Protection of Anaerobic Microbes from Processing Stressors Using Metal–Phenolic Networks
Authors: Gang Fan
Pris Wasuwanich
Mariela R Rodriguez-Otero
Ariel L Furst
Journal: Journal of the American Chemical Society (2021)
Discipline: CHEMISTRY
Keywords: gut microbiome; disease prevention; microbial biotherapeutics; anaerobes; self-assembling cellular coating
Affiliations: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States, Department of Chemical Engineering, University of Puerto Rico, Mayagüez 00681, Puerto Rico, Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States

Abstract:  The gut microbiome is essential to maintain overall health and prevent disease, which can occur when these microbes are not in homeostasis. Microbial biotherapeutics are important to combat these issues, but they must be alive at the time of delivery for efficacy. Many potentially therapeutic species are anaerobes and thus are difficult to manufacture because of the limited efficacy of existing protective methods, making their production nearly impossible. We have developed a self-assembling cellular coating to improve the viability and stability of the next-generation biotherapeutic Bacteroides thetaiotaomicron. We show protection from both harsh processing conditions and oxygen exposure, even in the absence of canonical cryoprotectants. This advance will increase the range of microbes that can be stably manufactured and facilitate the development of emerging strains of interest by ensuring their postproduction viability. 

Retracted: No

PubPeer comments:
  Fe(III)u2013TA films are readily formed on many biotic surfaces, including eukaryotes and yeast, and (as shown here for the first time) microbes. Yeasts are eukaryotic microbes. Also, the seminal paper describing the formation of Fe(III)-TA films demonstrated that the coating is readily formed on E. coli and yeast. The authors did not cite this work. https: www.science.org doi abs 10.1126 science.1237265 , 
 The article states that u201cFe(III)-TA film is readily formed on many biotic surfaces, including eukaryotes and yeast, and (for the first time shown here), microbesu201d, however that is not true. This coating was formed on E. coli and S. epidermis in the seminal 2013 Science paper (which was not cited by the authors), as well as in others. See: https: www.science.org doi abs 10.1126 science.1237265 , https: onlinelibrary.wiley.com doi 10.1002 adma.201700784 and https: www.sciencedirect.com science article abs pii S1748013221002164 (Also, yeast is technically a eukaryotic microbe.) The title and the main point of this article indicate that this coating protects anaerobes from oxygen, however the only indication for this is demonstrated in Figure S7, which is inconclusive. There were no detrimental effects demonstrated from oxygen exposure to this species, and thus one cannot say that the coating protects from oxygen exposure considering oxygen is detrimental in this instance. It is a poor control. The abstract claims the coating was developed to improve stability, yet stability is not investigated. Stability testing generally involves examining potency (in this case, viability) over time under different environmental conditions, such as temperature and relative humidity. The only harsh processing conditions examined was lyophilization without cryoprotectants and acid exposure (which technically isnu2019t a processing condition.) In some instances, viability was assessed by the onset of exponential growth (Teg), however the measurements were 8-12 hrs apart and thus this onset point was often missed. Additionally, this method is not a typical measure for viability, and the authors did not cite any method development work. They may have been attempting this method https: www.sciencedirect.com science article pii S0167701202002269?via%3Dihub, but did so inappropriately and Teg is not defined (typically, Teg is the time when OD=0.1 or another defined OD is reached). Accepted methods for viability testing are generally CFU mL or LIVE DEAD staining. CFU assays were used, however resulted in underestimated viability results due to the coating restricting cell division. Previous papers have indicated that MPNs inhibit cell division, but this is more likely due to their mechanical strength, not nutrient diffusion as the authors suggest, as MPNs have been reported to be porous and allow for diffusion. The authors were able to overcome this CFU assay underestimation by removing the cell coating. However, these viability results would have been more convincing if they were able to demonstrate comparable results using LIVE DEAD staining, but the authors only used a LIVE stain and no DEAD stain. This is especially needed for Fig S2, for example, which is a very important figure that was hidden in the SI. Fig S2 shows that there is little difference between Fe-GA coated cells and Fe-GA coating-removed cells, and that there are less CFUs of all coated cells than the control. Was there a control of the E. coli ascorbic acid uncoated cells to demonstrate that weak acid exposure decreases viability? Otherwise, it seems as though the coatings decrease viability, and further stress is why DEAD staining is required. Authors suggest that the addition of iron increases CFUs compared to control and in the discussion, they attribute this to iron being a micronutrient involved in bacterial growth. That is not a feasible explanation as that would affect the colony size, not the number of colonies. It is more likely a result in human error, since CFU assays are prone to error and cell clumping results in underestimated CFUs. Or, potentially, that addition of salts enable cells to be more homogeneously dispersed and result in less cell clumping and thus more CFUs compared to the control. Authors correlate the growth delay to the thickness of the coating, however these various thicknesses were not measured. Instead of repeating the coating process to increase thickness, authors varied the concentration of components. This affects MPN porosity and morphology. Previous reports have altered the thickness by repeating the coating process to maintain a similar morphology, yet that was not the method chosen here, and the morphology of the coatings were not examined. Additionally, thickness measurements were investigated by measuring the cell widths lengths with and without the coating, but the errors were larger than the measurements. Are their coating thicknesses comparable to previously reported thicknesses in the literature? Growth rates and max OD were examined of Fe(III)-TA encapsulated B. thetaiotamicron when treated with FaSSGF FaSSIF (Fig S13, S14) . Max ODs varied considerably, as did the growth rates, and this is not expected nor was addressed by the authors. Also, growth curves do not quantify the number of viable cells, but rather their overall fitness, and are thus not an accurate measure of viability. This is where CFU assays or LIVE DEAD staining is necessary. Especially when the uncoated cells have a shorter Teg (Fig S13, S14), which is more indicative of better viability than the max OD, and would also indicate that the coating does not provide protective benefits from the GI tract conditions if using this metric. Iu2019d like to reiterate that OD measurements at 48 hrs of growing after lyophilization are not quantitative for viability. Max OD should not be different, the Teg is related to the number of viable initial cells but itu2019s expected that the cells should all eventually reach the same max OD. If they donu2019t, as shown in some of the SI figures, then something is likely wrong with the culturing technique or, there is likely evaporation in the microwells which would affect the OD measurement. Whatu2019s most important is the number of viable cells present an t=0 once rehydrated. This is a very convoluted way to demonstrate that and may not be accurate. ,