Antibiotic
Ribosomal 23S rRNA (peptidyl transferase center). Hygromycin A inhibits protein synthesis by binding to the 23S rRNA of the 50S ribosomal subunit at the peptidyl transferase center. This blocks peptide bond formation, leading to bacterial cell death. It is selectively taken up by spirochete bacteria via a nucleoside transporter, contributing to its specificity. In eukaryotic cells, Hygromycin B (a different analog) is more commonly used as a selection antibiotic; Hygromycin A has a different target specificity.

Lyme disease, which is caused by the spirochete Borrelia burgdorferi, is on the rise. Current treatment relies on broad-spectrum antibiotics that perturb the gut microbiome. In a recent paper in Cell, Leimer et al. demonstrate the utility of a long-forgotten antibiotic, Hygromycin A, as a spirochete-specific antibacterial that is conducive to gut health[1].

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Hygromycin A is active against numerous spirochete bacteria, including Treponema pallidum, Leptospira biflexa, and Borreliella burgdorferi, with minimum inhibitory concentrations (MICs) typically in the range of 0.5-8 ug/mL. It also exhibits activity against Gram-positive and Gram-negative bacteria, as well as Mycobacteria. It has immunosuppressant and anthelmintic activity. It clears B. burgdorferi infection in mice with less disruption of the microbiome than clinical antibiotics.

Hygromycin A produces probiotics by killing Borrelia burgdorferi selectively and improving the makeup of the gut flora [1].

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In a mouse model of Lyme disease, Hygromycin A administered orally at 25-50 mg/kg twice daily for 5 days effectively clears B. burgdorferi infection from tissues, including the heart, bladder, and joints. It has been shown to be more effective than clinical antibiotics such as doxycycline or ceftriaxone in some models, with less disruption of the gut microbiome. It also shows anti-inflammatory activity in vivo.

For cell-free ribosome binding assays, purified 50S ribosomal subunits from E. coli or B. burgdorferi are incubated with 3H-labeled Hygromycin A (0.1 nM-10 uM) in binding buffer (10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 100 mM NH4Cl) at 37degC for 30 minutes. The reaction mixture is filtered through nitrocellulose membranes to separate bound from unbound antibiotic. Bound radioactivity is measured by liquid scintillation counting. The dissociation constant (Kd) is calculated by Scatchard analysis. The peptidyl transferase activity can be assessed using a fragment reaction assay. V. burgdorferi B31 is grown in BSK-H medium under microaerophilic conditions (5% CO2, 34degC) for 3-7 days.

In a recent Cell paper, Leimer et al., (2021) address the lack of a specific and/or selective antibiotic for B. burgdorferi with the surprising rediscovery of Hygromycin A, which was first discovered in 1953 and later forgotten due to its poor potency against most Gram-positive and Gram-negative organisms (Pittenger et al., 1953). Traditional screens for antibiotics are focused on compounds that present a broad-spectrum -cidal activity against multiple bacteria. Using a non-conventional approach, the authors instead focused on compounds with a selective-cidal activity against B. burgdorferi, and so they identified Hygromycin A, which is produced copiously by the soil bacterium Streptomyces hygroscopicus. They found that Hygromycin A was also effective against genospecies of Lyme-disease-causing spirochetes found in Eurasia, B. garinii, and B. afzeli. Adding to the importance of this study is the finding that Hygromycin A is also effective against Treponema pallidum, the agent of syphilis. This could be highly significant because, unlike those for Lyme disease, an increasing level of antibiotic resistance to some of the antibiotics used to treat syphilis is being observed (Stamm, 2010). [1]

The authors elegantly and systematically unravel a mechanistic understanding of the specificity of Hygromycin A for B. burgdorferi (and other spirochetes). Hygromycin A binds to 23S rRNA of the 70S subunit of the bacterial ribosome, which forms the core of the peptidyl transferase center, and effectively impairs protein synthesis (Polikanov et al., 2015). Given that the 23S rRNA sequence is highly conserved across bacterial species, why is the antibiotic so ineffective against most pathogens? Hydrophilic molecules such as Hygromycin A are normally prevented from entering bacterial cells due to the hydrophobic nature of the cell membrane barrier. Gram-negative bacteria have an inner and outer membrane surrounding the cell wall, while Gram-positive bacteria only have an inner membrane. Transmembrane multidrug efflux pumps also help to extrude any antibiotic that might breach the barrier (Peterson and Kaur, 2018). Using efflux and porin mutants of Escherichia coli, the authors demonstrate that Hygromycin A is excluded from entering Gram-negative and Gram-positive bacteria predominantly by the cytoplasmic membrane barrier. Although B. burgdorferi lacks typical lipopolysaccharide (LPS) that is found in other Gram-negative bacteria, it does retain an outer and inner membrane barrier. Unlike many other Gram-negative bacteria, B. burgdorferi lacks the enzymes for de novo synthesis of purines—quintessential building blocks of RNA and DNA. Instead, it encodes and is dependent upon a nucleoside transporter, Basic membrane protein D (BmpD), which is a periplasmic substrate binding protein and is part of the ATP-binding cassette (ABC)-type purine nucleoside transporter that functions to scavenge purine nucleosides from the host environment (Cuellar et al., 2020). Hygromycin A has a structural resemblance to a purine nucleoside, and it gets a free ride into the spirochete via BmpD, allowing it to preferentially halt protein synthesis in spirochetes (Figure 1). The study provides compelling evidence, using robust molecular tools, that BmpD—along with BmpD orthologs in other spirochetes—is the basis for the unique transport of Hygromycin A. This explains the selectivity of Hygromycin A for spirochetes.[1]

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Bacteria are seeded in 96-well plates at 10⁵-10⁶ cells/well and treated with Hygromycin A at serial dilutions (0.001-100 ug/mL). After 72 hours of incubation, viability is assessed by counting motile spirochetes using dark-field microscopy or by using a colorimetric tetrazolium dye (MTT). The minimum inhibitory concentration (MIC) is determined as the lowest concentration that inhibits growth by ≥90%. For mammalian cells, the compound is less toxic.

In a murine Lyme disease model, 6-8 week old female C3H/HeN mice are infected intradermally with 10⁵-10⁶ B. burgdorferi (N40 strain) at the base of the tail. Two weeks post-infection, when disseminated infection is established, mice are treated orally with Hygromycin A at doses of 25-50 mg/kg twice daily for 5 consecutive days. Control groups receive vehicle (PBS or 0.5% methylcellulose) or positive control antibiotics (doxycycline 50 mg/kg BID, ceftriaxone 100 mg/kg once daily). At the end of the treatment period (day 28), mice are euthanized, and tissues (heart, bladder, ear, joint) are collected. B. burgdorferi DNA is detected by qPCR targeting the flaB gene. Infection status is confirmed by culture in BSK-H medium. Gut microbiome composition is assessed by 16S rRNA sequencing of fecal samples collected before, during, and after treatment.

Hygromycin A is orally bioavailable, with moderate absorption in rodents. Following oral administration in mice at 25-50 mg/kg, peak plasma concentrations (Cmax) are achieved within 1-2 hours. The compound distributes to tissues, including the heart, bladder, and joints, where B. burgdorferi resides. The elimination half-life is approximately 2-4 hours. Due to selective uptake by spirochete-specific nucleoside transporters, tissue concentrations in infected areas may be higher than in plasma.

The intraperitoneal LD50 in mice was 1067 mg/kg. (Index of Actinomycete Antibiotics, edited by Hiroshi Umezawa et al., Tokyo: University of Tokyo Press, 1967, p. 656.) The intravenous LD50 in mice was 200 mg/kg. (CRC Handbook of Antibiotic Compounds, Vol. 1-332, edited by J. Berdy, Boca Raton, Florida: CRC Press, 1980, Vol. 6, p. 332.)

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Hygromycin A has low toxicity in mammals at therapeutic doses. In mice, oral doses up to 200 mg/kg/day for 5 days are well tolerated with no significant body weight loss or clinical signs of toxicity. Unlike clinical Lyme disease antibiotics (doxycycline, ceftriaxone), Hygromycin A causes less disruption of the gut microbiome. The compound is not genotoxic or carcinogenic in standard assays. The LD50 in mice is >1000 mg/kg. It is not approved for human use.

[1]. Hygromycin A in the Lymelight. Cell Host Microbe. 2021 Nov 10;29(11):1599-1601.

Hygromycin A is a hydroxycinnamic acid that functions as a metabolite. Hygromycin A has been reported in Streptomyces, Streptomyces norovirus, and other microorganisms for which relevant data exist. This work lays the foundation for preclinical studies to further develop hygromycin A as a selective treatment for Lyme disease, examining its efficacy, toxicity, and bioavailability. The authors note that oral administration of hygromycin A to mice significantly improved gut microbiota composition (Figure 1). The potential impact of this antibiotic on human gut microbiota composition remains to be observed. Changes in gut microbiota composition have been associated with antibiotic-resistant Lyme arthritis and post-treatment Lyme disease syndrome (sometimes referred to as chronic Lyme disease) (Morrissette et al., 2020). Whether these changes are a cause or consequence of these controversial clinical presentations is currently unclear; however, it is always beneficial to replace broad-spectrum antibiotics with effective selective antibiotics whenever possible. Notably, while the use of hygromycin A may limit its broad impact on the microbiota, its selective absorption limits its efficacy against Borrelia burgdorferi, and unlike doxycycline, it is ineffective against other tick-borne bacterial pathogens that may co-infect. The authors also suggest that potential uses of this antibiotic include controlling the prevalence of Borrelia burgdorferi in hosts and ticks by using baits carrying hygromycin A (Figure 1). They hypothesize that the specificity of hygromycin A and the fact that Borrelia burgdorferi carries only one copy of the BmpD gene required for growth make it unlikely that antibiotic resistance will spread rapidly in the host. However, we remind that similar hypotheses have been made for other antibiotics in the past, but resistance has ultimately emerged. It is noteworthy that antimicrobial resistance (AMR) is not a problem in Lyme disease because humans are the definitive host of Borrelia burgdorferi. Therefore, even if a resistant strain of Borrelia burgdorferi is developed in an individual, it will not spread beyond that individual. However, using antibiotics that are used to treat human diseases to treat reservoir hosts may promote the spread of resistant strains of Borrelia burgdorferi, which may ultimately affect treatment outcomes in humans. Despite these limitations, this study presents a potential viable alternative for treating Lyme disease and other spirochetal diseases. [1]

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Hygromycin A is also known as (−)-Hygromycin A, Totomycin, and has the molecular formula C23H29NO12 and molecular weight 511.5. It was originally isolated from Streptomyces hygroscopicus. It is an orally available antibiotic that selectively targets spirochete bacteria, including Borreliella burgdorferi, which causes Lyme disease. It clears B. burgdorferi infection in mice with lesser disruption of the microbiome than clinical antibiotics. It is not approved for human use. It is a research chemical for Lyme disease, antimicrobial, and immunomodulatory studies.

C23H29NO12

511.48

511.169

C, 54.01; H, 5.72; N, 2.74; O, 37.54

6379-56-2

6433481

Typically exists as White to light yellow solid at room temperature

Streptomyces hygroscopicus

-1.8

7

12

6

36

853

10

CC(=CC1=CC(=C(C=C1)OC2C(C(C(O2)C(=O)C)O)O)O)C(=O)NC3C(C(C4C(C3O)OCO4)O)O

YQYJSBFKSSDGFO-IIHALWDASA-N

InChI=1S/C23H29NO12/c1-8(22(32)24-13-14(27)16(29)21-20(15(13)28)33-7-34-21)5-10-3-4-12(11(26)6-10)35-23-18(31)17(30)19(36-23)9(2)25/h3-6,13-21,23,26-31H,7H2,1-2H3,(H,24,32)/b8-5+/t13-,14+,15-,16-,17+,18+,19-,20+,21-,23-/m1/s1

(E)-N-[(3aS,4R,5R,6S,7R,7aR)-4,6,7-trihydroxy-3a,4,5,6,7,7a-hexahydro-1,3-benzodioxol-5-yl]-3-[4-[(2S,3S,4S,5S)-5-acetyl-3,4-dihydroxyoxolan-2-yl]oxy-3-hydroxyphenyl]-2-methylprop-2-enamide

Hygromycin A; 6379-56-2; HYGROMYCIN; Homomycin; UNII-3YJY415DDI; HYGROMYCIN [MI]; (E)-N-[(3aS,4R,5R,6S,7R,7aR)-4,6,7-trihydroxy-3a,4,5,6,7,7a-hexahydro-1,3-benzodioxol-5-yl]-3-[4-[(2S,3S,4S,5S)-5-acetyl-3,4-dihydroxyoxolan-2-yl]oxy-3-hydroxyphenyl]-2-methylprop-2-enamide; 3YJY415DDI;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~195.51 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)