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6.11B: Biofilms - Biology

6.11B: Biofilms - Biology



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Learning Objectives

  • Describe biofilms

Biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface. These cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, also referred to as slime, is a polymeric conglomeration composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial, and hospital settings.

The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single cells that may float or swim in liquid. Microbes form a biofilm in response to many factors, including cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated.

Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists initially form a weak, reversible adhesion to the surface via van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili. Some species are not able to attach to a surface on their own but are able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing using such products as AHL. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development; this is the stage in which the biofilm is established and may change only in shape and size. The development of a biofilm may allow for an aggregate cell colony (or colonies) to be antibiotic-resistant.

In sum, the five stages of biofilm development are as follows:

  1. Initial attachment
  2. Irreversible attachment
  3. Maturation I
  4. Maturation II
  5. Dispersion

Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle. Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm dispersal. Biofilm matrix-degrading enzymes may be useful as anti-biofilm agents. Recent evidence has shown that one fatty acid messenger, cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces cyclo heteromorphic cells in several species of bacteria and the yeast Candida albicans. Nitric oxide has also been shown to trigger the dispersal of biofilms of several bacteria species at sub-toxic concentrations, so it shows potential for use in the treatment of patients that suffer from chronic infections caused by biofilms.

Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased a thousandfold. Lateral gene transfer is also greatly facilitated in biofilms and leads to a more stable structure.

However, biofilms are not always less susceptible to antibiotics. For instance, the biofilm form of Pseudomonas aeruginosa has no greater resistance to antimicrobials than do stationary-phase planktonic cells, although when the biofilm is compared to logarithmic-phase planktonic cells, the biofilm does show greater resistance to antimicrobials. This resistance to antibiotics in both stationary phase cells and biofilms may be due to the presence of persister cells.

Key Points

  • Microbes form a biofilm in response to many factors, including cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or exposure of planktonic cells to sub-inhibitory concentrations of antibiotics.
  • Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists initially adhere to the surface through weak, reversible adhesion via van der Waals forces.
  • If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili.

Key Terms

  • biofilm: an aggregate of microorganisms in which cells adhere to each other on a surface

Explain how microorganisms can be used in response to pollution incidents such as an oil spill.

a. «bioremediation» is the use of microbes to remove environmental contaminants from oil spill

b. some pollutants are metabolized/degraded by microorganisms

c. microorganisms can be eubacteria/archaeans

d. microorganisms are useful in bioremediation because they can multiply very quickly «by binary fission»

e. microorganisms can use pollutants/oil spills/crude oil as energy sources/carbon sources/electron acceptors in cellular respiration

f. eg: Pseudomonas used «in bioremediation»

g. Pseudomonas requires nutrients «such as potassium and urea» to metabolize the oil at a faster rate «so sprayed on to an oil spill to aid the bacteria in their work»


Starting a new face: from biochemistry to the biology of biofilms

After working with bacterial biofilms as model to understand the evolution of enzymes in complex environments, it was hard to go back to domesticated bacteria. In my new lab we will continue to bridge between biofilms' biology and evolutionary biochemistry.

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In April 2019, we published in Nature Microbiology my favourite part of the six amazing years I spent as a postdoctoral fellow in Dan Tawfik’s lab (Weizmann Institute of Science, Israel). The project was designed in 2013, aiming at contributing to a fundamental field in biology: evolutionary biochemistry.

Evolutionary biochemistry is, broadly-speaking, a multi-disciplinary field. The goal is to understand how changes in the DNA sequence (mutations), affect enzyme function, and in turn, organismal survival within a population. Due to their fast duplication times and technical advantages, bacteria are typically the most common organism used in the field. So, an evolutionary biochemist is a combination of a geneticist, ecologist, microbiologist and biochemist. I personally have always been inclined towards the biochemist side. I like observing how the biochemical properties of enzymes, such as binding affinities or catalytic turnovers correlate with bacterial survival. So, both my graduate and postdoctoral studies were spent in evolutionary biochemistry laboratories, where proteins are at the centre of attention.

In evolutionary biochemistry, however, very often, beneficial mutations obtained in laboratory conditions are different from those that are most commonly found in nature, amongst orthologues. At Danny’s laboratory we decided to start testing evolution in biofilms. We hypothesised that perhaps more complex bacterial growth states, albeit still in laboratory-controlled conditions, could better mimic natural selection. Biofilms are one of the most common states of bacteria in nature, and are fundamentally different from bacterial populations in shaking conditions (planktonic). Still, in evolutionary biochemistry, we often measure fitness in the planktonic state, rather than the common and natural state of bacteria, the biofilm.

In our Nature Microbiology paper, we measured the fitness of mutations in biofilm populations, and compared the results to the typical scenario, obtained with the shaking conditions. I will not elaborate on the findings of our paper here, you can also find them in the “behind the paper” blog from this community. I think it will suffice to say that during my postdoc I learned many important scientific and personal lessons. But perhaps one of the most relevant is my evolved fascination for biofilms and although deep down I am a biochemist, microbiology started gaining a significant place in my scientific interest.

With my new biofilms-centered approach, last July I started my own laboratory in the Department of Plant Pathology and Microbiology, at the Faculty of Agriculture of the Hebrew University of Jerusalem, Israel. Our lab’s main goal is to continue dissecting evolution in biofilms, from a strong biochemistry perspective. Biofilms are not only one of the most common states of bacteria in nature, but are also known to thrive better than planktonic populations in adverse environmental conditions. However, despite the clear adaptability potential of biofilms, we don’t know enough about the evolutionary mechanisms that render new enzymatic functions in these bacterial populations. By combining genomics, biochemistry and biofilm microbiology, our new lab will explore how metabolic functions can evolve in biofilm populations, mediating their high adaptability capacity. We also hope this research will guide future efforts to engineer new metabolic functions in biofilms, which have been granted as potentially better bio-reactors than planktonic populations.


Nanocrystals that eradicate bacterial biofilms

Credit: Pohang University of Science & Technology (POSTECH)

The COVID-19 pandemic is raising fears of new pathogens such as viruses or drug-resistant bacteria. On this note, a Korean research team has recently drawn attention for developing the technology for removing antibiotic-resistant bacteria by controlling the surface texture of nanomaterials.

A joint research team from POSTECH and UNIST has introduced mixed-FeCo-oxide-based surface-textured nanostructures (MTex) as highly efficient magneto-catalytic platform in the international journal Nano Letters. The team consisted of professors In Su Lee and Amit Kumar with Dr. Nitee Kumari of POSTECH's Department of Chemistry and Professor Yoon-Kyung Cho and Dr. Sumit Kumar of UNIST's Department of Biomedical Engineering.

First, the researchers synthesized smooth surface nanocrystals in which various metal ions were wrapped in an organic polymer shell and heated them at a very high temperature. While annealing the polymer shell, a high-temperature solid-state chemical reaction induced mixing of other metal ions on the nanocrystal surface, creating a number of few-nm-sized branches and holes on it. This unique surface texture was found to catalyze a chemical reaction that produced reactive oxygen species (ROS) that kills the bacteria. It was also confirmed to be highly magnetic and easily attracted toward the external magnetic field. The team had discovered a synthetic strategy for converting normal nanocrystals without surface features into highly functional mixed-metal-oxide nanocrystals.

Transmission electron microscope (TEM) image of Mtex. Credit: POSTECH

The research team named this surface topography—with branches and holes that resembles that of a plowed field—'MTex.' This unique surface texture has been verified to increase the mobility of nanoparticles to allow efficient penetration into biofilm matrix while showing high activity in generating reactive oxygen species (ROS) that are lethal to bacteria.

This system produces ROS over a broad pH range and can effectively diffuse into the biofilm and kill the embedded bacteria resistant to antibiotics. And since the nanostructures are magnetic, biofilm debris can be scraped out even from the hard-to-reach microchannels.

"This newly developed MTex shows high catalytic activity, distinct from the stable smooth-surface of the conventional spinel forms," explained Dr. Amit Kumar, one of the corresponding authors of the paper. "This characteristic is very useful in infiltrating biofilms even in small spaces and is effective in killing the bacteria and removing biofilms."

"This research allows to regulate the surface nanotexturization, which opens up possibilities to augment and control the exposure of active sites," remarked Professor In Su Lee who led the research. "We anticipate the nanoscale-textured surfaces to contribute significantly in developing a broad array of new enzyme-like properties at the nano-bio interface."


Acknowledgements

The authors thank D. Kearns (Indiana University) for the kind gift of the B. subtilis 2569 strain, and Y. Liu for the software instruction. Regular TEM characterization was performed at the National Center for Protein Science, Shanghai. Fluorescence microscopy was performed at the Molecular Imaging Core Facility of SLST, Shanghai Tech University. This work was funded by the Science and Technology Commission of Shanghai Municipality (17JC1403900), National Natural Science Foundation of China (No. 31570972), and 2016 Open Financial Fund of Qingdao National Laboratory for Marine Science and Technology (Grant No. QNLM2016ORP0403) for C. Zhong C.Zhong. also acknowledges start-up funding support from ShanghaiTech University and 1000 Youth Talents Program, granted by the Chinese Central Government. The work was also partially funded by the National Natural Science Foundation of China (No.31872728) for J.H., and the National Natural Science Foundation of China (NSFC: No. 31522017, No. 31470834, No. 31670869) for H.Y.


3 RESULTS AND DISCUSSION

In an initial study presented here, the minimum inhibitory concentrations (MIC) and biofilm eradication concentrations (MBEC) were evaluated for all parent phenols and their AM derivatives toward the Gram-negative bacterium P. aeruginosa and the Gram-positive bacterium S. epidermidis. AM derivatives were typically more potent than their corresponding parent phenols against planktonic cells, apart from 1/2k against S. epidermidis (Table 1). We have previously shown that the lipophilic phenols 1d (in particular) and 1e are unusually potent toward S. epidermidis (Walsh et al., 2020 ). The observation that 2d exhibits a lower potency compared to 1d toward both bacteria may simply be a case where the exceptional activity of the parent phenol is ineffectively expressed in its AM. On the high end (average over four pairings), AM derivatives were 66 times more potent than their phenolic counterparts toward S. epidermidis and 16.0 times more potent toward P. aeruginosa in the planktonic phase. These results are consistent with the cleavage of the AM group via intracellular esterase, resulting in cellular retention and intracellular concentration of the phenolic antimicrobial.

Minimum inhibitory concentration (mM)
S. epidermidis P. aeruginosa
Compound Phenol AM Phenol AM
1/2a 1.9 0.1 1.9 0.5
1/2b 3.9 0.9 7.8 1.9
1/2c 15.6 0.23 7.8 1.9
1/2d 0.3 1.9 1.5 3
1/2e 4.5 1.9 7.8 3
1/2f 15.62 0.7 31.2 1.3
1/2g 7.9 0.5 15.6 1.3
1/2h 15.6 1.9 15.6 3.8
1/2i 15.6 0.1 7.8 0.9
1/2j 2.5 0.25 6.2 0.9
1/2k 0.9 1.5 1.9 0.75
1/2l 0.9 0.7 1.9 1.5
1/2m 15.6 7.8 31.2 25
1/2n 0.23 0.12 7.8 0.9
1/2o 0.23 0.023 3.9 0.5
1p/3a 1.9 0.5 1.9 0.1
1q/3b 3.1 0.6 3.9 0.9
1r/3c 125 62.5 125 31.2
1s/4a 15.6 3.9 15.6 1.9
1t/4b 7.8 1.9 15.6 3.9

Against biofilms, AMs were again more potent than parent phenols with rare exception. Toward biofilms, AM derivatives (high-end average over four pairings) were 9.3 times more potent toward S. epidermidis and 15.0 times more potent against P. aeruginosa. These results provide further confirmation that the addition of an AM group can substantially increase the modest potency of small phenols toward both biofilms and planktonic cells. It should be emphasized here that the foregoing results strongly support the use of an AM-based prodrug approach for increasing antimicrobial activities, even though the present compounds are active at high micro/low millimolar concentrations. It is expected that the potencies of commercially successful antimicrobials will be similarly augmented by the use of this strategy.

AMs 2c, 2f, 2j, and 3b were the most potent compounds against S. epidermidis biofilms, while AMs 2f, 2k, 2j, and 3a were most effective in eradicating P. aeruginosa biofilms. Exceptions to the predominant trend are 1/2k, 1/2g, and 1/2e where parent and AM shared the same MBEC against S. epidermidis and compound 2d where the AM was less potent against both bacteria (Table 2). In the cases of 1/2d,1/2e, and 1/2k (a chlorophenol), the parent phenol already possessed superb activity, with 2d and 2e sharing a highly hydrophobic substituent at the 4-position (vide supra Walsh et al., 2020 ).

Minimum Biofilm Eradication Concentration (mM)
S. epidermidis P. aeruginosa
Compound Phenol AM Phenol AM
1/2a 31.2 6.2 62.5 12.5
1/2b 31.2 12.5 31.2 12.5
1/2c 31.2 3.1 62.5 6.2
1/2d 1.9 12.6 7.5 25
1/2e 6.2 6.2 50 25
1/2f 31.2 2.7 62.5 2.7
1/2g 15.6 15.6 62.5 15.6
1/2h 25 7.8 25 15.6
1/2i 62.5 6.2 31 12.5
1/2j 3.1 2.7 6.2 3.1
1/2k 6.2 6.2 12.5 3.1
1/2l 31 7.8 31.2 12.5
1/2m 50 12.6 50 15.6
1/2n 15.6 7.8 31.2 15
1/2o 31.2 7.8 62.5 15
1p/3a 62.5 25 31.2 1.5
1q/3b 6.2 3 12.5 6.2
1r/3c 62.5 31.2 31.2 15.6
1s/4a 50 15.6 100 15.6
1t/4b 25 7.8 25 12.5

As a control experiment, AM 3d, which lacks a phenolic OH, was among the least potent AM derivatives, with a MBEC of 24 mM toward both bacteria. It should be noted that many of these AMs possess electron rich aromatic nuclei (i.e., 2c [for S. epidermidis], 2f and 3a) whereas the other is the simple 4-chloro derivative 2j. The unexpectedly high activity of 2f prompted us to investigate capsaicin derivative 2h, to probe for a vanilloid receptor component. Unfortunately, 2h was in no way special in its activity.

It is also interesting to note that the most potent parent phenols did not consistently result in the most potent AMs against biofilms. Phenols 1d, 1e, 1k, 1j, and 1q were most potent toward S. epidermidis, while phenols 1d, 1h, 1k, 1j, and 1q were most potent toward P. aeruginosa (Tables 1 and 2). Out of these six compounds, the only AMs with top potency were 2k and 2j toward S. epidermidis, with 2j and 3b most active toward P. aeruginosa. Phenol 1f was among the least potent toward both bacteria while 2f was among the five most potent toward both bacteria.

AMs 2f and 2j were the most successful compounds against biofilms for both types of bacteria. Two isomers of 2f, 2g, and 3c were also evaluated. However, these isomers demonstrated a substantial decrease in potency compared to 2f. This trend was not seen in parent phenols where 1g was the most potent isomer toward S. epidermidis and 1r was the most potent isomer toward P. aeruginosa. In parent phenols, a dramatic difference in potency was not observed as it was with the prodrug derivatives (Table 3).

Minimum Biofilm Eradication Concentration (mM)
S. epidermidis P. aeruginosa
2a 6.2 3b 12.5
2f 2.7 1b 2.7
2g 15.6 10b 15.6
3a 25 11b 1.5
3c 31.2 12b 15.6

These results suggest that the positioning of functional groups around the aromatic ring can make a dramatic difference in potency for AM derivatives, which is also observed in the isomers 2a and 3a (Table 3). AM 2a has methyl groups is the 2 and 4 position while 3a has methyl groups in the 2 and 6 positions. Here, 2a is more potent toward S. epidermidis and 3a more potent toward P. aeruginosa (Table 3). This was also observed with the corresponding phenols (Table 2).

It was observed that all compounds exhibited a higher potency toward planktonic cells when compared to biofilms (Tables 1 and 2). Parent phenols were, on average, 26 times more potent toward S. epidermidis planktonic cells compared to biofilms and 10 times more potent toward P. aeruginosa in the planktonic state. AM derivatives were on average 55 times more potent toward planktonic S. epidermidis and 11 times more potent toward planktonic P. aeruginosa compared to the corresponding biofilm states. This was expected due to the higher susceptibility of planktonic cells. AMs also experienced a larger disparity in potencies between planktonic cells and biofilms than were seen with parent phenols. The latter observation is consistent with the ability for the cleaved iminodiacetate to concentrate within cells, a characteristic that the parent phenol lacks.

From a structural prospective, the AM series 3ac, wherein the phenolic OH occupies the 4-position, did not consistently show either a heightened or muted potency compared to 2ao, where involvement of the phenolic OH in chelation is expected (vide supra). It is nonetheless significant that both 3a and 3b exhibited a dramatic increase in potency toward P. aeruginosa. Interestingly, the “non-traditional” AMs 4a and b did not show enhanced potency toward either bacteria compared to the five most potent “traditional” AMs. This could be due to the lengthened distance of the chelating moiety from the aromatic ring. As before, however, aromatic chlorine substitution did lead to an enhancement of activity (4b vs. 4a, Table 4) as would be expected (Suter, 1941 ).

Minimum Biofilm Eradication Concentration (mM)
S. epidermidis P. aeruginosa
Compound Phenol AM Phenol AM
1p/3a 62.5 25 31.2 1.5
1q/3b 6.2 3 12.5 6.2
1r/3c 62.5 31.2 31.2 15.6
1s/4a 50 15.6 100 15.6
1t/4b 25 7.8 25 12.5

3.1 Comparison of AM prodrugs to commercial antibiotics

Gratifying as the aforementioned activity enhancements were for the prodrug AMs vis a vis their parent phenols, overall potencies seldom reached the micromolar range expected for modern antibiotics against planktonic cells. To document a direct comparison to the present AMs, three commercial antibiotics were selected for MBEC evaluation under the current assay toward S. epidermidis and P. aeruginosa biofilms (Table 4). Metronidazole is a nitroimidazole derivative that was selected for its use in treating a variety of bacterial infections and has been shown to exhibit activity toward biofilms of Helicobacter pylori (Yonezawa, Osaki, Hojo, & Kamiya, 2019 ) and C. difficile (Vuotto, Moura, Barbanti, Donelli, & Spigaglia, 2016 ). Metronidazole had a MBEC of 6.2 mM toward S. epidermidis and 50 mM toward P. aeruginosa biofilms. Tobramycin is an aminoglycoside that was chosen because it has been extensively studied for efficacy toward P. aeruginosa biofilms and has been clinically used in the treatment of cystic fibrosis (Høiby et al., 2019 ). Under our experimental protocol, tobramycin had a MBEC of 18 mM toward S. epidermidis and of 0.06 mM toward P. aeruginosa biofilms. Nitazoxanide is a broad spectrum antiparasitic and antiviral drug that has more recently been studied for effectiveness against bacteria (Carvalho, Lin, Jiang, & Nathan, 2009 Guttner, Windsor, Viiala, Dusci, & Marshall, 2003 Singh & Narayan, 2011 ). Nitazoxanide has also been shown to inhibit S. epidermidis biofilm formation (Tchouaffi-Nana et al., 2010 ). Nitazoxanide demonstrated an MBEC of 50 mM toward S. epidermidis and of 3.12 mM toward P. aeruginosa biofilms.

Against S. epidermidis biofilms, several AM compounds were more potent than metronidazole and tobramycin. A comprehensive table (Table S1) can be found in the supplementary. All 18 AM prodrugs exhibited a lower MBEC toward P. aeruginosa when compared to metronidazole although here, tobramycin exhibited the highest potency of all compounds evaluated. Similarly, all 18 AM derivatives were more potent toward S. epidermidis when compared to nitazoxanide. That several AMs were more potent toward both bacteria compared to metronidazole might be anticipated since metronidazole is used to treat anaerobic infections while both S. epidermidis and P. aeruginosa are both facultative anaerobes.

In an additional control study, a selected number of iminodiacetic acids 5a, 5g, 5k, and 5l were evaluated for potency. These are the cleaved form of the drug, produced after esterase cleavage (Figure 2). The free iminodiacetates were not expected to effectively permeate through the biofilms or cross the cell membrane as efficiently as their AM counterparts.

All iminodiacetates were significantly less potent than their corresponding AM prodrugs against biofilms (Table 5). AMs were, on average, 10 times more potent than the corresponding iminodiacetates against S. epidermidis and 16 times more potent against P. aeruginosa biofilms. This also supports that the AM form of the drug is able to penetrate and eradicate cells within a biofilm. In comparison to phenols 5a, 5g, 5k, and 5l, it was also observed that the corresponding iminodiacetates were often less potent (Tables 1 and 5).

Minimum Biofilm Eradication Concentration (mM)
S. epidermidis P. aeruginosa
Compound Parent phenol AM Iminodiacid (5) Parent phenol AM Iminodiacid (5)
1/2/5a 31.2 6.2 125 62.5 12.5 >250
1/2/5f 31.2 2.7 62.5 62.5 2.7 125
1/2/5k 6.2 6.2 62.5 12.5 3.1 125
1/2/5l 31 7.8 62.5 31.2 12.5 31.2

In effort to better explore additional synthetic options for derivations of iminodiacetate functionalized prodrugs, five variations of ester prodrugs (e.g., 6-10f) were synthesized and evaluated against planktonic cells and biofilms. This was done in an effort to explore alternative ester varieties as prodrugs. Eugenol (1f) was chosen as the phenolic scaffold since its AM (2f) was one of the five most potent toward biofilms in the initial series (Table 1, vide supra). The hemiacytal MEM derivative 10f was selected to increase hydrophilicity, since eugenol itself has low water solubility. The simple esters 6/7f were selected since common ester functionality should be more robust toward esterase than the acylal function present in AMs. Acylals 8/9f possess butyryl and pivaloyl esters in place of the acetate, respectively. These were selected for examining terminus variation of the iminodiacetate group. The bis(pivaloyloxy)methyl ester was also selected due to its use in prodrugs to improve bioavailability ( Brass, 2002 ).

Against planktonic cells, prodrug derivative 8f exhibited the highest potency against S. epidermidis, while 2f showed the highest potency toward P. aeruginosa (Table 6). The ethyl and allyl ester derivatives (6f, 7f) were the least potent prodrugs overall with MICs of 31.2 mM toward both bacteria. Against S. epidermidis 6f and 7f were also less potent than the parent phenol (1f) (Figure 3).

Minimum inhibitory concentration (mM)
S. epidermidis P. aeruginosa
1f 15.6 1f 31.2
2f 0.68 2f 1.3
6f 31.2 6f 31.2
7f 31.2 7f 31.2
8f 0.12 8f 3.9
9f 1.9 9f 15.6
10f 1.9 10f 31.2

Against biofilms, AM 2f had the highest potency against both bacteria (Table 7). Compound 7b was the least potent toward S. epidermidis while 7e was the least potent toward P. aeruginosa. This suggests that AM 2f is either more permeable to the biofilm or the ester bonds present in this group are more readily cleaved by esterase or both. It is also of interest that the hemiacytal 7b showed commendable activity toward S. epidermidis.

Minimum Biofilm Eradication Concentration (mM)
S. epidermidis P. aeruginosa
1f 31.2 1f 62.5
2f 2.75 2f 2.75
6f 31.2 6f 62.5
7f 50 7f 50
8f 15.6 8f 62.5
9f 20.6 9f 41.2
10f 7.8 10f 125

A CDC Biofilm reactor assay was also used to substantiate the comparative efficacy of eugenol (1f) with its corresponding AM derivative (2f) against P. aeruginosa (PA015542). Here, unlike the static condition of the 96-well plate, biofilms were grown in a high shear environment. This method increases the biofilm adherence to the surface on which it is grown and causes the biofilm to produce a more robust EPS (Gloag, Fabbri, Wozniak, & Stoodley, 2020 Stoodley, Cargo, Rupp, Wilson, & Klapper, 2002 ). This method was chosen because it has been standardized by the ASTM. Similar to the static 96-well plate assays, the potency of the AM derivative (2f) was greater than the parent compound (1f) using the CDC biofilm reactor. Eugenol (1f) demonstrated a mean log reduction of 1.68 ± 0.12 while its corresponding AM (2f) had a mean log reduction of 5.81 ± 0.53. A graph of these data can be found in the supplementary materials (Figure S1). This has shown that the AM (2f) is significantly more potent than the parent (1f) against biofilms grown in both static and high shear environments.

Once AMs have penetrated the biofilm's extracellular matrix and the membrane of indwelling cells, they are predicted to be acted upon by intracellular esterase, liberating the active form as the iminodiacetate. The resulting highly charged antimicrobial is then entrapped within the cell. The observed increase in potency supports that AMs are being acted upon by esterase once inside the cell, but additional experimentation was also performed to further support this hypothesis. According to KEGG genome annotations, P. aeruginosa (PAO1) and S. epidermidis (RP62A) contain 39 and 16 esterases, respectively, as well as other enzymes that have been shown to have esterase activity (Foster, 1996 Goullet & Picard, 1991 ). For example, EstA has been shown to possess esterase activity in Pseudomonas and is thought to be involved the hydrolysis of ester containing compounds on the cell surface or in the culture medium (Nicolay, Devleeschouwer, Vanderleyden, & Spaepen, 2012 Wilhelm, Gdynia, Tielen, Rosenau, & Jaeger, 2007 ). Here, esterase from porcine liver was used because it has been shown to readily cleave ester bonds in small organic molecules (Perez, Daniel, & Cohen, 2013 ) as well as antibiotics such as ampicillin and amoxicillin (Zhou et al., 2019 ). In order to determine whether esterase will cleave these AM groups, 2b was exposed to esterase in vitro and samples were viewed via mass spectrometry to determine if the liberated iminodiacetate 5b was present (Figure 4).

The AM derivative of 4-methoxyphenol (2b) was exposed to esterase in cold HEPES buffer, and LC-MS was performed to determine the amount of the liberated product, 2,2′-((2-hydroxy-5-methoxybenzyl)azanediyl)diacetate (5b), present. The exact mass of 2b is 413.1322 amu, with a predicted [M] − of 413.1322 amu and the exact mass of 5b is 267.0745 amu with predicted [M – 2H] 2− of 132.5366 amu. The exact mass of the protonated derivative of 5b is 269.0889 amu with a predicted [M + H] + of 268.0816. Both the mono- and di-anionic product are expected to be present in the esterase exposed samples, which were evaluated.

Spectra were taken of the pure AM compound 2b (A) and the liberated derivative 5b (B) (Figure 5). In frame B, both peaks for the mono and di-anionic species can be observed. The AM 2b was exposed to esterase in HEPES buffer for 8 (D), 16 (E), and 24 (F) min, extracted, and then analyzed via LCMS (Figure 5). The masses of 2b and 5b were found were within two decimals of the predicted masses for each compound. A control of 2b in HEPE without esterase was also included to ensure that HEPE does not influence the prodrug (C). This assay has shown that, in vitro, the AM derivatives are, in fact, acted upon by esterase. This suggests that the increase in potency is, in part, due to the AM being able to penetrate the biofilm and transform within the cell to release the iminodiacetate. This is further supported by the observation that the iminodiacetates 5a,5f,5k, and 5l are significantly less active than the corresponding AMs toward biofilms. Although an in vivo study has yet to be conducted, Calcein AM has been used extensively to successfully stain biofilms (Godoy-Santos, Pitts, Stewart, & Mantovani, 2019 Ohsumi et al., 2015 Tawakoli, Al-Ahmad, Hoth-Hannig, Hannig, & Hannig, 2013 Tawakoli et al., 2013 Wakamatsu et al., 2014 ) and the results presented herein strongly support the hypothesis that AM derived prodrugs will act via a similar mechanism.


Septic Arthritis in Anterior Cruciate Ligament Surgery

Charalampos G. Zalavras MD , Michael J. Patzakis MD , in The Anterior Cruciate Ligament (Second Edition) , 2018

Biofilm Formation

Biofilm formation is a key mechanism for persistence or recurrence of infection. The biofilm is an aggregation of microbial colonies enclosed within an extracellular polysaccharide matrix (glycocalyx) that adheres on the surface of implants or devitalized tissue. 55 The presence of avascular graft and metal fixation devices in ACL reconstruction creates conditions conducive to biofilm development if postoperative SA is not treated early and adequately. The biofilm protects the organism from antibiotics and host defense mechanisms, such as antibody formation and phagocytosis thus infection may exist in a subclinical state and eventually recur. In chronic musculoskeletal infections, removal of the biofilm by removal of implants and débridement of devitalized tissue is necessary for successful treatment of infection. 56


Dental Caries

Andréa G. Ferreira Zandoná , . R. Scott Eidson , in Sturdevant's Art and Science of Operative Dentistry , 2019

Ecologic Basis of Dental Caries: The Role of the Biofilm

Dental plaque is a term historically used to describe the soft, tenacious film accumulating on the surface of teeth. Dental plaque has been more recently referred to as the dental biofilm or simply the biofilm, which is a more complete and accurate description of its composition (bio) and structure (film). 5 The biofilm is composed mostly of bacteria, their by-products, extracellular matrix, and water ( Figs. 2.6, 2.7, 2.8, 2.9, and 2.10 ). Biofilm is not adherent food debris, as was widely and erroneously thought, nor does it result from the haphazard collection of opportunistic microorganisms. The accumulation of the biofilm on teeth is a highly organized and ordered sequence of events. Bacteria seem to occupy the same spatial niche on most individuals. A “hedgehog” formation has been recently characterized 209 because of the spine of radially oriented filaments. The filaments are a mass of Corynebacterium filaments with Streptococcus at the periphery. Actinomyces are usually found at the base of the biofilm suggesting that Corynebacterium attaches to a preexisting biofilm containing Actinomyces. In any case it is notable that each taxon is localized in a precise and well-defined spatial zone indicating that the microbes in the oral biofilm have a precise and well-tuned interaction 209 (see Fig. 2.6D ).

Fig. 2.6 . A, Composite diagram illustrating the relationship of biofilm (p) to the enamel in a smooth-surface initial (noncavitated) lesion. A relatively cell-free layer of precipitated salivary protein material, the acquired pellicle (ap) covers the perikymata ridges (pr). The biofilm bacteria attach to the pellicle. Overlapping perikymata ridges can be seen on the surface of enamel (see Fig. 2.7 ). ( Figs. 2.9 and 2.10 are photomicrographs of cross sections of biofilm.) The enamel is composed of rodlike structures (er) that course from the inner dentinoenamel junction (DEJ) to the surface of the crown. Striae of Retzius (sr) can be seen in cross sections of enamel. B, Higher power view of the cutout portion of enamel in A. Enamel rods interlock with each other in a head-to-tail orientation. The rod heads are visible on the surface as slight depressions on the perikymata ridges. The enamel rods comprise tightly packed crystallites. The orientation of the crystallites changes from being parallel to the rod in the head region to being perpendicular to the rod axis in the tail end. Striae of Retzius form a descending diagonal line, descending cervically. C, Drawings 1 through 5 illustrate the various stages in colonization during plaque formation on the shaded enamel block shown in B. The accumulated mass of bacteria on the tooth surface may become so thick that it is visible to the unaided eye. Such plaques are gelatinous and tenaciously adherent they readily take up disclosing dyes, aiding in their visualization for oral hygiene instruction. Thick plaque biofilms (4 and 5) are capable of great metabolic activity when sufficient nutrients are available. The gelatinous nature of the plaque limits outward diffusion of metabolic products and serves to prolong the retention of organic acid metabolic by-products. D, This illustrates how different taxons inhabit specific niches on the biofilm creating microenvironments. There is a fine-tuned synergy among the cells in the oral microbial communities. The environment and the biochemical gradients drive the selection process. This can be exemplified by the the role of Streptococcus. Where Streptococcus predominate they create an environment rich in CO2, lactate, and acetate, containing peroxide and having low oxygen. This environment is advantageous for the growth of bacteria such as Fusobacterium and Leptotrichia.

(From Welch JL, Rossetti BJ, Riekem CW, et al: Biogeography of a human oral microbiome at the micron scale, Proc Natl Acad Sci USA 9113(6):E791–E800, 2016. doi:10.1073/pnas.1522149113)

Fig. 2.7 . A, Scanning electron microscope view (600×) of overlapping perikymata (P) in sound enamel from unerupted molar. B, Higher power view (2300×) of overlapped site rotated 180 degrees. Surface of noncavitated enamel lesions has “punched-out” appearance.

(From Hoffman S: Histopathology of caries lesions. In Menaker L, editor: The biologic basis of dental caries, New York, 1980, Harper &amp Row.)

Fig. 2.8 . Representative 3-D rendering images of mixed-species biofilms in an environment with 1% (W/V) sucrose. The images show the evolution of the microcolonies over time and the arrangement with the EPS matrix.

(From Xiao J, Klein MI, Falsetta ML, et al: The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm, PLoS Pathog 8(4):e1002623, 2012. https://doi.org/10.1371/journal.ppat.1002623 )

Fig. 2.9 . Plaque biofilm formation at 1 week. Filamentous bacteria (f) appear to be invading cocci microcolonies. Plaque near gingival sulcus has fewer coccal forms and more filamentous bacteria (860×).

(From Listgarten MA, Mayo HE, Tremblay R: Development of dental plaque on epoxy resin crowns in man. A light and electron microscopic study, J Periodontol 46(1):10–26, 1975.)

Fig. 2.10 . At 3 weeks old, plaque biofilm is almost entirely composed of filamentous bacteria. Heavy plaque formers have spiral bacteria (a) associated with subgingival plaque (660×).

(From Listgarten MA, Mayo HE, Tremblay R: Development of dental plaque on epoxy resin crowns in man. A light and electron microscopic study, J Periodontol 46(1):10–26, 1975.)

Many of the organisms found in the mouth are not found elsewhere in nature. Survival of microorganisms in the oral environment depends on their ability to adhere to a surface. Free-floating organisms are cleared rapidly from the mouth by salivary flow and frequent swallowing. Although a few specialized organisms, primarily streptococci, are particularly able to adhere to oral surfaces such as the mucosa and tooth structure, over 700 different species of bacteria have been identified in the oral biofilm. Oral biofilm from healthy teeth have a higher diversity than from carious teeth. 134

Significant differences exist in the biofilm communities found in various habitats (ecologic environments) within the oral cavity ( Fig. 2.11A and B ). The organisms also have unique contributions to the ecosystem (see Fig. 2.11B ). Mature biofilm communities have tremendous metabolic potential and are capable of rapid anaerobic metabolism of any available carbohydrate ( Fig. 2.12 ). However, because of the highly structured bacterial microcolonies embedded in an exopolysaccharide (EPS)-rich matrix, there are acidic regions in the biofilm that are not neutralized by saliva buffers. 210

Fig. 2.11 . Approximate proportional distribution of predominant cultivable flora of five oral habitats.

(From Simón-Soro Á, Tomás I, Cabrera-Rubio R, et al: Microbial geography of the oral cavity, J Dent Res 92:616, 2013. DOI:10.1177/0022034513488119.)

Fig. 2.12 . A, Mature biofilm communities have tremendous metabolic potential and are capable of rapid anaerobic metabolism of any available carbohydrates. Classic studies by Stephan show this metabolic potential by severe pH drops at the plaque-enamel interface after glucose rinse. It is generally agreed that a pH of 5.5 is the threshold for enamel demineralization. Exposure to a glucose rinse for an extreme caries activity plaque results in a sustained period of demineralization (pH 5.5). Recording from a slight caries activity biofilm shows a much shorter period of demineralization. B, The frequency of sucrose exposure for cariogenic biofilm greatly influences the progress of tooth demineralization. The top line illustrates pH depression, patterned after Stephan's curves in A. Three meals per day results in three exposures of biofilm acids, each lasting approximately 1 hour. The biofilm pH depression is relatively independent of the quantity of sucrose ingested. Between-meal snacks or the use of sweetened breath mints results in many more acid attacks, as illustrated at the bottom. The effect of frequent ingestion of small quantities of sucrose results in a nearly continuous acid attack on the tooth surface. (The clinical consequences of this behavior can be seen in Fig. 2.37 .) C, In active caries, a progressive loss of mineral content subjacent to the cariogenic biofilm occurs. Inset illustrates that the loss is not a continuous process. Instead, alternating periods of mineral loss (demineralization) occur, with intervening periods of remineralization. The critical event for the tooth is cavitation of the surface, marked by the vertical dashed line. This event marks an acceleration in caries destruction of the tooth and irreversible loss of tooth structure. An intervention is usually required to arrest the lesion, often of the restorative nature.

(A, Adapted and redrawn from Stephan RM: Intra-oral hydrogen-ion concentration associated with dental caries activity, J Dent Res 23:257, 1944.)

Many distinct habitats may be identified on individual teeth, with each habitat containing a unique biofilm community ( Table 2.2 see Fig. 2.11A ). Although the pits and fissures on the crown may harbor a relatively simple population of streptococci, the root surface in the gingival sulcus may harbor a complex community dominated by filamentous and spiral bacteria. Even within the same anatomic location there can be a considerable difference in bacterial diversity. 134 For example the mesial surface of a molar may be carious and have a biofilm dominated by large populations of mutans streptococci (MS) and lactobacilli, whereas the distal surface may lack these organisms and be caries free. Generalization about biofilm communities is difficult.

TABLE 2.2 . Oral Habitats a

HabitatPredominant SpeciesEnvironmental Conditions Within Biofilm
MucosaS. mitisAerobic
S. sanguispH approximately 7
S. salivariusOxidation-reduction potential positive
TongueS. salivariusAerobic
S. mutanspH approximately 7
S. sanguisOxidation-reduction potential positive
Teeth (noncarious)S. sanguisAerobic
pH 5.5
Oxidation-reduction negative
Gingival creviceFusobacteriumAnaerobic
SpirochaetapH variable
ActinomycesOxidation-reduction very negative
Veillonella
Enamel cariesS. mutansAnaerobic
pH &lt5.5
Oxidation-reduction negative
Dentin cariesS. mutansAnaerobic
LactobacilluspH &lt5.5
Oxidation-reduction negative
Root cariesActinomycesAnaerobic
pH &lt5.5
Oxidation-reduction negative

Recent evidence indicates that there are no specific pathogens that correlate with dental caries, but rather microbial communities. 135 Nevertheless, the general activity of biofilm growth and maturation is predictable and sufficiently well known to be of therapeutic importance in the prevention of dental caries.

Professional tooth cleanings are intended to control the biofilm (plaque) and prevent caries (and periodontal) disease. However, after professional removal of all organic material and bacteria from the tooth surface, a new coating of organic material begins to accumulate immediately. Within 2 hours, a cell-free, organic film, the acquired enamel pellicle (AEP) (see Fig. 2.6A and C ), can cover the previously denuded area completely. The pellicle is formed primarily from the selective precipitation of various components of saliva, particularly selective enzymes. The functions of the pellicle are believed to be (1) to protect the enamel, (2) to reduce friction between teeth, and (3) to provide a matrix for remineralization. 6 Although the pellicle exhibits antibacterial activity due to the presence of several enzymes, it can also function as a facilitator of bacterial cononization. 136


Biology Professor Discovers Key Elements for Biofilm Spreading

A biology professor at the University of California, Merced, discovered mechanisms that allow a potentially fatal biofilm to spread and resist drugs.

The research was published during the summer in mBio, an open-access online journal by the American Society for Microbiology.

Professor Clarissa J. Nobile, who studies microbial communities, said the findings could help in developing treatments for fungal biofilm infections, specifically those formed by Candida albicans.

“There are no known biofilm-specific drugs on the market today for any microorganism,” Nobile said. The fungus is naturally found in the human gut and can cause yeast infections and oral thrush. Infections can also be caused by implanted medical devices, which provide surfaces for biofilms to form. The infections can be life-threatening.

Nobile pinpointed four core proteins — all members of a histone deacetylase complex — that control how the biofilm forms, and learned what happens when they’re changed.

Mutations in the genes encoding each of these four complex members cause the biofilm to be more resistant to agitation and less likely to spread to other parts of a body, and also more resistant to drugs. Drugs that inhibit histone deacetylase are most often used to as mood stabilizers in psychiatry and neurology, and are also being tested to combat cancer, Nobile said. It’s not yet known if they could be used against biofilms and whether there’d be side effects.

Nobile’s interest in biofilms began just as she entered graduate school at Columbia University. Her mother became extremely sick with a biofilm infection. Nobile was surprised to learn there weren’t any reliable treatments and generally little was known about it.

She received her Ph.D. in biology from Columbia in 2007 and served as a postdoctoral researcher at UCSF until she was appointed to a tenure-track position at UC Merced in January 2014.

The opportunity to forge innovative collaborations with UC Merced’s ambitious faculty members was among the reasons she took a position with the campus. Nobile works with Professor Miriam Barlow, who studies antibiotic resistance, and Professor David Ojcius, who studies infectious diseases, including valley fever.

With advances in research and technology, Nobile is looking at the microbiome — all the microorganisms, good and bad, that live within the human body. Increasingly, research is showing what an important role the microbiome plays in health and disease.

Microbes are able to communicate with each other, and Nobile believes these interactions will shed more light on infections and diseases.


Community dynamics: Microbiomes and biofilms

The human body is home to an extraordinary diversity of microbes, which are increasingly suggested to play pivotal roles in human health. Human microbiome sequencing projects have revealed intriguing correlations between specific patterns of microbial diversity and multiple aspects of host health. The establishment of microbial causal roles is gathering pace thanks to experimental manipulations, however the inter-cellular causal mechanisms frequently remain obscure.

The Brown lab is developing a framework to understand microbiome developmental biology – to understand when, where and how potential interactions come to be realized via demographic and regulatory interactions between expanding lineages of bacteria, and the consequences of these interactions for microbiome functioning in both health and in polymicrobial disease.

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McNally L, Brown SP* (2015) Building the microbiome in health and disease: niche construction and social conflict in bacteria. Phil Trans R Soc Lond B 370, e20140298

Single gene locus changes perturb complex microbial communities as much as apex predator loss. 2015. D McClean, L McNally, LI Salzberg, KM Devine, SP Brown, I Donohue. Nature communications6, 8235-8235

Estrela S, Whiteley M, Brown SP. 2015. The demographic determinants of human microbiome health. Trends Microbiology23, 134-141.

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