannot clear already-large resistant populations.

Two novel bacteriophage genera from a groundwater reservoir highlight subsurface environments as underexplored biotopes in bacteriophage ecology

Although bacteriophages are central entities in bacterial ecology and population dynamics, there is currently no literature on the genomes of bacteriophages isolated from groundwater. Using a collection of bacterial isolates from an aquifer as hosts, this study isolated, sequenced and characterised two bacteriophages native to the groundwater reservoir. Host phylogenetic analyses revealed that the phages targeted B. mycoides and a novel Pseudomonas species. These results suggest that both bacteriophages represent new genera, highlighting that groundwater reservoirs, and probably other subsurface environments as well, are underexplored biotopes in terms of the presence and ecology of bacteriophages.

Wide lag time distributions break a trade-off between reproduction and survival in bacteria

Many microorganisms face a fundamental trade-off between reproduction and survival: Rapid growth boosts population size but makes microorganisms sensitive to external stressors. Here, we show that starved bacteria encountering new resources can break this trade-off by evolving phenotypic heterogeneity in lag time. We quantify the distribution of single-cell lag times of populations of starved Escherichia coli and show that population growth after starvation is primarily determined by the cells with shortest lag due to the exponential nature of bacterial population dynamics. As a consequence, cells with long lag times have no substantial effect on population growth resumption. However, we observe that these cells provide tolerance to stressors such as antibiotics. This allows an isogenic population to break the trade-off between reproduction and survival. We support this argument with an evolutionary model which shows that bacteria evolve wide lag time distributions when both rapid growth resumption and survival under stressful conditions are under selection. Our results can explain the prevalence of antibiotic tolerance by lag and demonstrate that the benefits of phenotypic heterogeneity in fluctuating environments are particularly high when minorities with extreme phenotypes dominate population dynamics.

Temporal Dynamics of Soil Virus and Bacterial Populations in Agricultural and Early Plant Successional Soils.

As reported in many aquatic environments, recent studies in terrestrial ecosystems implicate a role for viruses in shaping the structure, function, and evolution of prokaryotic soil communities. However, given the heterogeneity of soil and the physical constraints (i.e., pore-scale hydrology and solid-phase adsorption of phage and host cells) on the mobility of viruses and bacteria, phage-host interactions likely differ from those in aquatic systems. In this study, temporal changes in the population dynamics of viruses and bacteria in soils under different land management practices were examined. The results showed that bacterial abundance was significantly and positively correlated to both virus and inducible prophage abundance. Bacterial and viral abundance were also correlated with soil organic carbon and nitrogen content as well as with C:N ratio. The seasonal variability in viral abundance increased with soil organic carbon content. The prokaryotic community structure was influenced more by land use than by seasonal variation though considerable variation was evident in the early plant successional and grassland sites. The free extracellular viral communities were also separated by land use, and the forest soil viral assemblage exhibiting the most seasonal variability was more distinct from the other sites. Viral assemblages from the agricultural soils exhibited the least seasonal variability. Similar patterns were observed for inducible prophage viral assemblages. Seasonal variability of viral assemblages was greater in mitomycin-C (mitC) induced prophages than in extracellular viruses irrespective of land use and management. Taken together, the data suggest that soil viral production and decay are likely balanced but there was clear evidence that the structure of viral assemblages is influenced by land use and by season.

Interaction of Staphylococcus aureus and Host Cells upon Infection of Bronchial Epithelium during Different Stages of Regeneration.

The primary barrier that protects our lungs against infection by pathogens is a tightly sealed layer of epithelial cells. When the integrity of this barrier is disrupted as a consequence of chronic pulmonary diseases or viral insults, bacterial pathogens will gain access to underlying tissues. A major pathogen that can take advantage of such conditions is Staphylococcus aureus, thereby causing severe pneumonia. In this study, we investigated how S. aureus responds to different conditions of the human epithelium, especially nonpolarization and fibrogenesis during regeneration using an in vitro infection model. The infective process was monitored by quantification of the epithelial cell and bacterial populations, fluorescence microscopy, and mass spectrometry. The results uncover differences in bacterial internalization and population dynamics that correlate with the outcome of infection. Protein profiling reveals that, irrespective of the polarization state of the epithelial cells, the invading bacteria mount similar responses to adapt to the intracellular milieu. Remarkably, a bacterial adaptation that was associated with the regeneration state of the epithelial cells concerned the early upregulation of proteins controlled by the redox-responsive regulator Rex when bacteria were confronted with a polarized cell layer. This is indicative of the modulation of the bacterial cytoplasmic redox state to maintain homeostasis early during infection even before internalization. Our present observations provide a deeper insight into how S. aureus can take advantage of a breached epithelial barrier and show that infected epithelial cells have limited ability to respond adequately to staphylococcal insults.

Growth Dynamics of Bacterial Populations in a Two-Compartment Biofilm Bioreactor Designed for Continuous Surfactin Biosynthesis.

Biofilm bioreactors are promising systems for continuous biosurfactant production since they provide process stability through cell immobilization and avoid foam formation. In this work, a two-compartment biofilm bioreactor was designed consisting of a stirred tank reactor and a trickle-bed reactor containing a structured metal packing for biofilm formation. A strong and poor biofilm forming B. subtilis 168 strain due to restored exopolysaccharides (EPS) production or not were cultivated in the system to study the growth behavior of the planktonic and biofilm population for the establishment of a growth model. A high dilution rate was used in order to promote biofilm formation on the packing and wash out unwanted planktonic cells. Biofilm development kinetics on the packing were assessed through a total organic carbon mass balance. The EPS+ strain showed a significantly improved performance in terms of adhesion capacity and surfactin production. The mean surfactin productivity of the EPS+ strain was about 37% higher during the continuous cultivation compared to the EPS- strain. The substrate consumption together with the planktonic cell and biofilm development were properly predicted by the model (α = 0.05). The results show the efficiency of the biofilm bioreactor for continuous surfactin production using an EPS producing strain.

Fluorescent reporter plasmids for single-cell and bulk-level composition assays in E. faecalis.

Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available–particularly for common model organisms like E. coli–fewer options exist for other species, including E. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available for E. faecalis, we exchanged the original fluorescent reporter in a previously developed plasmid (pBSU101) with one of eight fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single E. faecalis cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from E. faecalis populations in microwell plate (liquid) cultures during different phases of aerobic growth. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations under aerobic growth conditions. Finally, we incorporated codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in E. faecalis.