STAADIUM™ Inhibitor Substrates

Changing the Composition of Microbiota Using Enzyme-Triggered Growth Inhibitors

At BIOSYNTH we developed a range of small molecules to provide a generic toolbox for the modulation of microbiota. Such compounds are needed in diagnostics to suppress background flora in enrichment media but also in therapies to change population compositions where disturbed microbiota are a cause of disease. We synthesized a number of compounds containing three functional elements: an enzyme labile group to facilitate species selectivity and a biocide that is released upon cleavage by species selective enzyme activity and additional elements and to enhance chemical stability.
STAADIUM™ enzyme-responsive inhibitors are non-inhibitory in their original state and exert their growth inhibition only after the enzymatic release of the biocide that acts through its metal chelating activity. Addition of metal ions is used to modulate the activity and prevent effects on the non-target populations.

STAADIUM™ GalactoZides are the first members of a new generation of antimicrobial compounds

At Biosynth we designed the beta-galactosidase-responsive inhibitor STAADIUM™ GalactoZide II and tested it on beta-galactosidase negative Salmonella enteritidis and beta-galactosidase positive Escherichia coli. The growth inhibition was determined by measuring optical density in broth and colony size and number on agar.

GalactoZide Experiment

Figure 1: STAADIUM™ GalactoZide II elimiates E. coli from mixed cell cultures. A mixture of test organisms Salmonella enteritidis (beta-galactosidase negative) and Escherichia coli ATCC 25922 (beta-galactosidase positive) were incubated without inhibitors (A) and with STAADIUM™GalactoZide II (B) in their growth media. A chromogenic indicator was also added to the media to stain E. coli colonies in blue.

The compound proved effective against E. coli (20% of OD compared to control in broth and pin-point colonies compared to 1.5 – 2 mm colonies of control after o/n growth) and did not affect the growth of S. enteritidis (same as control). In mixed cultures of initially 90 % E. coli and 10 % S. enteritidis the ratio shifted to 50% E. coli and 50% S. enteritidis in presence of the enzyme-activated inhibitor.

effect of GalactoZide in mixed cell suspensionFigure 2: Enrichment of Salmonella enteritidis in broth culture. In the experiment a cell suspension was prepared with 10% S.e. and 90% E.coli in saline. Tube cultures with NB were inoculated at low density (ca. 150 CFU/mL) and incubated at 37°C for 20 h. After the incubation time the culture was plated on agar with chromogenic indicator for E. coli. Numbers of colored colonies (E. coli) and white S. enteritidis colonies were counted.


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GalactoZide I - Z-4006_P00

GalactoZide II - Z-4002_P00

PeptiZide L-Ala - Z-4001_P00

GlucuroniZide - Z-4000_P00

PeptiZide L-Pyr - Z-4005_P00

Reduction of the bacterial background in microbiological tests

In food and drinking water control, hygiene monitoring and clinical diagnostics predominant bacteria such as E.coli overgrow the target bacteria. As a result the detection limit for the target bacteria is diminished.

Enzyme-activated inhibitors have the potential
to revolutionize microbiological assays

As described, by adding STAADIUM™ GalactoZide to solid growth media inhibits the growth of unwanted E. coli background in a standard microbiological growth test. As a result, colonies of the bacteria of interest on agar plates are detected more easily and the detection limits are improved. Adding enzyme-activated inhibitors to growth media can also lower the incubation time of a plate assay considerably in some cases since distinct colonies are visible much earlier.
At Biosynth we designed more enzyme-activated inhibitors to target more bacteria that are typically seen as background flora in microbiological tests. These enzyme-triggered inhibitors can be applied to control background problems with high accuracy. In contrast to antibiotics, enzyme-activated inhibitors discriminate bacterial strains by their well characterized enzyme activity. 

sterile culture

The STAADIUM™ Platform:

In addition to the STAADIUM™ GalactoZides, we designed the L-alanine aminopeptidase responsive inhibitor STAADIUM™ PeptiZide L-Ala that targets specifically gram negative bacteria. Another example, STAADIUM™ GlucuroniZide uses E. coli's specific glucuronidase activity and allows to inhibit the growth of this bacteria. The following list summarizes new Biosynth products and examples of possible targets:


GalactoZide I E.coli (EHEC) / coliforms Cat.No. Z-4006_P00
GalactoZide II* E.coli (EHEC) / coliforms Cat.No. Z-4002_P00
PeptiZide L-Ala Gram negatives Cat.No. Z-4001_P00
GlucuroniZide E. coli Cat.No. Z-4000_P00
PhosphoZide S. aureus (MRSA) Cat.No. Z-4004_P00
PeptiZide L-Pyr Citrobacter spp. Cat.No. Z-4005_P00


* Please note:  STAADIUM™ GalactoZide II is not compatible with certain pH indicators such as Neutral red. The inhibitor may also change the color of indigo stains. In some cases, STAADIUM™ GalactoZide II was found to suppress H2S staining.
If one or more of these conditions apply to your experimental setup, we recommend using STAADIUM™ GalactoZide I instead. STAADIUM™ GalactoZide I has a slightly lower inhibitory activity on coliforms as compared to STAADIUM™ GalactoZide II but does not show any of the above-mentioned side effects.


In summary, our product line of enzyme-responsive inhibitors STAADIUM™ GalactoZide I and II, PhosphoZide, PeptiZide and GlucuroniZide do inhibit specifically sub-populations of microbiota, while the non-targeted population is not affected. Such enzyme-responsive inhibitors are useful tools to control unwanted background for increased sensitivity of in vitro applications in food control, environmental safety, hygiene and clinical diagnostics.

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Watch our STAADIUM™ GalactoZide Teaser on YouTube

GalactoZide YouTube


The microbiome represents an essential part of every higher organism

Over millions of years of co-evolution, microbial communities (microbiota) have developed that live in close association with their hosts (e.g., animals or plants). It is now generally recognized that microbiota colonizing humans and animals play a key role in the host’s health and disease state. The human gut, for example, harbors one of the most complex microbiota, and disturbance of the normal gut microbiology have been implicated in many health and disease issues.

These findings have spurred intensive research to find ways of influencing microbiota to result in human health benefits. Early attempts to alter microbiota composition primarily involved the use of probiotic bacteria (e.g., Lactobacilli or Bifidobacteria) or prebiotic food components (e.g., nondigestable oligosaccharides) as food or feed additives. However, these approaches are limited in that they target undefined clinical indications by unknown mechanisms of action and often do not evoke the desired positive health effect. In case of probiotics, there is also a potential risk for transfer of drug-resistant genes or harmful infection and the problem of inconsistent product quality, leading to differing results.
Another approach is based on a medical procedure known as fecal microbiota transplantation (FMT). This procedure involves the infusion of a fecal suspension from a healthy donor into the gastrointestinal (GI) tract of a patient to restore the intestinal microbiota. It was found that FMT can have some therapeutic effects on gastrointestinal disorders, e.g. infections caused by Clostridium difficile. However, the efficacy of FMT remains in question. In addition, FMT is cumbersome, unpleasant and carries the risk of spreading infectious diseases.

E-coli cells

A more recent strategy to modulate the microbiota composition and function is based on the use of antimicrobial peptides (AMPs). AMPs are promising alternatives to conventional antibiotics because of their natural, broad-spectrum antimicrobial properties and low propensity for development of induced resistance. AMPs, for example, have been demonstrated to positively modulate the intestinal microbiota and to alter the oral microbiota for caries prevention. However, AMPs have disadvantages that limit their use, including hemolytic activity toward human cells, rapid turnover in the human body, reduced activity due to their fragile chemical nature and/or sensitivity to the environment, limited scope of application and/or high cost of production.

Enzyme-activated inhibitors open the way
for new therapies and healthcare products

Intensive efforts have been made to develop small-molecule antimicrobials as alternatives to existing antimicrobial compounds. Scientists were compelled to look for new options for identifying novel antimicrobials. Among other things, they explored strategies for exploiting beneficial and commensal bacteria in microbiota, including the development of generic systems based on the principle of microbial auto-inhibition (i.e. the self-inhibition of a given microbial species by an inhibitory substance generated by the species itself).

With STAADIUM™ enzyme-triggered inhibitors a class of antimicrobial compounds becomes available that is capable of changing microbiota composition and function by selectively inhibiting distinct microbial species.
This provides a tool for use in therapy, in particular for use in the treatment of:

  • gastrointestinal diseases, such as inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, irritable bowel syndrome (IBS), enterocolitis, and colorectal cancer
  • bacterial infections, such as Clostridium difficile infections (CDI)
  • cardiovascular diseases, such as cerebrovascular disease, myocardial infarction, and stroke
  • autoimmune diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, multiple sclerosis, and Celiac disease
  • neurological disorders, such as Alzheimer's disease, autistic spectrum disorders, and Parkinson's disease
  • metabolic diseases, such as metabolic syndrome, autoimmune type 1 diabetes (T1D), and insulin resistant type 2 diabetes (T2D)
  • neoplastic diseases, such as ulcers, adenocarcinomas, gastric B-cell lymphomas, and colorectal carcinoma
  • conditions caused by dysbiosis of vaginal microbiota (VMB), such as vaginal discharge, poor pregnancy outcomes, pelvic inflammatory disease, post-operative infections, endometritis following elective abortions, and sexually transmitted diseases (STDs)  and
  • other disorders like obesity, chronic fatigue syndrome, and atherosclerosis


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The STAADIUM™ Principle

We provide more information about the mode of actions of enzyme-activated inhibitors in our brief handout.