Human feces were streaked onto MRS Agar adjusted to pH 2.5, 3.0, and 6.4, respectively, and medium supplemented with 1.0% (w/v) bile salts. Two aciduric strains, identified as Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 (based on 16S rDNA and recA sequences), were non-hemolytic and did not hydrolyze mucin. The surface of Lactobacillus reuteri HFI-LD5 cells has a weak negative charge, whereas Lactobacillus rhamnosus HFI-K2 has acidic and basic properties, and produces exopolysaccharides (EPS). None of the strains produce bacteriocins. Both strains are resistant to several antibiotics, including sulfamethoxazole-trimethoprim and sulphonamides. The ability of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 to grow at pH 2.5 suggests that they will survive passage through the stomach. EPS production may assist in binding to intestinal mucus, especially in the small intestinal tract, protect epithelial cells, and stimulate the immune system. Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 may be used as probiotics, especially in the treatment of small intestinal bacterial overgrowth (SIBO).

The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass–function relationships and aid in the development of biofilm control strategies.

In this study, we report on the formation and resilience of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 biofilms cultivated in a CO evolution measurement system (CEMS) and exposed to biologically relevant, fasting-state gastrointestinal fluids under continuous flow conditions. For comparative purposes, planktonic and sessile populations of L. reuteri HFI-LD5 and L. rhamnosus HFI-K2 were each exposed to fasting-state gastric fluid (FSGF, pH 2.0) for 2 h, fasting-state intestinal fluid (FSIF, pH 7.5) for 6 h, and simulated colonic fluid (SCoF, pH 7.0) for 24 h. Planktonic cell numbers of L. reuteri HFI-LD5 declined from 6.6 log CFU/mL to 3.2 log CFU/mL and L. rhamnosus HFI-K2 from 6.6 log CFU/mL to undetectable levels after exposure to FSGF. Limited loss in viability was observed when free-floating cells were exposed to FSIF and SCoF. Sessile populations of both strains survived and recovered from the sequential exposure to all three gastric fluids despite observed detachment of biofilm biomass and a temporary decrease in metabolic activity to below detection limits, as recorded by changes in whole-biofilm CO production rates. The planktonic cell-focused gut microbiome-related research has most likely caused an underestimation in the overall survival ability of microorganisms in the gastrointestinal tract. Sessile cells of L. reuteri HFI-LD5 were metabolically inactive when exposed to gastric (FSGF) and intestinal (FSIF) fluids, suggesting that biofilms are formed in the small intestinal tract as survival mechanism. In the case of L. rhamnosus HFI-K2, cells were released from biofilms when suddenly exposed to pH 2.0.

Listeria monocytogenes biofilms are ubiquitous in the food-processing environment, where they frequently show resistance against treatment with disinfectants such as peracetic acid (PAA) due to sub-lethal damage resulting in biofilm persistence or the formation of secondary biofilms. L. monocytogenes serovar ½a EGD-e biofilms were cultivated under continuous flow conditions at 10 °C, 22 °C, and 37 °C and exposed to industrially relevant PAA concentrations. The effect of PAA on biofilm metabolic activity and biomass was monitored in real-time using the CEMS-BioSpec system, in addition to daily measurement of biofilm-derived planktonic cell production. Biofilm-derived planktonic cell yields proved to be consistent with high yields during biofilm establishment (≥106 CFU.mL−1). The exposure of biofilms to the minimum inhibitory PAA concentration (0.16%) resulted in only a brief disruption in whole-biofilm metabolic activity and biofilm biomass accumulation. The recovered biofilm accumulated more biomass and greater activity, but cell yields remained similar. Increasing concentrations of PAA (0.50%, 1.5%, and 4.0%) had a longer-lasting inhibitory effect. Only the maximum dose resulted in a lasting inhibition of biofilm activity and biomass–a factor that needs due consideration in view of dilution in industrial settings. Better disinfection monitoring tools and protocols are required to adequately address the problem of Listeria biofilms in the food-processing environment, and more emphasis should be placed on biofilms serving as a “factory” for cell proliferation rather than only a survival mechanism.

<p>The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass–function relationships and aid in the development of biofilm control strategies. </p>