Antibiotic resistance is a severe problem stemming from the overuse of antibiotics. Manuka honey, a unique honey from New Zealand, may treat bacterial infections as an alternative to traditional antibiotics. A transgenic line of Caenorhabditis elegans: VZ892 was utilized to determine changes in GFP expression upon exposure to S. aureus and honey treatment. An imaging, longevity, and developmental assay all showed promise that manuka honey was able to help to reduce the severity of the S. aureus infection in C. elegans. Manuka honey may be effective against S. aureus infections.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
University of Pittsburgh at Greensburg the Biological Sciences Division
","authors":[{"affiliations":["Pitt-Greensburg, Greensburg, PA, US"],"departments":[""],"credit":["methodology","validation","writing_originalDraft","writing_reviewEditing"],"email":"rileyjolesko@gmail.com","firstName":"Riley","lastName":"Lesko","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Pitt-Greensburg, Greensburg, PA, US"],"departments":[""],"credit":["supervision","writing_reviewEditing","writing_originalDraft","project"],"email":"osl5@pitt.edu","firstName":"Olivia","lastName":"Long","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0008-8713-4429"},{"affiliations":["Pitt-Greensburg, Greensburg, PA, US"],"departments":[""],"credit":["investigation","conceptualization","validation","writing_reviewEditing"],"email":"KLM256@pitt.edu","firstName":"Kelsey","lastName":"Murphy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"Funding was obtained through the Beta Beta Beta Biological Honor Society Research Grant
","image":{"url":"https://portal.micropublication.org/uploads/4f90989a3481a3753c89b395b05179b8.png"},"imageCaption":"A) GFP fluorescence imaging of VZ892 adult animals. Exposure to S. aureus results in increased levels of fluorescence with bright accumulations along the intestinal tract of the worms. Treatment with manuka honey helped return fluorescence back to the original diffuse baseline.
B) Graph of the average corrected total fluorescence from imaging. After conducting a one-tailed T-test there was significance between the control group and worms exposed to S. aureus, P= 0.0006. There was significance between worms exposed to S. aureus and worms that were exposed to S. aureus then treated, P= 0.0055. There was no significance between control groups and treatment groups, P= 0.1109.
C) Kaplan-Myer survival curve for VZ892 animals. The VZ892 control group had 50% lethality of 192 hrs. VZ892 exposed to S. aureus had 50% lethality of 24 hrs. VZ892 exposed to S. aureus and treatment had 50% lethality of 72 hrs. Log rank (Mantel-Cox) test showed overall significance of P=0.0025 when comparing all three conditions.
D) Kaplan- Myer survival curve for N2 animals. The N2 control group had 50% lethality of 180 hrs. N2 exposed to S. aureus had 50% lethality of 48 hrs. N2 exposed to S. aureus and treatment had 50% lethality of 84 hrs. Log rank (Mantel-Cox) test showed overall significance of P=<0.0001 when comparing all three conditions.
E) There was a significance in the development of C. elegans exposed to S. aureus when compared to their respective control group, N2 P=0.0002, VZ892 P=0.0005. There was a significance in the development between C. elegans exposed to S. aureus and the groups given treatment, N2 P=0.0009, VZ892 P=0.0015. Significance was determined using a one-tailed T-test between each group.
","imageTitle":"Manuka Honey’s effects on S. aureus infection in C. elegans stress response, longevity, and development
","methods":"C. elegans care
Both N2 and VZ892 C. elegans strains were purchased from the Caenorhabditis Genetics Center (CGC). The strains were maintained on 60mm plates of NGM Lite from USBiological Life Sciences seeded with 50 μl of E. coli OP50 (NGM/OP50). All worms were grown and kept in incubators kept at 21 °C. Worms were transferred to fresh NGM OP50 seeded plates with a sterilized platinum pick as needed to prevent starvation. All experiments utilized 2 day old adult worms, to prevent the S. aureus from stunting the worm's growth.
S. aureus solution
A single S. aureus (ATCC 14775) colony was isolated and grown in a Tryptic soy broth culture. This was then placed in a shaking incubator at 37 °C for 24 hours. A fresh culture from a single isolate was made fresh as needed for seeding experimental plates.
S. aureus plates
Worms were infected with S. aureus by exposing them to a lawn grown on a Tryptic Soy Agar plate. The lawn was grown by seeding 35mm plates with 5ul of the diluted (50% overnight S. aureus culture and 50% fresh TSB) S. aureus solution. Plates would then be placed in a 37 °C incubator for 24 hours. Plates were used once a thick lawn of S. aureus was visible.
Manuka honey solution
A 135% w/v solution 20 UMF manuka honey was made with distilled water was added and vortexed until there was no honey visibly stuck to the sides or lid of the conical. The solution was made fresh as needed. Plates of manuka honey were made to expose the worms to the treatment. 50ul of the solution was placed on a NGM lite plate with a lawn of OP50 and spread with a hockey stick The honey plates were then left to sit for 24 hours under a biological safety cabinet to dry.
Infecting C. elegans with S. aureus and treating with manuka honey
2 day old adult worms were placed on OP50 control plates or TSA plates with a lawn of S. aureus for 24 hours. After this exposure half of the worms on the S. aureus plates would be transferred to a manuka honey plate to be treated while the rest were placed on a plate with OP50 and no treatment. The control worms were transferred to another control plate containing only OP50. Exposure to treatment or no treatment would last for 24 hours and then worms that had now been exposed and undergone treatment were used in imaging, development, and longevity assays.
Imaging Assay
Slides were prepared for imaging with a 3% agarose pad. On the pads, 25 μL of 100 mM Sodium azide was added to paralyze the worms. Worms were then added to the slides, a cover slip was placed over them and they were imaged at 100x at a wavelength of 480nm fluorescent microscope. Three trials were run with 15 worms in each experimental group.
Longevity Assay
After 24 hour exposure to S. aureus twenty worms were placed on plates with OP50 and counted daily. Death was determined through lack of response upon prodding the worms with a sterilized pick. Any worms that died were removed from the plate and their deaths were tallied. The assay continued until each worm was dead, and was run in triplicate. Kaplan-Meier survival curve. Log rank test for trends. Chi square value
Developmental Assay
After 24 hour exposure to S. aureus twenty adult worms were transferred to OP50 plates and allowed to lay eggs for 5 hours. All adults were then removed and the eggs were left to develop for 48 hours at 21 °C. After 48 hours the offspring were then staged and counted.
","reagents":"Reagent Table:
STRAIN | GENOTYPE | AVAILABLE FROM |
C. elegans wild isolate | Caenorhabditis Genetics Center (CGC) | |
CGC |
Antibiotic resistance is a major public health concern, with Staphylococcus aureus strains evolving rapidly and becoming less responsive to standard treatments (Aslam et al., 2018; Myles and Datta, 2012). In the United States, there are approximately 2.8 million antibiotic resistance cases per year, causing the death of over 35,000 people (Myles and Datta, 2012). In an effort to help fight antibiotic resistance other antibacterial treatments that do not result in resistance are being explored. Manuka honey is one of these potential treatments. It is a honey only produced in New Zealand from the nectar of the Manuka tree, Leptospermum scoparium (Sultanbawa, 2014). The honey is famous for its strong antibiotic and antifungal properties, rivaling that of other honeys (Almasaudi et al., 2017). The quality of the honey is measured by MGO concentrations. These concentrations are rated by UMF unique manuka factor. The lower the manuka factor, the lower the MGO concentrations and therefore the lower the antibiotic properties (Atrott and Henle, 2009). The complex interactions between the MGO and peroxides in the honey result in pathogens being unable to develop resistance to Manuka honey (Sultanbawa, 2014). It has been shown to be effective in treating S. aureus infections, reducing the amount of growth, outperforming other honey samples (Almasaudi et al., 2017). Manuka honey's ability to treat S. aureus infections has never been explored in a C. elegans model.
This study uses Caenorhabditis elegans, a transparent nematode. C. elegans are microscopic worm, and a popular model organism for scientific research. C. elegans has a short life, lasting only two or three weeks (Apfeld and Alper, 2018). They go from an egg to a phenotypically distinct larval stage called the L4 stage, to an adult (Corsi, 2006). They are especially hardy in the lab and can be maintained easily and cost-effectively on plates of media, seeded with a lawn of Escherichia coli. Previous research has utilized the worms as a model for both MSSA and MRSA infections (Thompson and Brown, 2014). The bacteria consumed by the worm colonize their intestines (Sifri et al., 2003). This causes significant degradation of the digestive system and, eventually, of the entire worm, resulting in death (Irazoqui et al., 2010). This research aims to explore C. elegans as a model for S. aureus infections and their responsiveness to manuka honey treatment. (looking for connection to next paragraph)
Both the wild-type C. elegans strain N2 and the stress-reporter strain VZ892 (expressing HLH-30::GFP) were used to evaluate the effects of Manuka honey on infection outcomes (Martina et al., 2021). The VZ892 strain is particularly susceptible to S. aureus infections and expresses GFP when under stress. This allows for a reliable measure of how the infection harms the organism. Worms were divided into three treatment groups: control (no S. aureus, no honey), S. aureus only, and S. aureus with manuka honey treatment that had a UMF value of 20. Assays included GFP fluorescence imaging with the VZ892, lifespan, and developmental progression.
To utilize the stress reporter strain VZ892, an imaging assay was performed, allowing for the amount of GFP expressed to be quantified. Higher levels of GFP, or pinpoint fluorescence within the digestive system of C. elegans, could indicate stress caused by S. aureus infection. VZ892 in the control groups had a mean fluorescence of 55,768.3 ACTF, and the fluorescence was diffusely distributed throughout the worms' bodies (A, left-hand panel). S. aureus exposure resulted in a 102.2% increase to 112,770 ACTF. Along with the significant increase in fluorescence, it was no longer diffuse; instead, pinpoint fluorescence concentrated heavily on the worm's digestive system, as seen in (A, middle panel). After S. aureus-exposed worms were treated with manuka honey (A, right-hand panel) the fluorescence decreased by 38.5% to 69,296 ACTF, with no significant difference between it and the no S. aureus control values after running a one-tailed T-test P= 0.1109. Furthermore, upon exposure to Manuka honey, the GFP fluorescence returned to a diffuse pattern, similar to that observed in the control group.
To assess worm survival, a longevity assay was performed. It revealed that the lifespans of both the VZ892 (Figure 1-C) and N2 (Figure 1-D) strains were dramatically shortened upon exposure to S. aureus relative to controls, confirming previous data (Sifri et al., 2003). The VZ892 LT50 decreased 85.7% from 168 hours to 24 hours. The N2 LT50 was similarly affected, decreasing 73.3% from 180 hours to 48 hours. When worms exposed to S. aureus were treated with manuka honey, their lifespans improved, but did not return to baseline. With treatment, the VZ892 strain LT50 improved by 200% to 72 hours, and the N2 strain LT50 improved by 75% to 84 hours.
Previous data suggest that upon exposure to S. aureus, C. elegans development is repressed, showing slow growth and development (Sifri et al., 2003). To determine whether manuka honey could help restore development, a developmental assay was performed to assess the percentage of worms that progressed to the L4 larval stage or to adulthood at a normal rate. Figure 1-E shows that both VZ892 and N2 controls had similarly high rates of worms normally progressing through development, 94.42% for VZ892 and 98.15% for N2 after 48 hours. Upon exposure to S. aureus, these rates significantly dropped to 62.26% for VZ892 and 76.74% for N2. S. aureus exposure results in significantly slower development as compared to worms in the control group. With manuka honey treatment, the development increased for VZ892 to 82.01% and for N2 to 90.30%. This indicates that S. aureus exposure reduces the developmental rate of C. elegans and that the treatment mitigates this effect, restoring development to levels closer to controls.
These findings support the use of C. elegans as a simple model for screening natural therapeutics, such as manuka honey. Additionally, these results suggest that UMF 20 manuka honey can reduce S. aureus-induced stress and pathogenesis in the worms, demonstrating that manuka honey may be a promising alternative or additive to antibiotics. Further studies are warranted to explore dose dependence with the different UMF values of manuka honey, long-term effects, and impact on other resistant S. Aureus strains such as MRSA.
","references":[{"reference":"Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, et al., Baloch. 2018. Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance Volume 11: 1645-1658.
","pubmedId":"","doi":"10.2147/IDR.S173867"},{"reference":"Almasaudi SB, Al-Nahari AAM, Abd El-Ghany ESM, Barbour E, Al Muhayawi SM, Al-Jaouni S, et al., Harakeh. 2017. Antimicrobial effect of different types of honey on Staphylococcus aureus. Saudi Journal of Biological Sciences 24: 1255-1261.
","pubmedId":"","doi":"10.1016/j.sjbs.2016.08.007"},{"reference":"Apfeld J, Alper S. 2018. What Can We Learn About Human Disease from the Nematode C. elegans? Methods in Molecular Biology, Disease Gene Identification : 53-75.
","pubmedId":"","doi":"10.1007/978-1-4939-7471-9_4"},{"reference":"Atrott J, Henle T. 2009. Methylglyoxal in Manuka Honey - Correlation with Antibacterial Properties. Czech Journal of Food Sciences 27: S163-S165.
","pubmedId":"","doi":"10.17221/911-CJFS"},{"reference":"Corsi AK. 2006. A biochemist’s guide to Caenorhabditis elegans. Analytical Biochemistry 359: 1-17.
","pubmedId":"","doi":"10.1016/j.ab.2006.07.033"},{"reference":"Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. 2010. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathogens 6: e1000982.
","pubmedId":"","doi":"10.1371/journal.ppat.1000982"},{"reference":"Martina JA, Guerrero‐Gómez D, Gómez‐Orte E, Antonio Bárcena J, Cabello J, Miranda‐Vizuete A, Puertollano R. 2020. A conserved cysteine‐based redox mechanism sustains TFEB/HLH‐30 activity under persistent stress. The EMBO Journal 40: 10.15252/embj.2020105793.
","pubmedId":"","doi":"10.15252/embj.2020105793"},{"reference":"Myles IA, Datta SK. 2012. Staphylococcus aureus: an introduction. Seminars in Immunopathology 34: 181-184.
","pubmedId":"","doi":"10.1007/s00281-011-0301-9"},{"reference":"Sifri CD, Begun J, Ausubel FM, Calderwood SB. 2003. Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis. Infection and Immunity 71: 2208-2217.
","pubmedId":"","doi":"10.1128/IAI.71.4.2208-2217.2003"},{"reference":"Sultanbawa, Y. (2014) Leptospermum (Manuka) Honey: Accepted Natural Medicine. (2014). In Honey in Traditional and Modern Medicine (pp. 113–121) 10.1201/b15608-11
","pubmedId":"","doi":""},{"reference":"Thompson T, Brown PD. 2014. Comparison of antibiotic resistance, virulence gene profiles, and pathogenicity of methicillin-resistant and methicillin-susceptible Staphylococcus aureus using a Caenorhabditis elegans infection model. Pathogens and Global Health 108: 283-291.
","pubmedId":"","doi":"10.1179/2047773214Y.0000000155"}],"title":"The Effectiveness of Manuka Honey in Treating Staphylococcus aureus in C. elegans
","reviews":[{"reviewer":{"displayName":"Jessica Sowa"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Karen Yook (Ad)"},"openAcknowledgement":false,"submitted":null},{"curator":{"displayName":"Karen Yook (Ad)"},"openAcknowledgement":false,"submitted":null},{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"397c4994-6630-482f-b30d-f5226afafd78","decision":"revise","abstract":"Antibiotic resistance is a severe problem stemming from the overuse of antibiotics. Manuka honey, a unique honey from New Zealand, may treat bacterial infections as an alternative to traditional antibiotics. A transgenic line of Caenorhabditis elegans: HLH‐30::3xFLAG::eGFP was utilized as a stress reporter strain to quantify changes in GFP expression associated with host response to S. aureus infection and honey treatment. An imaging, longevity, and developmental assay all showed promise that manuka honey was able to help to reduce the host stress response due to S. aureus infection in C. elegans. Manuka honey may be effective against S. aureus infections.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
University of Pittsburgh at Greensburg the Biological Sciences Division
","authors":[{"affiliations":["University of Pittsburgh at Greensburg"],"departments":[""],"credit":["methodology","validation","writing_originalDraft","writing_reviewEditing"],"email":"rileyjolesko@gmail.com","firstName":"Riley","lastName":"Lesko","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Pittsburgh at Greensburg"],"departments":[""],"credit":["supervision","writing_reviewEditing","writing_originalDraft","project"],"email":"osl5@pitt.edu","firstName":"Olivia","lastName":"Long","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0008-8713-4429"},{"affiliations":["University of Pittsburgh at Greensburg"],"departments":[""],"credit":["investigation","conceptualization","validation","writing_reviewEditing"],"email":"KLM256@pitt.edu","firstName":"Kelsey","lastName":"Murphy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"Funding was obtained through the Beta Beta Beta Biological Honor Society Research Grant
","image":{"url":"https://portal.micropublication.org/uploads/4f90989a3481a3753c89b395b05179b8.png"},"imageCaption":"A) GFP fluorescence imaging of hlh-30::3xFLAG::eGFP (referred to as strain VZ892) adult animals. Exposure to S. aureus results in increased levels of fluorescence with bright accumulations along the intestinal tract of the worms. Treatment with manuka honey helped return fluorescence back to the original diffuse baseline.
B) Graph of the average corrected total fluorescence from imaging. After conducting a one-tailed T-test there was significance between the control group and worms exposed to S. aureus, P= 0.0006. There was significance between worms exposed to S. aureus and worms that were exposed to S. aureus then treated, P= 0.0055. There was no significance between control groups and treatment groups, P= 0.1109.
C) Kaplan-Myer survival curve for VZ892 animals. The VZ892 control group had 50% lethality of 192 hrs. VZ892 exposed to S. aureus had 50% lethality of 24 hrs. VZ892 exposed to S. aureus and treatment had 50% lethality of 72 hrs. Log rank (Mantel-Cox) test showed overall significance of P=0.0025 when comparing all three conditions.
D) Kaplan- Myer survival curve for N2 animals. The N2 control group had 50% lethality of 180 hrs. N2 exposed to S. aureus had 50% lethality of 48 hrs. N2 exposed to S. aureus and treatment had 50% lethality of 84 hrs. Log rank (Mantel-Cox) test showed overall significance of P=<0.0001 when comparing all three conditions.
E) A statistically significant difference in was observed in the development of both C. elegans exposed to S. aureus when compared to their respective control group (N2 P=0.0002, VZ892 P=0.0005) and between C. elegans exposed to S. aureus and the groups given treatment (N2 P=0.0009, VZ892 P=0.0015). Significance was determined using a one-tailed T-test between each group.
","imageTitle":"Manuka Honey’s effects on S. aureus infection in C. elegans stress response, longevity, and development
","methods":"C. elegans care
Both N2 and VZ892 C. elegans strains were purchased from the Caenorhabditis Genetics Center (CGC). The strains were maintained on 60mm plates of NGM Lite from USBiological Life Sciences seeded with 50 μl of E. coli OP50 (NGM/OP50). All worms were grown and kept in incubators kept at 21 °C. Worms were transferred to fresh NGM OP50 seeded plates with a sterilized platinum pick as needed to prevent starvation. All experiments utilized 2 day old adult worms, to prevent the S. aureus from stunting the worm's growth.
S. aureus solution
A single S. aureus (ATCC 14775) colony was isolated and grown in a Tryptic soy broth culture. This was then placed in a shaking incubator at 37 °C for 24 hours. A fresh culture from a single isolate was made fresh as needed for seeding experimental plates.
S. aureus plates
Worms were infected with S. aureus by exposing them to a lawn grown on a Tryptic Soy Agar plate. The lawn was grown by seeding 35mm plates with 5ul of the diluted (50% overnight S. aureus culture and 50% fresh TSB) S. aureus solution. Plates would then be placed in a 37 °C incubator for 24 hours. Plates were used once a thick lawn of S. aureus was visible.
Manuka honey solution
A 135% w/v solution 20 UMF manuka honey was made with distilled water was added and vortexed until there was no honey visibly stuck to the sides or lid of the conical. The solution was made fresh as needed. Plates of manuka honey were made to expose the worms to the treatment. 50ul of the solution was placed on a NGM lite plate with a lawn of OP50 and spread with a hockey stick The honey plates were then left to sit for 24 hours under a biological safety cabinet to dry.
Infecting C. elegans with S. aureus and treating with manuka honey
2 day old adult worms were placed on OP50 control plates or TSA plates with a lawn of S. aureus for 24 hours. After this exposure half of the worms on the S. aureus plates would be transferred to a manuka honey plate to be treated while the rest were placed on a plate with OP50 and no treatment. The control worms were transferred to another control plate containing only OP50. Exposure to treatment or no treatment would last for 24 hours and then worms that had now been exposed and undergone treatment were used in imaging, development, and longevity assays.
Imaging Assay
Slides were prepared for imaging with a 3% agarose pad. On the pads, 25 μL of 100 mM Sodium azide was added to paralyze the worms. Worms were then added to the slides, a cover slip was placed over them. Slides were imaged utilized using a Zeiss Primo Star microscope, images were taken with Motic cam pro and images were captured using the Motic Image Plus 3.0 software at 100x total magnification utilizing a wavelength of 480nm fluorescent microscope. Images of the entire worm were then analyzed with ImageJ following previously described protocol (Fitzpatrick, 2014). Three trials were run with 15 worms in each experimental group.
Longevity Assay
After 24 hour exposure to S. aureus twenty worms were placed on plates with OP50 and counted daily. Death was determined through lack of response upon prodding the worms with a sterilized pick. Any worms that died were removed from the plate and their deaths were tallied. The assay continued until each worm was dead, and was run in triplicate. Kaplan-Meier survival curve. Log rank test for trends. Chi square value
Developmental Assay
Twenty adult worms were transferred to plates with S. aureus with and without treatment and allowed to lay eggs for 5 hours. All adults were then removed and the eggs were left to develop for 48 hours at 21 °C. After 48 hours the offspring were then staged and counted.
","reagents":"Reagent Table:
STRAIN | GENOTYPE | AVAILABLE FROM |
C. elegans wild isolate | Caenorhabditis Genetics Center (CGC) | |
hlh-30::3xFLAG::eGFP | CGC |
Antibiotic resistance is a major public health concern, with Staphylococcus aureus strains evolving rapidly and becoming less responsive to standard treatments (Aslam et al., 2018; Myles and Datta, 2012). In the United States, there are approximately 2.8 million antibiotic resistance cases per year, causing the death of over 35,000 people (Myles and Datta, 2012). In an effort to help fight antibiotic resistance other antibacterial treatments that do not result in resistance are being explored. Manuka honey is one of these potential treatments. It is a honey only produced in New Zealand from the nectar of the Manuka tree, Leptospermum scoparium (Sultanbawa, 2014). The honey is famous for its strong antibiotic and antifungal properties, rivaling that of other honeys (Almasaudi et al., 2017). The quality of the honey is measured by Methylglyoxal (MGO) concentrations (Sultanbawa, 2014). These concentrations are rated by UMF unique manuka factor. The lower the manuka factor, the lower the MGO concentrations and therefore the lower the antibiotic properties (Atrott and Henle, 2009). The complex interactions between the MGO and peroxides in the honey result in pathogens being unable to develop resistance to Manuka honey (Sultanbawa, 2014). It has been shown to be effective in treating S. aureus infections, reducing the amount of growth, outperforming other honey samples (Almasaudi et al., 2017). Manuka honey's ability to treat S. aureus infections has never been explored in a C. elegans model.
This study uses Caenorhabditis elegans, a transparent nematode. C. elegans are microscopic worm, and a popular model organism for scientific research. C. elegans has a short life, lasting only two or three weeks (Apfeld and Alper, 2018). From an egg they progress through several larval stages until they reach a phenotypically distinct L4 stage after which they mature to an adult worm (Corsi, 2006). They are especially hardy in the lab and can be maintained easily and cost-effectively on plates of media, seeded with a lawn of Escherichia coli. Previous research has utilized the worms as a model for both MSSA and MRSA infections (Thompson and Brown, 2014). The bacteria consumed by the worm colonize their intestines (Sifri et al., 2003). This causes significant degradation of the digestive system and, eventually, of the entire worm, resulting in death (Irazoqui et al., 2010). This research aims to explore C. elegans hlh-30::3xFLAG::eGFP, strain VZ892, as a model for S. aureus infections and their responsiveness to manuka honey treatment. (Martina et al., 2021)
Both the wild-type C. elegans strain N2 and the stress-reporter strain VZ892 (expressing HLH-30::GFP) were used to evaluate the effects of Manuka honey on infection outcomes (Martina et al., 2021). VZ892 contains a GFP-tagged version of the endogenous hlh-30 locus and is considered phenotypically similar to wild-type. This strain serves as a reporter of cellular stress and immune activation. Upon exposure to stressors such as S. aureus, HLH-30 translocates and GFP fluorescence increases, allowing for visualization and quantification of host responses (Colino-Lage et al., 2024). Worms were divided into three treatment groups: control (no S. aureus, no honey), S. aureus only, and S. aureus with Manuka honey treatment (UMF 20). Assays included GFP fluorescence imaging, lifespan, and developmental progression.
To utilize the stress reporter strain VZ892, an imaging assay was performed, allowing for the amount of GFP expressed to be quantified. Localized GFP fluorescence within the intestinal tract suggests activation of host defense responses at the primary site of S. auerus bacterial colonization. VZ892 in the control groups had a mean fluorescence of 55,768.3 ACTF, and the fluorescence was diffusely distributed throughout the worms' bodies (A, left-hand panel). S. aureus exposure resulted in a 102.2% increase to 112,770 ACTF. Along with the significant increase in fluorescence, it was no longer diffuse; instead, pinpoint fluorescence concentrated heavily on the worm's digestive system, as seen in (A, middle panel). After S. aureus-exposed worms were treated with manuka honey (A, right-hand panel) the fluorescence decreased compared to the group without treatment by 38.5% to 69,296 ACTF. No significant difference was found between the treatment group values and the values from the no S. aureus control group after running a one-tailed T-test P= 0.1109. Furthermore, upon exposure to Manuka honey, the GFP fluorescence returned to a diffuse pattern, similar to that observed in the control group.
To assess worm survival, a longevity assay was performed. It revealed that the lifespans of both the VZ892 (Figure 1-C) and N2 (Figure 1-D) strains were dramatically shortened upon exposure to S. aureus relative to controls, confirming previous data (Sifri et al., 2003). The VZ892 LT50 decreased 85.7% from 168 hours to 24 hours. The N2 LT50 was similarly affected, decreasing 73.3% from 180 hours to 48 hours. When worms exposed to S. aureus were treated with manuka honey, their lifespans improved, but did not return to baseline. With treatment, the VZ892 strain LT50 improved by 200% to 72 hours, and the N2 strain LT50 improved by 75% to 84 hours.
Previous data suggest that upon exposure to S. aureus, C. elegans development is repressed, showing slow growth and development (Sifri et al., 2003). To determine whether manuka honey could help restore development, a developmental assay was performed to assess the percentage of worms that progressed to the L4 larval stage or to adulthood at a normal rate. Figure 1-E shows that both VZ892 and N2 controls had similarly high rates of worms normally progressing through development, 94.42% for VZ892 and 98.15% for N2 after 48 hours. Upon exposure to S. aureus, these rates significantly dropped to 62.26% for VZ892 and 76.74% for N2. S. aureus exposure results in significantly slower development as compared to worms in the control group. With manuka honey treatment, the development increased for VZ892 to 82.01% and for N2 to 90.30%. This indicates that S. aureus exposure reduces the developmental rate of C. elegans and that the treatment mitigates this effect, restoring development to levels closer to controls.
These findings support the use of C. elegans as a simple model for screening natural therapeutics, such as manuka honey. Additionally, these results suggest that UMF 20 manuka honey can reduce S. aureus-induced stress and pathogenesis in the worms, demonstrating that manuka honey may be a promising alternative or additive to antibiotics. Further studies are warranted to explore dose dependence with the different UMF values of manuka honey, long-term effects, and impact on other resistant S. aureus strains such as MRSA.
","references":[{"reference":"Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, et al., Baloch. 2018. Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance Volume 11: 1645-1658.
","pubmedId":"","doi":"10.2147/IDR.S173867"},{"reference":"Almasaudi SB, Al-Nahari AAM, Abd El-Ghany ESM, Barbour E, Al Muhayawi SM, Al-Jaouni S, et al., Harakeh. 2017. Antimicrobial effect of different types of honey on Staphylococcus aureus. Saudi Journal of Biological Sciences 24: 1255-1261.
","pubmedId":"","doi":"10.1016/j.sjbs.2016.08.007"},{"reference":"Apfeld J, Alper S. 2018. What Can We Learn About Human Disease from the Nematode C. elegans? Methods in Molecular Biology, Disease Gene Identification : 53-75.
","pubmedId":"","doi":"10.1007/978-1-4939-7471-9_4"},{"reference":"Atrott J, Henle T. 2009. Methylglyoxal in Manuka Honey - Correlation with Antibacterial Properties. Czech Journal of Food Sciences 27: S163-S165.
","pubmedId":"","doi":"10.17221/911-CJFS"},{"reference":"Colino-Lage, H., Guerrero-Gómez, D., Gómez-Orte, E., González, X., Martina, J. A., Dansen, T. B., Ayuso, C., Askjaer, P., Puertollano, R., Irazoqui, J. E., Cabello, J., & Miranda-Vizuete, A. 2024. Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism. Free Radical Biology and Medicine 223: 369-383.
","pubmedId":"","doi":"10.1016/j.freeradbiomed.2024.07.027"},{"reference":"Corsi AK. 2006. A biochemist’s guide to Caenorhabditis elegans. Analytical Biochemistry 359: 1-17.
","pubmedId":"","doi":"10.1016/j.ab.2006.07.033"},{"reference":"Fitzpatrick, M. (2014). Measuring cell fluorescence using ImageJ. The Open Lab Book v1.0. https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html
","pubmedId":"","doi":""},{"reference":"Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. 2010. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathogens 6: e1000982.
","pubmedId":"","doi":"10.1371/journal.ppat.1000982"},{"reference":"Martina JA, Guerrero‐Gómez D, Gómez‐Orte E, Antonio Bárcena J, Cabello J, Miranda‐Vizuete A, Puertollano R. 2021. A conserved cysteine‐based redox mechanism sustains TFEB/HLH‐30 activity under persistent stress. The EMBO Journal 40: 10.15252/embj.2020105793.
","pubmedId":"","doi":"10.15252/embj.2020105793"},{"reference":"Myles IA, Datta SK. 2012. Staphylococcus aureus: an introduction. Seminars in Immunopathology 34: 181-184.
","pubmedId":"","doi":"10.1007/s00281-011-0301-9"},{"reference":"Sifri CD, Begun J, Ausubel FM, Calderwood SB. 2003. Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis. Infection and Immunity 71: 2208-2217.
","pubmedId":"","doi":"10.1128/IAI.71.4.2208-2217.2003"},{"reference":"Sultanbawa, Y. (2014). Chapter 6: Leptospermum (Manuka) Honey: Accepted Natural Medicine. In L. Boukraa (Ed.), Honey in Traditional and Modern Medicine (pp. 113–121). CRC Press. https://doi.org/10.1201/b15608
","pubmedId":"","doi":"10.1201/b15608"},{"reference":"Thompson T, Brown PD. 2014. Comparison of antibiotic resistance, virulence gene profiles, and pathogenicity of methicillin-resistant and methicillin-susceptible Staphylococcus aureus using a Caenorhabditis elegans infection model. Pathogens and Global Health 108: 283-291.
","pubmedId":"","doi":"10.1179/2047773214Y.0000000155"}],"title":"The Effectiveness of Manuka Honey in Treating Staphylococcus aureus in C. elegans
","reviews":[{"reviewer":{"displayName":"Jessica Sowa"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":"1775766774170"}]},{"id":"e3bf9d57-21d3-4445-8d2b-557bee9f4229","decision":"accept","abstract":"Antibiotic resistance is a severe problem stemming from the overuse of antibiotics. Manuka honey, a unique honey from New Zealand, may serve as an alternative to traditional antibiotics for treating bacterial infections. A transgenic line of Caenorhabditis elegans, HLH-30::3xFLAG::eGFP, was utilized as a stress reporter strain to quantify changes in GFP expression associated with host response to S. aureus infection and honey treatment. Imaging, longevity, and developmental assays all demonstrated that manuka honey reduced the host stress response to S. aureus infection in C. elegans. These findings suggest that manuka honey may be effective against S. aureus infections.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
This work was conducted within the Biological Sciences Department at the University of Pittsburgh at Greensburg.
","authors":[{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["methodology","validation","writing_originalDraft","writing_reviewEditing"],"email":"rileyjolesko@gmail.com","firstName":"Riley","lastName":"Lesko","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["supervision","writing_reviewEditing","writing_originalDraft","project"],"email":"osl5@pitt.edu","firstName":"Olivia","lastName":"Long","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0008-8713-4429"},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["investigation","conceptualization","validation","writing_reviewEditing"],"email":"KLM256@pitt.edu","firstName":"Kelsey","lastName":"Murphy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Funding was obtained through the Beta Beta Beta Biological Honor Society Research Grant
","image":{"url":"https://portal.micropublication.org/uploads/4f90989a3481a3753c89b395b05179b8.png"},"imageCaption":"A) Green fluorescent protein (GFP) fluorescence imaging of hlh-30::3xFLAG::eGFP (referred to as strain VZ892) adult animals. Exposure to S. aureus resulted in increased fluorescence, with bright accumulations along the intestinal tract of the worms. Treatment with manuka honey helped return fluorescence to the original diffuse baseline.
B) Graph of the average corrected total fluorescence (ACTF) from imaging. After conducting a one-tailed t-test, there was a significant difference between the control group and worms exposed to S. aureus (P = 0.0006). There was also a significant difference between worms exposed to S. aureus and worms exposed to S. aureus followed by treatment (P = 0.0055). There was no significant difference between the control and treatment groups (P = 0.1109).
C) Kaplan-Meier survival curve for VZ892 animals. The control group exhibited 50% lethality at 192 hrs. Animals exposed to S. aureus exhibited 50% lethality at 24 hrs. VZ892 animals exposed to S. aureus and manuka honey treatment exhibited 50% lethality at 72 hrs. A log-rank (Mantel-Cox) test showed a significant difference among all conditions (P = 0.0025).
D) Kaplan-Meier survival curve for N2 animals. The control group exhibited 50% lethality at 180 hrs. Animals exposed to S. aureus exhibited 50% lethality at 48 hrs. N2 exposed to S. aureus and manuka honey treatment had 50% lethality at 84 hrs. A log-rank (Mantel-Cox) test showed a significant difference among all conditions (P < 0.0001).
E) A statistically significant difference in development was observed in worms exposed to S. aureus compared to their respective control groups (N2 P = 0.0002, VZ892 P = 0.0005), as well as between S. aureus exposed groups and manuka honey treatment groups (N2 P = 0.0009, VZ892 P = 0.0015). Significance was determined using a one-tailed t-test.
","imageTitle":"Manuka honey’s effects on S. aureus infection in C. elegans stress response, longevity, and development
","methods":"C. elegans care
N2 and VZ892 C. elegans strains were obtained from the Caenorhabditis Genetics Center (CGC). Strains were maintained at 21°C on 60mm plates of NGM Lite (USBiological Life Sciences) seeded with 50μL of E. coli OP50 (NGM/OP50). Worms were transferred to fresh NGM/OP50 plates using a sterilized platinum pick as needed to prevent starvation. All experiments utilized 2-day-old adult worms to avoid the confounding effects of S. aureus on worms' development.
S. aureus solution
A single S. aureus (ATCC 14775) colony was isolated and grown in a tryptic soy broth (TSB). Cultures were incubated shaking at 37°C for 24 hours. Fresh cultures were prepared from a single colony as needed for seeding experimental plates.
S. aureus plates
Worms were infected with S. aureus by exposure to bacterial lawns grown on tryptic soy agar plates. Lawns were prepared by seeding 35mm plates with 5μL of the diluted culture (50% overnight S. aureus culture and 50% fresh TSB). Plates were incubated at 37 °C for 24 hours or until a lawn was visible.
Manuka honey solution
A 135% (w/v) solution of 20 UMF manuka honey and distilled water was vortexed until fully dissolved, with no visible residue stuck to the sides of the container. The solution was made fresh as needed. For treatment plates, 50μL of the solution was spread onto an NGM Lite plate seeded with OP50 using a sterile spreader. Plates were allowed to dry for 24 hours in a biological safety cabinet.
Infecting C. elegans with S. aureus and treating with manuka honey
Two-day-old adult worms were placed on OP50 control plates or TSA plates containing S. aureus lawns for 24 hours. Following exposure, half of the worms from S. aureus plates were transferred to manuka honey treatment plates, while the remaining worms were transferred to OP50 plates. Exposure to treatment or control conditions was maintained for 24 hours, after which worms were imaged, assessed for longevity, and subjected to developmental assays.
Imaging Assay
Slides were prepared using a 3% agarose pad. A 25μL drop of 100 mM sodium azide was added to the agarose pad to which worms were placed. Imaging was performed using a Zeiss Primo Star microscope with a Moticcam Pro camera. Images were captured using the Motic Image Plus 3.0 software at 100x total magnification with a 480nm fluorescence filter. Images of the entire worm were then analyzed with ImageJ following the previously described protocol (Fitzpatrick, 2014). Three independent trials were conducted with fifteen worms per experimental group.
Longevity Assay
After 24-hour exposure to S. aureus, twenty worms were transferred to OP50 plates and counted daily. Death was determined through lack of response upon prodding the worms with a sterilized pick. Dead worms were removed and recorded daily. The assay continued until all worms were deceased and was performed in triplicate.
Developmental Assay
Twenty adult animals were transferred to plates containing S. aureus with or without treatment and allowed to lay eggs for 5 hours. Adults were removed, and eggs were left to develop for 48 hours at 21 °C. After 48 hours, offspring were staged and counted to assess developmental progression. Developmental assays were completed in triplicate.
","reagents":"Reagent Table:
STRAIN | GENOTYPE | AVAILABLE FROM |
C. elegans wild isolate | Caenorhabditis Genetics Center (CGC) | |
hlh-30::3xFLAG::eGFP | CGC |
Antibiotic resistance is a major public health concern, with Staphylococcus aureus strains evolving rapidly and becoming less responsive to standard treatments (Aslam et al., 2018; Myles and Datta, 2012). In the United States, there are approximately 2.8 million antibiotic-resistant cases per year, resulting in over 35,000 deaths (Myles and Datta, 2012). In an effort to combat antibiotic resistance, alternative antibacterial treatments that do not promote resistance are being explored. Manuka honey is one such potential treatment. It is produced exclusively in New Zealand from the nectar of the manuka tree, Leptospermum scoparium (Sultanbawa, 2014). This honey is well known for its strong antibacterial and antifungal properties, rivaling those of other honeys (Almasaudi et al., 2017). The quality of the manuka honey is measured by methylglyoxal (MGO) concentrations (Sultanbawa, 2014), which is rated using the Unique Manuka Factor (UMF) scale. Lower UMF values correspond to lower MGO concentrations and, therefore, reduced antibacterial activity (Atrott and Henle, 2009). The complex interactions between MGO and peroxides in honey prevent pathogens from developing resistance to manuka honey (Sultanbawa, 2014). Manuka honey has been shown to be effective in treating S. aureus infections by reducing the bacterial growth (Almasaudi et al., 2017). However, its effects have not been explored in a C. elegans model.
This study utilized Caenorhabditis elegans, a transparent nematode widely used as a model organism. C. elegans is a microscopic worm with a short lifespan of approximately two to three weeks (Apfeld and Alper, 2018). From the egg stage, it progresses through several larval stages before reaching the phenotypically distinct L4 stage, after which it matures into an adult (Corsi, 2006). These worms are especially robust in the lab and can be easily and cost-effectively maintained on media plates seeded with a lawn of Escherichia coli. Previous studies have used C. elegans as a model for both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively) infections (Thompson and Brown, 2014). The bacteria consumed by the worm colonize the intestinal tract (Sifri et al., 2003), leading to degradation of the digestive system and eventual death of the organism (Irazoqui et al., 2010).
This research investigates the use of C. elegans hlh-30::3xFLAG::eGFP (strain VZ892) as a model for S. aureus infection and its responsiveness to manuka honey treatment (Martina et al., 2021). Both the wild-type C. elegans strain N2 and the stress reporter strain VZ892 were used to evaluate the effects of manuka honey on infection outcomes (Martina et al., 2021). VZ892 contains a GFP-tagged version of the endogenous hlh-30 locus and is phenotypically similar to wild-type. This strain serves as a reporter of cellular stress and immune activation. Upon exposure to stressors such as S. aureus, HLH-30 translocates, and GFP fluorescence increases, enabling visualization and quantification of the host response (Colino-Lage et al., 2024). Worms were divided into three groups: control (no S. aureus, no manuka honey treatment), S. aureus only, and S. aureus with manuka honey treatment (UMF 20). Assays included GFP fluorescence imaging, lifespan analysis, and developmental progression.
To utilize the stress reporter strain VZ892, an imaging assay was performed to quantify GFP expression. Localized GFP fluorescence within the intestinal tract suggests activation of host defense responses at the primary site of S. aureus colonization. VZ892 worms in the control groups had a mean fluorescence of 55,768.3 ACTF, with fluorescence diffusely distributed throughout the worms' bodies (Figure 1A, left panel). Exposure to S. aureus resulted in a 102.2% increase in fluorescence to 112,770 ACTF. In addition to this increase, fluorescence became localized, with intense pinpoint accumulations in the digestive system (Figure 1A, middle panel). Following treatment with manuka honey (Figure 1A, right panel), fluorescence decreased by 38.5% to 69,296 ACTF as compared to infected worms. No significant difference was found between the treatment group and the control group following a one-tailed t-test (P = 0.1109). Furthermore, upon exposure to manuka honey, the GFP fluorescence returned to a diffuse pattern similar to that observed in control worms.
To assess survival, a longevity assay was performed. Lifespans of both the VZ892 (Figure 1C) and N2 (Figure 1D) strains were significantly reduced following exposure to S. aureus, consistent with previous findings (Sifri et al., 2003). The VZ892 LT50 decreased 85.7% from 168 hours to 24 hours. Similarly, the N2 LT50 decreased by 73.3% from 180 hours to 48 hours.
Treatment with manuka honey improved lifespans but did not restore them to baseline levels. With treatment, the VZ892 strain LT50 improved to 72 hours (a 200% increase), and the N2 strain LT50 improved to 84 hours (a 75% increase).
Previous studies suggest that exposure to S. aureus represses development in C. elegans, resulting in delayed growth (Sifri et al., 2003). To determine whether manuka honey could mitigate this effect, a developmental assay was conducted to assess the percentage of worms reaching the L4 larval stage or adulthood within a normal timeframe. Figure 1E shows that both VZ892 and N2 control groups had high rates of normal development (94.42% and 98.15%, respectively). Exposure to S. aureus significantly reduced these rates to 62.26% for VZ892 and 76.74% for N2. Treatment with manuka honey improved development to 82.01% for VZ892 and 90.30% for N2. These results indicate that S. aureus exposure slows the development in C. elegans and that manuka honey treatment partially restores normal developmental progression.
These findings support the use of C. elegans as a model for screening natural therapeutics such as manuka honey. Additionally, the results suggest that UMF 20 manuka honey can reduce S. aureus-induced stress and pathogenesis in this model, indicating its potential as an alternative or adjunct to traditional antibiotics. Further studies are warranted to explore dose dependence across different UMF values, long-term effects, and efficacy against resistant S. aureus strains such as MRSA.
","references":[{"reference":"Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, et al., Baloch. 2018. Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance Volume 11: 1645-1658.
","pubmedId":"","doi":"10.2147/IDR.S173867"},{"reference":"Almasaudi SB, Al-Nahari AAM, Abd El-Ghany ESM, Barbour E, Al Muhayawi SM, Al-Jaouni S, et al., Harakeh. 2017. Antimicrobial effect of different types of honey on Staphylococcus aureus. Saudi Journal of Biological Sciences 24: 1255-1261.
","pubmedId":"","doi":"10.1016/j.sjbs.2016.08.007"},{"reference":"Apfeld J, Alper S. 2018. What Can We Learn About Human Disease from the Nematode C. elegans? Methods in Molecular Biology, Disease Gene Identification : 53-75.
","pubmedId":"","doi":"10.1007/978-1-4939-7471-9_4"},{"reference":"Atrott J, Henle T. 2009. Methylglyoxal in Manuka Honey - Correlation with Antibacterial Properties. Czech Journal of Food Sciences 27: S163-S165.
","pubmedId":"","doi":"10.17221/911-CJFS"},{"reference":"Colino-Lage, H., Guerrero-Gómez, D., Gómez-Orte, E., González, X., Martina, J. A., Dansen, T. B., Ayuso, C., Askjaer, P., Puertollano, R., Irazoqui, J. E., Cabello, J., & Miranda-Vizuete, A. 2024. Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism. Free Radical Biology and Medicine 223: 369-383.
","pubmedId":"","doi":"10.1016/j.freeradbiomed.2024.07.027"},{"reference":"Corsi AK. 2006. A biochemist’s guide to Caenorhabditis elegans. Analytical Biochemistry 359: 1-17.
","pubmedId":"","doi":"10.1016/j.ab.2006.07.033"},{"reference":"Fitzpatrick, M. 2014. Measuring cell fluorescence using ImageJ. The Open Lab Book v1.0. https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html
","pubmedId":"","doi":""},{"reference":"Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. 2010. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathogens 6: e1000982.
","pubmedId":"","doi":"10.1371/journal.ppat.1000982"},{"reference":"Martina JA, Guerrero‐Gómez D, Gómez‐Orte E, Antonio Bárcena J, Cabello J, Miranda‐Vizuete A, Puertollano R. 2021. A conserved cysteine‐based redox mechanism sustains TFEB/HLH‐30 activity under persistent stress. The EMBO Journal 40: 10.15252/embj.2020105793.
","pubmedId":"","doi":"10.15252/embj.2020105793"},{"reference":"Myles IA, Datta SK. 2012. Staphylococcus aureus: an introduction. Seminars in Immunopathology 34: 181-184.
","pubmedId":"","doi":"10.1007/s00281-011-0301-9"},{"reference":"Sifri CD, Begun J, Ausubel FM, Calderwood SB. 2003. Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis. Infection and Immunity 71: 2208-2217.
","pubmedId":"","doi":"10.1128/IAI.71.4.2208-2217.2003"},{"reference":"Sultanbawa, Y. 2014. Chapter 6: Leptospermum (Manuka) Honey: Accepted Natural Medicine. In L. Boukraa (Ed.), Honey in Traditional and Modern Medicine (pp. 113–121). CRC Press. https://doi.org/10.1201/b15608
","pubmedId":"","doi":"10.1201/b15608"},{"reference":"Thompson T, Brown PD. 2014. Comparison of antibiotic resistance, virulence gene profiles, and pathogenicity of methicillin-resistant and methicillin-susceptible Staphylococcus aureus using a Caenorhabditis elegans infection model. Pathogens and Global Health 108: 283-291.
","pubmedId":"","doi":"10.1179/2047773214Y.0000000155"}],"title":"The Effectiveness of Manuka Honey in Treating Staphylococcus aureus in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"75ce51ab-1338-4918-9608-7b4f7166ada3","decision":"publish","abstract":"Antibiotic resistance is a severe problem stemming from the overuse of antibiotics. Manuka honey, a unique honey from New Zealand, may serve as an alternative to traditional antibiotics for treating bacterial infections. A transgenic line of Caenorhabditis elegans, hlh-30::3xFLAG::eGFP, was utilized as a stress reporter strain to quantify changes in GFP expression associated with host response to S. aureus infection and honey treatment. Imaging, longevity, and developmental assays all demonstrated that manuka honey reduced the host stress response to S. aureus infection in C. elegans. These findings suggest that manuka honey may be effective against S. aureus infections.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
This work was conducted within the Biological Sciences Department at the University of Pittsburgh at Greensburg.
","authors":[{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["methodology","validation","writing_originalDraft","writing_reviewEditing"],"email":"rileyjolesko@gmail.com","firstName":"Riley","lastName":"Lesko","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["supervision","writing_reviewEditing","writing_originalDraft","project"],"email":"osl5@pitt.edu","firstName":"Olivia","lastName":"Long","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0008-8713-4429"},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["investigation","conceptualization","validation","writing_reviewEditing"],"email":"KLM256@pitt.edu","firstName":"Kelsey","lastName":"Murphy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Funding was obtained through the Beta Beta Beta Biological Honor Society Research Grant
","image":{"url":"https://portal.micropublication.org/uploads/4f90989a3481a3753c89b395b05179b8.png"},"imageCaption":"A) Green fluorescent protein (GFP) fluorescence imaging of hlh-30::3xFLAG::eGFP (referred to as strain VZ892) adult animals. Exposure to S. aureus resulted in increased fluorescence, with bright accumulations along the intestinal tract of the worms. Treatment with manuka honey helped return fluorescence to the original diffuse baseline.
B) Graph of the average corrected total fluorescence (ACTF) from imaging. After conducting a one-tailed t-test, there was a significant difference between the control group and worms exposed to S. aureus (P = 0.0006). There was also a significant difference between worms exposed to S. aureus and worms exposed to S. aureus followed by treatment (P = 0.0055). There was no significant difference between the control and treatment groups (P = 0.1109).
C) Kaplan-Meier survival curve for VZ892 animals. The control group exhibited 50% lethality at 192 hrs. Animals exposed to S. aureus exhibited 50% lethality at 24 hrs. VZ892 animals exposed to S. aureus and manuka honey treatment exhibited 50% lethality at 72 hrs. A log-rank (Mantel-Cox) test showed a significant difference among all conditions (P = 0.0025).
D) Kaplan-Meier survival curve for N2 animals. The control group exhibited 50% lethality at 180 hrs. Animals exposed to S. aureus exhibited 50% lethality at 48 hrs. N2 exposed to S. aureus and manuka honey treatment had 50% lethality at 84 hrs. A log-rank (Mantel-Cox) test showed a significant difference among all conditions (P < 0.0001).
E) A statistically significant difference in development was observed in worms exposed to S. aureus compared to their respective control groups (N2 P = 0.0002, VZ892 P = 0.0005), as well as between S. aureus exposed groups and manuka honey treatment groups (N2 P = 0.0009, VZ892 P = 0.0015). Significance was determined using a one-tailed t-test.
","imageTitle":"Manuka honey’s effects on S. aureus infection in C. elegans stress response, longevity, and development
","methods":"C. elegans care
N2 and VZ892 C. elegans strains were obtained from the Caenorhabditis Genetics Center (CGC). Strains were maintained at 21 °C on 60 mm plates of NGM Lite (USBiological Life Sciences) seeded with 50 μL of E. coli OP50 (NGM/OP50). Worms were transferred to fresh NGM/OP50 plates using a sterilized platinum pick as needed to prevent starvation. All experiments utilized 2-day-old adult worms to avoid the confounding effects of S. aureus on worms' development.
S. aureus solution
A single S. aureus (ATCC 14775) colony was isolated and grown in a tryptic soy broth (TSB). Cultures were incubated shaking at 37 °C for 24 hours. Fresh cultures were prepared from a single colony as needed for seeding experimental plates.
S. aureus plates
Worms were infected with S. aureus by exposure to bacterial lawns grown on tryptic soy agar plates. Lawns were prepared by seeding 35 mm plates with 5 μL of the diluted culture (50% overnight S. aureus culture and 50% fresh TSB). Plates were incubated at 37 °C for 24 hours or until a lawn was visible.
Manuka honey solution
A 135% (w/v) solution of 20 UMF manuka honey and distilled water was vortexed until fully dissolved, with no visible residue stuck to the sides of the container. The solution was made fresh as needed. For treatment plates, 50 μL of the solution was spread onto an NGM Lite plate seeded with OP50 using a sterile spreader. Plates were allowed to dry for 24 hours in a biological safety cabinet.
Infecting C. elegans with S. aureus and treating with manuka honey
Two-day-old adult worms were placed on OP50 control plates or TSA plates containing S. aureus lawns for 24 hours. Following exposure, half of the worms from S. aureus plates were transferred to manuka honey treatment plates, while the remaining worms were transferred to OP50 plates. Exposure to treatment or control conditions was maintained for 24 hours, after which worms were imaged, assessed for longevity, and subjected to developmental assays.
Imaging Assay
Slides were prepared using a 3% agarose pad. A 25 μL drop of 100 mM sodium azide was added to the agarose pad to which worms were placed. Imaging was performed using a Zeiss Primo Star microscope with a Moticcam Pro camera. Images were captured using the Motic Image Plus 3.0 software at 100x total magnification with a 480 nm fluorescence filter. Images of the entire worm were then analyzed with ImageJ version 1.54g following the previously described protocol (Fitzpatrick, 2014). Three independent trials were conducted with fifteen worms per experimental group.
Longevity Assay
After 24-hour exposure to S. aureus, twenty worms were transferred to OP50 plates and counted daily. Death was determined through lack of response upon prodding the worms with a sterilized pick. Dead worms were removed and recorded daily. The assay continued until all worms were deceased and was performed in triplicate.
Developmental Assay
Twenty adult animals were transferred to plates containing S. aureus with or without treatment and allowed to lay eggs for 5 hours. Adults were removed, and eggs were left to develop for 48 hours at 21 °C. After 48 hours, offspring were staged and counted to assess developmental progression. Developmental assays were completed in triplicate.
","reagents":"Strains:
STRAIN | GENOTYPE | AVAILABLE FROM |
C. elegans wild isolate | Caenorhabditis Genetics Center (CGC) | |
hlh-30::3xFLAG::eGFP | CGC |
Antibiotic resistance is a major public health concern, with Staphylococcus aureus strains evolving rapidly and becoming less responsive to standard treatments (Aslam et al., 2018; Myles and Datta, 2012). In the United States, there are approximately 2.8 million antibiotic-resistant cases per year, resulting in over 35,000 deaths (Myles and Datta, 2012). In an effort to combat antibiotic resistance, alternative antibacterial treatments that do not promote resistance are being explored. Manuka honey is one such potential treatment. It is produced exclusively in New Zealand from the nectar of the manuka tree, Leptospermum scoparium (Sultanbawa, 2014). This honey is well known for its strong antibacterial and antifungal properties, rivaling those of other honeys (Almasaudi et al., 2017). The quality of the manuka honey is measured by methylglyoxal (MGO) concentrations (Sultanbawa, 2014), which is rated using the Unique Manuka Factor (UMF) scale. Lower UMF values correspond to lower MGO concentrations and, therefore, reduced antibacterial activity (Atrott and Henle, 2009). The complex interactions between MGO and peroxides in honey prevent pathogens from developing resistance to manuka honey (Sultanbawa, 2014). Manuka honey has been shown to be effective in treating S. aureus infections by reducing the bacterial growth (Almasaudi et al., 2017). However, its effects have not been explored in a C. elegans model.
This study utilized Caenorhabditis elegans, a transparent nematode widely used as a model organism. C. elegans is a microscopic worm with a short lifespan of approximately two to three weeks (Apfeld and Alper, 2018). From the egg stage, it progresses through several larval stages before reaching the phenotypically distinct L4 stage, after which it matures into an adult (Corsi, 2006). These worms are especially robust in the lab and can be easily and cost-effectively maintained on media plates seeded with a lawn of Escherichia coli. Previous studies have used C. elegans as a model for both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively) infections (Thompson and Brown, 2014). The bacteria consumed by the worm colonize the intestinal tract (Sifri et al., 2003), leading to degradation of the digestive system and eventual death of the organism (Irazoqui et al., 2010).
This research investigates the use of C. elegans hlh-30::3xFLAG::eGFP (strain VZ892) as a model for S. aureus infection and its responsiveness to manuka honey treatment (Martina et al., 2021). Both the wild-type C. elegans strain N2 and the stress reporter strain VZ892 were used to evaluate the effects of manuka honey on infection outcomes (Martina et al., 2021). VZ892 contains a GFP-tagged version of the endogenous hlh-30 locus and is phenotypically similar to wild-type. This strain serves as a reporter of cellular stress and immune activation. Upon exposure to stressors such as S. aureus, hlh-30 translocates, and GFP fluorescence increases, enabling visualization and quantification of the host response (Colino-Lage et al., 2024). Worms were divided into three groups: control (no S. aureus, no manuka honey treatment), S. aureus only, and S. aureus with manuka honey treatment (UMF 20). Assays included GFP fluorescence imaging, lifespan analysis, and developmental progression.
To utilize the stress reporter strain VZ892, an imaging assay was performed to quantify GFP expression. Localized GFP fluorescence within the intestinal tract suggests activation of host defense responses at the primary site of S. aureus colonization. VZ892 worms in the control groups had a mean fluorescence of 55,768.3 ACTF, with fluorescence diffusely distributed throughout the worms' bodies (Figure 1A, left panel). Exposure to S. aureus resulted in a 102.2% increase in fluorescence to 112,770 ACTF. In addition to this increase, fluorescence became localized, with intense pinpoint accumulations in the digestive system (Figure 1A, middle panel). Following treatment with manuka honey (Figure 1A, right panel), fluorescence decreased by 38.5% to 69,296 ACTF as compared to infected worms. No significant difference was found between the treatment group and the control group following a one-tailed t-test (P = 0.1109). Furthermore, upon exposure to manuka honey, the GFP fluorescence returned to a diffuse pattern similar to that observed in control worms.
To assess survival, a longevity assay was performed. Lifespans of both the VZ892 (Figure 1C) and N2 (Figure 1D) strains were significantly reduced following exposure to S. aureus, consistent with previous findings (Sifri et al., 2003). The VZ892 LT50 decreased 85.7% from 168 hours to 24 hours. Similarly, the N2 LT50 decreased by 73.3% from 180 hours to 48 hours.
Treatment with manuka honey improved lifespans but did not restore them to baseline levels. With treatment, the VZ892 strain LT50 improved to 72 hours (a 200% increase), and the N2 strain LT50 improved to 84 hours (a 75% increase).
Previous studies suggest that exposure to S. aureus represses development in C. elegans, resulting in delayed growth (Sifri et al., 2003). To determine whether manuka honey could mitigate this effect, a developmental assay was conducted to assess the percentage of worms reaching the L4 larval stage or adulthood within a normal timeframe. Figure 1E shows that both VZ892 and N2 control groups had high rates of normal development (94.42% and 98.15%, respectively). Exposure to S. aureus significantly reduced these rates to 62.26% for VZ892 and 76.74% for N2. Treatment with manuka honey improved development to 82.01% for VZ892 and 90.30% for N2. These results indicate that S. aureus exposure slows the development in C. elegans and that manuka honey treatment partially restores normal developmental progression.
These findings support the use of C. elegans as a model for screening natural therapeutics such as manuka honey. Additionally, the results suggest that UMF 20 manuka honey can reduce S. aureus-induced stress and pathogenesis in this model, indicating its potential as an alternative or adjunct to traditional antibiotics. Further studies are warranted to explore dose dependence across different UMF values, long-term effects, and efficacy against resistant S. aureus strains such as MRSA.
","references":[{"reference":"Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, et al., Baloch. 2018. Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance Volume 11: 1645-1658.
","pubmedId":"","doi":"10.2147/IDR.S173867"},{"reference":"Almasaudi SB, Al-Nahari AAM, Abd El-Ghany ESM, Barbour E, Al Muhayawi SM, Al-Jaouni S, et al., Harakeh. 2017. Antimicrobial effect of different types of honey on Staphylococcus aureus. Saudi Journal of Biological Sciences 24: 1255-1261.
","pubmedId":"","doi":"10.1016/j.sjbs.2016.08.007"},{"reference":"Apfeld J, Alper S. 2018. What Can We Learn About Human Disease from the Nematode C. elegans? Methods in Molecular Biology, Disease Gene Identification : 53-75.
","pubmedId":"","doi":"10.1007/978-1-4939-7471-9_4"},{"reference":"Atrott J, Henle T. 2009. Methylglyoxal in Manuka Honey - Correlation with Antibacterial Properties. Czech Journal of Food Sciences 27: S163-S165.
","pubmedId":"","doi":"10.17221/911-CJFS"},{"reference":"Colino-Lage, H., Guerrero-Gómez, D., Gómez-Orte, E., González, X., Martina, J. A., Dansen, T. B., Ayuso, C., Askjaer, P., Puertollano, R., Irazoqui, J. E., Cabello, J., & Miranda-Vizuete, A. 2024. Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism. Free Radical Biology and Medicine 223: 369-383.
","pubmedId":"","doi":"10.1016/j.freeradbiomed.2024.07.027"},{"reference":"Corsi AK. 2006. A biochemist’s guide to Caenorhabditis elegans. Analytical Biochemistry 359: 1-17.
","pubmedId":"","doi":"10.1016/j.ab.2006.07.033"},{"reference":"Fitzpatrick, M. 2014. Measuring cell fluorescence using ImageJ. The Open Lab Book v1.0. https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html
","pubmedId":"","doi":""},{"reference":"Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. 2010. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathogens 6: e1000982.
","pubmedId":"","doi":"10.1371/journal.ppat.1000982"},{"reference":"Martina JA, Guerrero‐Gómez D, Gómez‐Orte E, Antonio Bárcena J, Cabello J, Miranda‐Vizuete A, Puertollano R. 2021. A conserved cysteine‐based redox mechanism sustains TFEB/HLH‐30 activity under persistent stress. The EMBO Journal 40: 10.15252/embj.2020105793.
","pubmedId":"","doi":"10.15252/embj.2020105793"},{"reference":"Myles IA, Datta SK. 2012. Staphylococcus aureus: an introduction. Seminars in Immunopathology 34: 181-184.
","pubmedId":"","doi":"10.1007/s00281-011-0301-9"},{"reference":"Sifri CD, Begun J, Ausubel FM, Calderwood SB. 2003. Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis. Infection and Immunity 71: 2208-2217.
","pubmedId":"","doi":"10.1128/IAI.71.4.2208-2217.2003"},{"reference":"Sultanbawa, Y. 2014. Chapter 6: Leptospermum (Manuka) Honey: Accepted Natural Medicine. In L. Boukraa (Ed.), Honey in Traditional and Modern Medicine (pp. 113–121). CRC Press. https://doi.org/10.1201/b15608
","pubmedId":"","doi":"10.1201/b15608"},{"reference":"Thompson T, Brown PD. 2014. Comparison of antibiotic resistance, virulence gene profiles, and pathogenicity of methicillin-resistant and methicillin-susceptible Staphylococcus aureus using a Caenorhabditis elegans infection model. Pathogens and Global Health 108: 283-291.
","pubmedId":"","doi":"10.1179/2047773214Y.0000000155"}],"title":"The Effectiveness of Manuka Honey in Treating Staphylococcus aureus in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"ba14df2a-a901-4f00-b6a7-324011d716b8","decision":"publish","abstract":"Antibiotic resistance is a severe problem stemming from the overuse of antibiotics. Manuka honey, a unique honey from New Zealand, may serve as an alternative to traditional antibiotics for treating bacterial infections. A transgenic line of Caenorhabditis elegans, hlh-30::3xFLAG::eGFP, was utilized as a stress reporter strain to quantify changes in GFP expression associated with host response to S. aureus infection and honey treatment. Imaging, longevity, and developmental assays all demonstrated that manuka honey reduced the host stress response to S. aureus infection in C. elegans. These findings suggest that manuka honey may be effective against S. aureus infections.
","acknowledgements":"Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
This work was conducted within the Biological Sciences Department at the University of Pittsburgh at Greensburg.
","authors":[{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["methodology","validation","writing_originalDraft","writing_reviewEditing"],"email":"rileyjolesko@gmail.com","firstName":"Riley","lastName":"Lesko","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["supervision","writing_reviewEditing","writing_originalDraft","project"],"email":"osl5@pitt.edu","firstName":"Olivia","lastName":"Long","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0008-8713-4429"},{"affiliations":["University of Pittsburgh at Greensburg, Greensburg, Pennsylvania, United States"],"departments":[""],"credit":["investigation","conceptualization","validation","writing_reviewEditing"],"email":"KLM256@pitt.edu","firstName":"Kelsey","lastName":"Murphy","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"Funding was obtained through the Beta Beta Beta Biological Honor Society Research Grant
","image":{"url":"https://portal.micropublication.org/uploads/4f90989a3481a3753c89b395b05179b8.png"},"imageCaption":"A) Green fluorescent protein (GFP) fluorescence imaging of hlh-30::3xFLAG::eGFP (referred to as strain VZ892) adult animals. Exposure to S. aureus resulted in increased fluorescence, with bright accumulations along the intestinal tract of the worms. Treatment with manuka honey helped return fluorescence to the original diffuse baseline.
B) Graph of the average corrected total fluorescence (ACTF) from imaging. After conducting a one-tailed t-test, there was a significant difference between the control group and worms exposed to S. aureus (P = 0.0006). There was also a significant difference between worms exposed to S. aureus and worms exposed to S. aureus followed by treatment (P = 0.0055). There was no significant difference between the control and treatment groups (P = 0.1109).
C) Kaplan-Meier survival curve for VZ892 animals. The control group exhibited 50% lethality at 192 hrs. Animals exposed to S. aureus exhibited 50% lethality at 24 hrs. VZ892 animals exposed to S. aureus and manuka honey treatment exhibited 50% lethality at 72 hrs. A log-rank (Mantel-Cox) test showed a significant difference among all conditions (P = 0.0025).
D) Kaplan-Meier survival curve for N2 animals. The control group exhibited 50% lethality at 180 hrs. Animals exposed to S. aureus exhibited 50% lethality at 48 hrs. N2 exposed to S. aureus and manuka honey treatment had 50% lethality at 84 hrs. A log-rank (Mantel-Cox) test showed a significant difference among all conditions (P < 0.0001).
E) A statistically significant difference in development was observed in worms exposed to S. aureus compared to their respective control groups (N2 P = 0.0002, VZ892 P = 0.0005), as well as between S. aureus exposed groups and manuka honey treatment groups (N2 P = 0.0009, VZ892 P = 0.0015). Significance was determined using a one-tailed t-test.
","imageTitle":"Manuka honey’s effects on S. aureus infection in C. elegans stress response, longevity, and development
","methods":"C. elegans care
N2 and VZ892 C. elegans strains were obtained from the Caenorhabditis Genetics Center (CGC). Strains were maintained at 21 °C on 60 mm plates of NGM Lite (USBiological Life Sciences) seeded with 50 μL of E. coli OP50 (NGM/OP50). Worms were transferred to fresh NGM/OP50 plates using a sterilized platinum pick as needed to prevent starvation. All experiments utilized 2-day-old adult worms to avoid the confounding effects of S. aureus on worms' development.
S. aureus solution
A single S. aureus (ATCC 14775) colony was isolated and grown in a tryptic soy broth (TSB). Cultures were incubated shaking at 37 °C for 24 hours. Fresh cultures were prepared from a single colony as needed for seeding experimental plates.
S. aureus plates
Worms were infected with S. aureus by exposure to bacterial lawns grown on tryptic soy agar plates. Lawns were prepared by seeding 35 mm plates with 5 μL of the diluted culture (50% overnight S. aureus culture and 50% fresh TSB). Plates were incubated at 37 °C for 24 hours or until a lawn was visible.
Manuka honey solution
A 135% (w/v) solution of 20 UMF manuka honey and distilled water was vortexed until fully dissolved, with no visible residue stuck to the sides of the container. The solution was made fresh as needed. For treatment plates, 50 μL of the solution was spread onto an NGM Lite plate seeded with OP50 using a sterile spreader. Plates were allowed to dry for 24 hours in a biological safety cabinet.
Infecting C. elegans with S. aureus and treating with manuka honey
Two-day-old adult worms were placed on OP50 control plates or TSA plates containing S. aureus lawns for 24 hours. Following exposure, half of the worms from S. aureus plates were transferred to manuka honey treatment plates, while the remaining worms were transferred to OP50 plates. Exposure to treatment or control conditions was maintained for 24 hours, after which worms were imaged, assessed for longevity, and subjected to developmental assays.
Imaging Assay
Slides were prepared using a 3% agarose pad. A 25 μL drop of 100 mM sodium azide was added to the agarose pad to which worms were placed. Imaging was performed using a Zeiss Primo Star microscope with a Moticcam Pro camera. Images were captured using the Motic Image Plus 3.0 software at 100x total magnification with a 480 nm fluorescence filter. Images of the entire worm were then analyzed with ImageJ version 1.54g following the previously described protocol (Fitzpatrick, 2014). Three independent trials were conducted with fifteen worms per experimental group.
Longevity Assay
After 24-hour exposure to S. aureus, twenty worms were transferred to OP50 plates and counted daily. Death was determined through lack of response upon prodding the worms with a sterilized pick. Dead worms were removed and recorded daily. The assay continued until all worms were deceased and was performed in triplicate.
Developmental Assay
Twenty adult animals were transferred to plates containing S. aureus with or without treatment and allowed to lay eggs for 5 hours. Adults were removed, and eggs were left to develop for 48 hours at 21 °C. After 48 hours, offspring were staged and counted to assess developmental progression. Developmental assays were completed in triplicate.
","reagents":"Strains:
STRAIN | GENOTYPE | AVAILABLE FROM |
C. elegans wild isolate | Caenorhabditis Genetics Center (CGC) | |
hlh-30::3xFLAG::eGFP | CGC |
Antibiotic resistance is a major public health concern, with Staphylococcus aureus strains evolving rapidly and becoming less responsive to standard treatments (Aslam et al., 2018; Myles and Datta, 2012). In the United States, there are approximately 2.8 million antibiotic-resistant cases per year, resulting in over 35,000 deaths (Myles and Datta, 2012). In an effort to combat antibiotic resistance, alternative antibacterial treatments that do not promote resistance are being explored. Manuka honey is one such potential treatment. It is produced exclusively in New Zealand from the nectar of the manuka tree, Leptospermum scoparium (Sultanbawa, 2014). This honey is well known for its strong antibacterial and antifungal properties, rivaling those of other honeys (Almasaudi et al., 2017). The quality of the manuka honey is measured by methylglyoxal (MGO) concentrations (Sultanbawa, 2014), which is rated using the Unique Manuka Factor (UMF) scale. Lower UMF values correspond to lower MGO concentrations and, therefore, reduced antibacterial activity (Atrott and Henle, 2009). The complex interactions between MGO and peroxides in honey prevent pathogens from developing resistance to manuka honey (Sultanbawa, 2014). Manuka honey has been shown to be effective in treating S. aureus infections by reducing the bacterial growth (Almasaudi et al., 2017). However, its effects have not been explored in a C. elegans model.
This study utilized Caenorhabditis elegans, a transparent nematode widely used as a model organism. C. elegans is a microscopic worm with a short lifespan of approximately two to three weeks (Apfeld and Alper, 2018). From the egg stage, it progresses through several larval stages before reaching the phenotypically distinct L4 stage, after which it matures into an adult (Corsi, 2006). These worms are especially robust in the lab and can be easily and cost-effectively maintained on media plates seeded with a lawn of Escherichia coli. Previous studies have used C. elegans as a model for both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively) infections (Thompson and Brown, 2014). The bacteria consumed by the worm colonize the intestinal tract (Sifri et al., 2003), leading to degradation of the digestive system and eventual death of the organism (Irazoqui et al., 2010).
This research investigates the use of C. elegans hlh-30::3xFLAG::eGFP (strain VZ892) as a model for S. aureus infection and its responsiveness to manuka honey treatment (Martina et al., 2021). Both the wild-type C. elegans strain N2 and the stress reporter strain VZ892 were used to evaluate the effects of manuka honey on infection outcomes (Martina et al., 2021). VZ892 contains a GFP-tagged version of the endogenous hlh-30 locus and is phenotypically similar to wild-type. This strain serves as a reporter of cellular stress and immune activation. Upon exposure to stressors such as S. aureus, hlh-30 translocates, and GFP fluorescence increases, enabling visualization and quantification of the host response (Colino-Lage et al., 2024). Worms were divided into three groups: control (no S. aureus, no manuka honey treatment), S. aureus only, and S. aureus with manuka honey treatment (UMF 20). Assays included GFP fluorescence imaging, lifespan analysis, and developmental progression.
To utilize the stress reporter strain VZ892, an imaging assay was performed to quantify GFP expression. Localized GFP fluorescence within the intestinal tract suggests activation of host defense responses at the primary site of S. aureus colonization. VZ892 worms in the control groups had a mean fluorescence of 55,768.3 ACTF, with fluorescence diffusely distributed throughout the worms' bodies (Figure 1A, left panel). Exposure to S. aureus resulted in a 102.2% increase in fluorescence to 112,770 ACTF. In addition to this increase, fluorescence became localized, with intense pinpoint accumulations in the digestive system (Figure 1A, middle panel). Following treatment with manuka honey (Figure 1A, right panel), fluorescence decreased by 38.5% to 69,296 ACTF as compared to infected worms. No significant difference was found between the treatment group and the control group following a one-tailed t-test (P = 0.1109). Furthermore, upon exposure to manuka honey, the GFP fluorescence returned to a diffuse pattern similar to that observed in control worms.
To assess survival, a longevity assay was performed. Lifespans of both the VZ892 (Figure 1C) and N2 (Figure 1D) strains were significantly reduced following exposure to S. aureus, consistent with previous findings (Sifri et al., 2003). The VZ892 LT50 decreased 85.7% from 168 hours to 24 hours. Similarly, the N2 LT50 decreased by 73.3% from 180 hours to 48 hours.
Treatment with manuka honey improved lifespans but did not restore them to baseline levels. With treatment, the VZ892 strain LT50 improved to 72 hours (a 200% increase), and the N2 strain LT50 improved to 84 hours (a 75% increase).
Previous studies suggest that exposure to S. aureus represses development in C. elegans, resulting in delayed growth (Sifri et al., 2003). To determine whether manuka honey could mitigate this effect, a developmental assay was conducted to assess the percentage of worms reaching the L4 larval stage or adulthood within a normal timeframe. Figure 1E shows that both VZ892 and N2 control groups had high rates of normal development (94.42% and 98.15%, respectively). Exposure to S. aureus significantly reduced these rates to 62.26% for VZ892 and 76.74% for N2. Treatment with manuka honey improved development to 82.01% for VZ892 and 90.30% for N2. These results indicate that S. aureus exposure slows the development in C. elegans and that manuka honey treatment partially restores normal developmental progression.
These findings support the use of C. elegans as a model for screening natural therapeutics such as manuka honey. Additionally, the results suggest that UMF 20 manuka honey can reduce S. aureus-induced stress and pathogenesis in this model, indicating its potential as an alternative or adjunct to traditional antibiotics. Further studies are warranted to explore dose dependence across different UMF values, long-term effects, and efficacy against resistant S. aureus strains such as MRSA.
","references":[{"reference":"Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, et al., Baloch. 2018. Antibiotic resistance: a rundown of a global crisis. Infection and Drug Resistance Volume 11: 1645-1658.
","pubmedId":"","doi":"10.2147/IDR.S173867"},{"reference":"Almasaudi SB, Al-Nahari AAM, Abd El-Ghany ESM, Barbour E, Al Muhayawi SM, Al-Jaouni S, et al., Harakeh. 2017. Antimicrobial effect of different types of honey on Staphylococcus aureus. Saudi Journal of Biological Sciences 24: 1255-1261.
","pubmedId":"","doi":"10.1016/j.sjbs.2016.08.007"},{"reference":"Apfeld J, Alper S. 2018. What Can We Learn About Human Disease from the Nematode C. elegans? Methods in Molecular Biology, Disease Gene Identification : 53-75.
","pubmedId":"","doi":"10.1007/978-1-4939-7471-9_4"},{"reference":"Atrott J, Henle T. 2009. Methylglyoxal in Manuka Honey - Correlation with Antibacterial Properties. Czech Journal of Food Sciences 27: S163-S165.
","pubmedId":"","doi":"10.17221/911-CJFS"},{"reference":"Colino-Lage, H., Guerrero-Gómez, D., Gómez-Orte, E., González, X., Martina, J. A., Dansen, T. B., Ayuso, C., Askjaer, P., Puertollano, R., Irazoqui, J. E., Cabello, J., & Miranda-Vizuete, A. 2024. Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism. Free Radical Biology and Medicine 223: 369-383.
","pubmedId":"","doi":"10.1016/j.freeradbiomed.2024.07.027"},{"reference":"Corsi AK. 2006. A biochemist’s guide to Caenorhabditis elegans. Analytical Biochemistry 359: 1-17.
","pubmedId":"","doi":"10.1016/j.ab.2006.07.033"},{"reference":"Fitzpatrick, M. 2014. Measuring cell fluorescence using ImageJ. The Open Lab Book v1.0. https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html
","pubmedId":"","doi":""},{"reference":"Irazoqui JE, Troemel ER, Feinbaum RL, Luhachack LG, Cezairliyan BO, Ausubel FM. 2010. Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus. PLoS Pathogens 6: e1000982.
","pubmedId":"","doi":"10.1371/journal.ppat.1000982"},{"reference":"Martina JA, Guerrero‐Gómez D, Gómez‐Orte E, Antonio Bárcena J, Cabello J, Miranda‐Vizuete A, Puertollano R. 2021. A conserved cysteine‐based redox mechanism sustains TFEB/HLH‐30 activity under persistent stress. The EMBO Journal 40: 10.15252/embj.2020105793.
","pubmedId":"","doi":"10.15252/embj.2020105793"},{"reference":"Myles IA, Datta SK. 2012. Staphylococcus aureus: an introduction. Seminars in Immunopathology 34: 181-184.
","pubmedId":"","doi":"10.1007/s00281-011-0301-9"},{"reference":"Sifri CD, Begun J, Ausubel FM, Calderwood SB. 2003. Caenorhabditis elegans as a Model Host for Staphylococcus aureus Pathogenesis. Infection and Immunity 71: 2208-2217.
","pubmedId":"","doi":"10.1128/IAI.71.4.2208-2217.2003"},{"reference":"Sultanbawa, Y. 2014. Chapter 6: Leptospermum (Manuka) Honey: Accepted Natural Medicine. In L. Boukraa (Ed.), Honey in Traditional and Modern Medicine (pp. 113–121). CRC Press. https://doi.org/10.1201/b15608
","pubmedId":"","doi":"10.1201/b15608"},{"reference":"Thompson T, Brown PD. 2014. Comparison of antibiotic resistance, virulence gene profiles, and pathogenicity of methicillin-resistant and methicillin-susceptible Staphylococcus aureus using a Caenorhabditis elegans infection model. Pathogens and Global Health 108: 283-291.
","pubmedId":"","doi":"10.1179/2047773214Y.0000000155"}],"title":"The Effectiveness of Manuka Honey in Treating Staphylococcus aureus in C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes 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