Sweet and sour synergy: exploring the antibacterial and antibiofilm activity of acetic acid and vinegar combined with medical-grade honeys.

Oxymel, a combination of honey and vinegar, has been used as a remedy for wounds and infections in historical and traditional medical settings. While honey is now clinically used to treat infected wounds, this use of a complex, raw natural product (NP) mixture is unusual in modern western medicine. Research into the antimicrobial activity of NPs more usually focuses on finding a single active compound. The acetic acid in vinegar is known to have antibacterial activity at low concentrations and is in clinical use to treat burn wound infections. Here, we investigated the potential for synergistic activity of different compounds present in a complex ingredient used in historical medicine (vinegar) and in an ingredient mixture (oxymel). We conducted a systematic review to investigate published evidence for antimicrobial effects of vinegars against human pathogenic bacteria and fungi. No published studies have explicitly compared the activity of vinegar with that of a comparable concentration of acetic acid. We then characterized selected vinegars by HPLC and assessed the antibacterial and antibiofilm activity of the vinegars and acetic acid, alone and in combination with medical-grade honeys, against Pseudomonas aeruginosa and Staphylococcus aureus. We found that some vinegars have antibacterial activity that exceeds that predicted by their acetic acid content alone, but that this depends on the bacterial species being investigated and the growth conditions (media type, planktonic vs. biofilm). Pomegranate vinegars may be particularly interesting candidates for further study. We also conclude that there is potential for acetic acid, and some vinegars, to show synergistic antibiofilm activity with manuka honey.

Transcriptome Analysis of Pseudomonas aeruginosa Biofilm Infection in an Ex Vivo Pig Model of the Cystic Fibrosis Lung.

Pseudomonas aeruginosa is the predominant cause of chronic biofilm infections that form in the lungs of people with cystic fibrosis (CF). These infections are highly resistant to antibiotics and persist for years in the respiratory tract. One of the main research challenges is that current laboratory models do not accurately replicate key aspects of a P. aeruginosa biofilm infection, highlighted by previous RNA-sequencing studies. We compared the P. aeruginosa PA14 transcriptome in an ex vivo pig lung (EVPL) model of CF and a well-studied synthetic cystic fibrosis sputum medium (SCFM). P. aeruginosa was grown in the EVPL model for 1, 2, and 7 days, and in vitro in SCFM for 1 and 2 days. The RNA was extracted and sequenced at each time point. Our findings demonstrate that expression of antimicrobial resistance genes was cued by growth in the EVPL model, highlighting the importance of growth environment in determining accurate resistance profiles. The EVPL model created two distinct growth environments: tissue-associated biofilm and the SCFM surrounding tissue, each cuing a transcriptome distinct from that seen in SCFM in vitro. The expression of quorum sensing associated genes in the EVPL tissue-associated biofilm at 48 h relative to in vitro SCFM was similar to CF sputum versus in vitro conditions. Hence, the EVPL model can replicate key aspects of in vivo biofilm infection that are missing from other current models. It provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance that quickly drives P. aeruginosa into a chronic-like infection phenotype. IMPORTANCE Pseudomonas aeruginosa lung infections that affect people with cystic fibrosis are resistant to most available antimicrobial treatments. The lack of a laboratory model that captures all key aspects of these infections hinders not only research progression but also clinical diagnostics. We used transcriptome analysis to demonstrate how a model using pig lungs can more accurately replicate key characteristics of P. aeruginosa lung infection, including mechanisms of antibiotic resistance and infection establishment. Therefore, this model may be used in the future to further understand infection dynamics to develop novel treatments and more accurate treatment plans. This could improve clinical outcomes as well as quality of life for individuals affected by these infections.

Oxygen saturation-dependent effects on blood transverse relaxation at low fields.

OBJECTIVE: Blood oxygenation can be measured using magnetic resonance using the paramagnetic effect of deoxy-haemoglobin, which decreases the [Formula: see text] relaxation time of blood. This [Formula: see text] contrast has been well characterised at the [Formula: see text] fields used in MRI (1.5 T and above). However, few studies have characterised this effect at lower magnetic fields. Here, the feasibility of blood oximetry at low field based on [Formula: see text] changes that are within a physiological relevant range is explored. This study could be used for specifying requirements for construction of a monitoring device based on low field permanent magnet systems. METHODS: A continuous flow circuit was used to control parameters such as oxygen saturation and temperature in a sample of blood. It flowed through a variable field magnet, where CPMG experiments were performed to measure its [Formula: see text]. In addition, the oxygen saturation was monitored by an optical sensor for comparison with the [Formula: see text] changes. RESULTS: These results show that at low [Formula: see text] fields, the change in blood [Formula: see text] due to oxygenation is small, but still detectable. The data measured at low fields are also in agreement with theoretical models for the oxy-deoxy [Formula: see text] effect. CONCLUSION: [Formula: see text] changes in blood due to oxygenation were observed at fields as low as 0.1 T. These results suggest that low field NMR relaxometry devices around 0.3 T could be designed to detect changes in blood oxygenation.

An ex vivo cystic fibrosis model recapitulates key clinical aspects of chronic Staphylococcus aureus infection.

Staphylococcus aureus is the most prevalent organism isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. In mice, and many experiments using cell lines, the bacterium invades cells or interstitium, and forms abscesses. This is at odds with the limited available clinical data: interstitial bacteria are rare in CF biopsies and abscesses are highly unusual. Bacteria instead appear to localize in mucus plugs in the lumens of bronchioles. We show that, in an established ex vivo model of CF infection comprising porcine bronchiolar tissue and synthetic mucus, S. aureus demonstrates clinically significant characteristics including colonization of the airway lumen, with preferential localization as multicellular aggregates in mucus, initiation of a small colony variant phenotype and increased antibiotic tolerance of tissue-associated aggregates. Tissue invasion and abscesses were not observed. Our results may inform ongoing debates relating to clinical responses to S. aureus in people with CF.

Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa.

In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and drug development pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production.

Bacteria produce a wide variety of exoproducts that favourably modify their environment and increase their fitness. These are often termed 'public goods' because they are costly for individuals to produce and can be exploited by non-producers (cheats). The outcome of conflict over public goods is dependent upon the prevailing environment and the phenotype of the individuals in competition. Many bacterial species use quorum sensing (QS) signalling molecules to regulate the production of public goods. QS, therefore, determines the cooperative phenotype of individuals, and influences conflict over public goods. In addition to their regulatory functions, many QS molecules have additional properties that directly modify the prevailing environment. This leads to the possibility that QS molecules could influence conflict over public goods indirectly through non-signalling effects, and the impact of this on social competition has not previously been explored. The Pseudomonas aeruginosa QS signal molecule PQS is a powerful chelator of iron which can cause an iron starvation response. Here, we show that PQS stimulates a concentration-dependent increase in the cooperative production of iron scavenging siderophores, resulting in an increase in the relative fitness of non-producing siderophore cheats. This is likely due to an increased cost of siderophore output by producing cells and a concurrent increase in the shared benefits, which accrue to both producers and cheats. Although PQS can be a beneficial signalling molecule for P. aeruginosa, our data suggest that it can also render a siderophore-producing population vulnerable to competition from cheating strains. More generally, our results indicate that the production of one social trait can indirectly affect the costs and benefits of another social trait.

An ex vivo lung model to study bronchioles infected with Pseudomonas aeruginosa biofilms.

A key aim in microbiology is to determine the genetic and phenotypic bases of bacterial virulence, persistence and antimicrobial resistance in chronic biofilm infections. This requires tractable, high-throughput models that reflect the physical and chemical environment encountered in specific infection contexts. Such models will increase the predictive power of microbiological experiments and provide platforms for enhanced testing of novel antibacterial or antivirulence therapies. We present an optimized ex vivo model of cystic fibrosis lung infection: ex vivo culture of pig bronchiolar tissue in artificial cystic fibrosis mucus. We focus on the formation of biofilms by Pseudomonas aeruginosa. We show highly repeatable and specific formation of biofilms that resemble clinical biofilms by a commonly studied laboratory strain and ten cystic fibrosis isolates of this key opportunistic pathogen.

Bacterial cooperation in the wild and in the clinic: are pathogen social behaviours relevant outside the laboratory?

Individual bacterial cells can communicate via quorum sensing, cooperate to harvest nutrients from their environment, form multicellular biofilms, compete over resources and even kill one another. When the environment that bacteria inhabit is an animal host, these social behaviours mediate virulence. Over the last decade, much attention has focussed on the ecology, evolution and pathology of bacterial cooperation, and the possibility that it could be exploited or destabilised to treat infections. But how far can we really extrapolate from theoretical predictions and laboratory experiments to make inferences about 'cooperative' behaviours in hosts and reservoirs? To determine the likely importance and evolution of cooperation 'in the wild', several questions must be addressed. A recent paper that reports the dynamics of bacterial cooperation and virulence in a field experiment provides an excellent nucleus for bringing together key empirical and theoretical results which help us to frame - if not completely to answer - these questions.

Development of an ex vivo porcine lung model for studying growth, virulence, and signaling of Pseudomonas aeruginosa.

Research into chronic infection by bacterial pathogens, such as Pseudomonas aeruginosa, uses various in vitro and live host models. While these have increased our understanding of pathogen growth, virulence, and evolution, each model has certain limitations. In vitro models cannot recapitulate the complex spatial structure of host organs, while experiments on live hosts are limited in terms of sample size and infection duration for ethical reasons; live mammal models also require specialized facilities which are costly to run. To address this, we have developed an ex vivo pig lung (EVPL) model for quantifying Pseudomonas aeruginosa growth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. In a first test of our model, we show that lasR mutants, which do not respond to 3-oxo-C(12)-homoserine lactone (HSL)-mediated QS, exhibit reduced virulence factor production in EVPL. We also show that lasR mutants grow as well as or better than a corresponding wild-type strain in EVPL. lasR mutants frequently and repeatedly arise during chronic CF lung infection, but the evolutionary forces governing their appearance and spread are not clear. Our data are not consistent with the hypothesis that lasR mutants act as social "cheats" in the lung; rather, our results support the hypothesis that lasR mutants are more adapted to the lung environment. More generally, this model will facilitate improved studies of microbial disease, especially studies of how cells of the same and different species interact in polymicrobial infections in a spatially structured environment.

Social role specialization promotes cooperation between parents.

Biparental care of offspring is a widespread social behavior, and various ecological, life-history, and demographic factors have been proposed to explain its evolution and maintenance. Raising offspring generally requires several types of care (e.g., feeding, brooding, and defense), and males and females often specialize in providing different types of care. However, theoretical models of care often assume that care is a single variable and hence that a unit of care by the mother is interchangeable with a unit of care by the father. We hypothesize that the ability of one parent to provide all types of care may be limited by nonadditive costs or by sex-based asymmetries in the costs of particular care types. Using an individual-based simulation, we show that synergistic costs of investing in two tasks or negligible sex-based cost asymmetries select for task specialization and biparental care. Biparental care persists despite intense sexual selection and sex-biased mortality, suggesting that previous models make overly restrictive predictions of the conditions under which cooperation can be maintained. Our model provides a mechanistic underpinning for published models that show that the synergistic benefits of individuals cooperating can stabilize cooperation, both in the context of parental care and in other social scenarios.

How Does Airway Surface Liquid Composition Vary in Different Pulmonary Diseases, and How Can We Use This Knowledge to Model Microbial Infections?

Growth environment greatly alters many facets of pathogen physiology, including pathogenesis and antimicrobial tolerance. The importance of host-mimicking environments for attaining an accurate picture of pathogen behaviour is widely recognised. Whilst this recognition has translated into the extensive development of artificial cystic fibrosis (CF) sputum medium, attempts to mimic the growth environment in other respiratory disease states have been completely neglected. The composition of the airway surface liquid (ASL) in different pulmonary diseases is far less well characterised than CF sputum, making it very difficult for researchers to model these infection environments. In this review, we discuss the components of human ASL, how different lung pathologies affect ASL composition, and how different pathogens interact with these components. This will provide researchers interested in mimicking different respiratory environments with the information necessary to design a host-mimicking medium, allowing for better understanding of how to treat pathogens causing infection in these environments.

Differential development of antibiotic resistance and virulence between Acinetobacter species.

The two species that account for most cases of Acinetobacter-associated bacteremia in the United Kingdom are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and Acinetobacter baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii, and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30, respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defense systems and harbored a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm, and infect both in vivo and in in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesize that the fact that it only harbors a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram-negative bacteria. IMPORTANCE: Acinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived bloodstream infections in England and Wales. In contrast to the well-studied and often highly drug-resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not, and could inform future novel treatment strategies.

Selflessness is sexy: reported helping behaviour increases desirability of men and women as long-term sexual partners.

BACKGROUND: Despite its short-term costs, behaviour that appears altruistic can increase an individual's inclusive fitness by earning direct (selfish) and/or indirect (kin-selected) benefits. An evolved preference for other-regarding or helping behaviour in potential mates has been proposed as an additional mechanism by which these behaviours can yield direct fitness benefits in humans. RESULTS: We asked 32 heterosexual women and 35 heterosexual men to rate the attractiveness of members of the opposite sex in the presence and the absence of information about helping behaviours. Reports of helping behaviour were associated with a significant increase in the attractiveness of both men and women as potential long-term sexual partners. Altruism also increased the attractiveness of men as potential partners for short-term flings, but to a lesser extent than when the same men were being considered for long-term relationships. Altruism did not affect the attractiveness of women as partners for short-term flings. CONCLUSIONS: Our results unite two important areas of evolutionary theory - social evolution and sexual selection - and extend the list of means by which helping behaviours, which appear at first glance to be costly to the actor, can in fact earn direct fitness benefits. Helping behaviours may be attractive because they signal 'good genes' and/or because they are perceived as a signal of likely provision of non-genetic benefits (e.g. parental care). Exactly why helping behaviours in a non-mating context might be attractive to potential mates, and whether they are honest signals of mate quality, remains to be elucidated.

Combining SNAPs with antibiotics shows enhanced synergistic efficacy against S. aureus and P. aeruginosa biofilms.

Biofilm infections are associated with a high mortality risk for patients. Antibiotics perform poorly against biofilm communities, so high doses and prolonged treatments are often used in clinical settings. We investigated the pairwise interactions of two synthetic nano-engineered antimicrobial polymers (SNAPs). The g-D50 copolymer was synergistic with penicillin and silver sulfadiazine against planktonic Staphylococcus aureus USA300 in synthetic wound fluid. Furthermore, the combination of g-D50 and silver sulfadiazine showed a potent synergistic antibiofilm activity against S. aureus USA300 using in vitro and ex vivo wound biofilm models. The a-T50 copolymer was synergistic with colistin against planktonic Pseudomonas aeruginosa in synthetic cystic fibrosis medium, and this pair showed a potent synergistic antibiofilm activity against P. aeruginosa in an ex vivo cystic fibrosis lung model. SNAPs thus have the potential for increased antibiofilm performance in combination with certain antibiotics to shorten prolonged treatments and reduce dosages against biofilm infection.

Increased Central Auditory Gain and Decreased Parvalbumin-Positive Cortical Interneuron Density in the Df1/+ Mouse Model of Schizophrenia Correlate With Hearing Impairment.

Background: Hearing impairment is a risk factor for schizophrenia. Patients with 22q11.2 deletion syndrome have a 25% to 30% risk of schizophrenia, and up to 60% also have varying degrees of hearing impairment, primarily from middle-ear inflammation. The Df1/+ mouse model of 22q11.2 deletion syndrome recapitulates many features of the human syndrome, including schizophrenia-relevant brain abnormalities and high interindividual variation in hearing ability. However, the relationship between brain abnormalities and hearing impairment in Df1/+ mice has not been examined. Methods: We measured auditory brainstem responses, cortical auditory evoked potentials, and/or cortical parvalbumin-positive (PV+) interneuron density in over 70 adult mice (32 Df1/+, 39 wild-type). We also performed longitudinal auditory brainstem response measurements in an additional 20 animals (13 Df1/+, 7 wild-type) from 3 weeks of age. Results: Electrophysiological markers of central auditory excitability were elevated in Df1/+ mice. PV+ interneurons, which are implicated in schizophrenia pathology, were reduced in density in the auditory cortex but not the secondary motor cortex. Both auditory brain abnormalities correlated with hearing impairment, which affected approximately 60% of adult Df1/+ mice and typically emerged before 6 weeks of age. Conclusions: In the Df1/+ mouse model of 22q11.2 deletion syndrome, abnormalities in central auditory excitability and auditory cortical PV+ immunoreactivity correlate with hearing impairment. This is the first demonstration of cortical PV+ interneuron abnormalities correlating with hearing impairment in a mouse model of either schizophrenia or middle-ear inflammation.

Mechanism of Action of Oxazoline-Based Antimicrobial Polymers Against Staphylococcus aureus: In Vivo Antimicrobial Activity Evaluation.

Antimicrobial-resistant pathogens have reached alarming levels, becoming one of the most pressing global health issues. Hence, new treatments are necessary for the fight against antimicrobial resistance. Synthetic nanoengineered antimicrobial polymers (SNAPs) have emerged as a promising alternative to antimicrobial peptides, overcoming some of their limitations while keeping their key features. Herein, a library of amphiphilic oxazoline-based SNAPs using cationic ring-opening polymerization (CROP) is designed. Amphipathic compounds with 70% cationic content exhibit the highest activity against clinically relevant Staphylococcus aureus isolates, maintaining good biocompatibility in vitro and in vivo. The mechanism of action of the lead compounds against S. aureus is assessed using various microscopy techniques, indicating cell membrane disruption, while the cell wall remains unaffected. Furthermore, a potential interaction of the compounds with bacterial DNA is shown, with possible implications on bacterial division. Finally, one of the compounds exhibits high efficacy in vivo in an insect infection model.

Single-Sided Magnet System for Quantitative MR Relaxometry and Preclinical In-Vivo Monitoring.

OBJECTIVE: We have developed a single-sided magnet system that allows Magnetic Resonance relaxation and diffusion parameters to be measured. METHODS: A single-sided magnet system has been developed, using an array of permanent magnets. The magnet positions are optimised to produce a B0 magnetic field with a spot that is relatively homogenous and can project into a sample. NMR relaxometry experiments are used to measure quantitative parameters such as T2, T1 and apparent diffusion coefficient (ADC) on samples on the benchtop. To explore preclinical application, we test whether it can detect changes during acute global cerebral hypoxia in an ovine model. RESULTS: The magnet produces a 0.2 T field projected into the sample. Measurements of benchtop samples show that it can measure T1, T2 and ADC, producing trends and values that are in line with literature measurements. In-vivo studies show a decrease in T2 during cerebral hypoxia that recovers following normoxia. CONCLUSION: The single-sided MR system has the potential to allow non-invasive measurements of the brain. We also demonstrate that it can operate in a pre-clinical environment, allowing T2 to be monitored during brain tissue hypoxia. SIGNIFICANCE: MRI is a powerful technique for non-invasive diagnosis in the brain, but its application has been limited by the requirements for magnetic field strength and homogeneity that imaging methods have. The technology described in this study provides a portable alternative to acquiring clinically significant MR parameters without the need for traditional imaging equipment.

An assessment of the evidence for antibacterial activity of stinging nettle (Urtica dioica) extracts.

Stinging nettles (Urtica spp.) have been used in a diverse range of traditional and historical medicines from around the world for the treatment of skin diseases, wounds, urinary disorders, respiratory diseases, bone and joint pain, anaemia and other circulatory problems, as well as in cosmetic preparations for skin and haircare. As part of an interdisciplinary exploration of nettle-based remedies, we performed a systematic review of published evidence for the antimicrobial activity of Urtica spp. extracts against bacteria and fungi that commonly cause skin, soft tissue and respiratory infections. We focussed on studies in which minimum inhibitory concentration (MIC) assays of U. dioica were conducted on the common bacterial opportunistic pathogens Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumoniae and Staphylococcus aureus . No studies used fresh leaves (all were dried prior to use), and no studies prepared nettles in weak acid (corresponding to vinegar) or in fats/oils, which are common combinations in historical and traditional preparations. We addressed this gap by conducting new antibacterial tests of extracts of fresh U. dioica leaves prepared in vinegar, butter or olive oil against P. aeruginosa and S. aureus . Our systematic review and additional experimental data leads us to conclude that there is no strong evidence for nettles containing molecules with clinically useful antimicrobial activity. It seems most likely that the utility of nettles in traditional topical preparations for wounds may simply be as a 'safe' absorbent medium for keeping antibacterial (vinegar) or emollient (oils) ingredients at the treatment site.

Phase 1 safety trial of a natural product cocktail with antibacterial activity in human volunteers.

New antibiotics are urgently needed to reduce the health burden of antibiotic-resistant bacterial infection. Natural products (NPs) derived from plants and animals are a current focus of research seeking to discover new antibacterial molecules with clinical potential. A cocktail of NPs based on a medieval remedy for eye infection eliminated biofilms of several highly antibiotic-resistant bacterial species in laboratory studies, and had a promising safety profile in vitro and in a mouse model. A necessary prelude to refining this remedy into a defined, synthetic mixture suitable for testing with wound infections is to firstly establish safety when applied to healthy human skin. We aimed to assess skin-related outcomes of the preparation in a sample of healthy volunteers. This prospective, single arm, non-randomised Phase I clinical trial consisted of a single patch test intervention with 48-h follow-up. Volunteers were staff, students and members of the public recruited from the University of Warwick and surrounding locality. Adults aged 18-79 years, with no history of severe immunity-related disease, diabetes, recent infection, or known pregnancy were eligible. A 100 µl application of a filter-sterilised NP mixture, comprising ground garlic, onion, white wine and bovine bile, was applied to skin on the upper arm and covered with a dressing. The primary outcome was skin-related adverse events over 48 h. Digital photographs were captured where bothersome, salve-related events were reported. 109 volunteers, aged 18-77 years, were recruited between June and July 2021. Sample mean age was 37.6 (SD 16.1) years, and 63 (58%) participants were female. Outcome data were obtained for 106/109 (97%); two participants were lost to follow-up and one removed the skin patch after nine hours due to a bothersome garlic odour. Twenty-one (19.8%) participants reported any patch-test related sign or symptom; of these 14 (13.2%) participants reported minor events related to the salve, including itchiness, redness, or garlic odour. No serious events were reported. We found no evidence of serious skin-related adverse events related to the NP preparation.Trial registration: International Standard Randomised Controlled Trial Number (ISRCTN10773579). Date registered: 08/01/2021.