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Microbiological features distinguishing Lyme disease and relapsing fever spirochetes

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Microbiological features distinguishing Lyme disease and relapsing fever spirochetes

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Summary

The recent proposal of splitting the genus Borrelia into two genera in the newly formed family of Borreliaceae, i. e. Borrelia and Borreliella has motivated us to reflect upon how these organisms has been characterized and differentiated. This article therefore aims to take a closer look on the biology and virulence attributes of the two suggested genera, i. e. those causing Lyme borreliosis and relapsing fever borreliosis. Both genera have much in common with similar infection biological features. They are both characterized as bacterial zoonoses, transmitted by hematophagous arthropods with almost identical microbiological appearance. Nevertheless, a closer look at the genotypic and phenotypic characteristics clearly reveals several differences that might motivate the suggested split. On the other hand, a change of this well-established classification within the genus Borrelia might impose an economical burden as well as a great confusion in society, including medical and scientific societies as well as the general population.

Keywords: Borrelia, Borreliella, Lyme borreliosis, Relapsing fever, Taxonomy

Introduction

Recent molecular analysis of Borrelia spirochetes has suggested a new taxonomic classification. Thus, spirochetes within the family Borreliaceae [ 1], which belongs to the order Spirochetales, has recently been reclassified and now been suggested to contain two different genera; Borrelia, and Borreliella [ 2]. The genealogical separation of relapsing fever and Lyme disease spirochetes has been debated and questioned among researchers, clinicians and stake holders within the borreliosis community [ 3, 4]. The suggested split is based on an in-depth molecular analysis of phylogenetic and phenotypic characteristics, i. e. genome and proteome comparison of the increasingly available omics data. The results from the comparative analyses partly support this separation, which will be further discussed in this review.

Although relapsing fever (RF) was described during antiquity it acquired its current name much later. About 430 BC Hippocrates described an epidemic fever that was probably related to RF. Several outbreaks of an "epidemic fever" during the following centuries have been attributed to RF mainly because of the disease symptoms [ 5]. The first description of clinical features associated to RF was documented in the middle of the eighteenth century from an outbreak in Ireland. This epidemic spread all over the British Isles and the disease was for the first time labelled "relapsing fever" in 1843 [ 5, 6]. The disease was then further disseminated to the European continent and the USA. The outbreaks continued for decades and plagued large parts of the world [ 5]: however, it is important to remember that at this time other microorganisms could have been the etiologic agent for this recurrent fever, including Rickettsiae, malarial parasites or various viruses. None of these historical descriptions are definite but are of course interesting from an epidemiological point of view. The suspected RF infection was most often found in poor and overpopulated areas, and therefore the body louse was assumed to spread the disease. The importance of the body louse, Pediculus humanus humanus in transmitting RF was later shown experimentally by inoculating monkeys with crushed lice from human RF patients [ 6]. In 1868 the RF microorganism was discovered and described by the German doctor Otto Obermeier and initially designated " Spirocheta Obermeieri," but finally denoted its current name Borrelia recurrentis [ 5].

Lyme disease is of much later date, i. e. the date of when the disease was defined and its etiologic agent was proven and described. The Lyme borreliosis era started when several cases of arthritis were reported and described in 1977 in the town of Old Lyme, Connecticut, USA. This came from an epidemic of oligoarthritis in children, which first was misdiagnosed as being juvenile arthritis [ 7]. Interestingly, the patients often developed an expanding skin rash prior to onset of arthritis. Therefore, these findings led to the suspicion that the diagnosis was incorrect. This pathognomonic skin rash, termed erythema migrans, was first reported by a Swedish dermatologist Arvid Afzelius in the year 1909 [ 8]. He suggested that a tick bite caused this particular type of skin lesion. Similar to the finding by Afzelius, Steere et al. also believed that ticks were involved in the transmission of this novel disease [ 9]. Therefore, it was proposed that the symptoms resulted from transmission of an infectious agent by an arthropod vector. Subsequently, a spirally formed bacteria could be isolated from the American Deer tick, Ixodes scapularis and a similar organism was later isolated from the blood and cerebrospinal fluid from patients with erythema migrans [ 9- 12]. Further microbiological and biochemical characterization of this microorganism suggested that the spirochete-like organism belonged to the genus Borrelia. Thus, these investigators now suggested this novel microorganism to be a Borrelia spirochete because of its microbiological and microscopically resemblance to RF Borrelia. This Borrelia-like spirochete was then named Borrelia burgdorferi after one of its co-discoverers, Willy Burgdorfer [ 12].

Basic characteristics

The typical symptom of Lyme borreliosis is the characteristic skin rash denoted erythema migrans. Untreated, the disease can in certain individuals disseminate and cause symptoms in the nervous system or joints as well as the more persistent skin disorder acrodermatitis chronica athrophicans. For a more thorough description of the various clinical symptoms of Lyme borreliosis see Review by Stanek et al. [ 13]. Nevertheless, Lyme borreliosis is characterized by the deleterious effects on specific tissues, including the skin, central nervous system, joints and the heart. In contrast to the tissue specificity of Lyme disease, relapsing fever (RF) borreliosis initially starts as a blood-borne disease and is characterised by a high concentration of spirochetes in the blood and intermittent attacks of high fever. In essence, the main disease manifestation is a recurring fever that coincides with high numbers of spirochetes in the blood. The severity of the infection ranges from asymptomatic to serious.

Despite the suggestion to separate the spirochetes of the new family Borreliaceae into two genera, they still have several general microbiological features in common. They are characteristically spiral shaped, approximately 25-30 µm long and 1 µm thick [ 14]. A second typical feature of the organisms is the locomotor organ that contains a bundle of flagellae attached to the poles. The flagellae are located in the periplasmic space and wrapped around the cell cylinder, giving it its characteristic shape [ 14- 16]. The outer membrane is Gram-negative, meaning that it has a diderm structure that contains a cytoplasmic-and an outer membrane separated by the periplasm [ 17, 18]. The spirochaetal outer membrane is regarded as more fluid than other Gram-negative bacteria with a 45-62% protein, 23-50% lipid and 3-4% carbohydrate content [ 14]. Another important difference compared to other Gram-negative bacteria is the absence of lipopolysaccharides on the outer surface. Instead, Borrelia spirochetes have a large amount of surface located lipoproteins and as much as ~8% of the open reading frames in Borreliae encodes for lipoproteins [ 14, 18, 19]. Many of the surface located lipoproteins are thought to be important for the life style of the Borreliae and therefore important factor(s) during infection.

Virulence factors

Lyme borreliosis

Several factors important for the pathogenesis of the various species within the family of Borreliaceae has been described and characterised both in vitro and in vivo. Virulence factors are defined as any specialized virulence trait that separates an infectious bacteria from a non-virulent variant. Typically, those factors are involved in adhesion, colonization, invasion and toxin production. Several virulence factors important for Lyme borreliosis has been described and investigated during the last few decades. The investigation of Lyme borreliosis virulence traits took off with the publication of the B. burgdorferi genome by Fraser et al. [ 18], and with the subsequent full genomic annotation in 2000 [ 20]. Many unique virulence factors that operate during Lyme borreliosis have been characterized and defined. These factors include various outer surface molecules that interact with the mammalian host. Among these are several cell surface outer membrane proteins (OMPs), which can act as adhesins or receptors for different molecules and aid in attachment, transmission and immunological escape of the pathogen. It is furthermore well known that Borrelia spp. express different parts of the OMP repertoire depending on if it resides in a mammalian or a tick host.

Several important adhesins have been characterized that are essential during Lyme borreliosis. Among those are the DbpA and DbpB surface located proteins, which bind to the proteoglycan decorin [ 21- 23]. Additionally, expression of DbpA and DbpB was shown to be up-regulated at 35 °C versus 23 °C and it was further shown that the presence of antibodies against DbpA could prevent a B. burgdorferi infection indicating a possible use as a vaccine. Disappointingly, in vivo challenge studies showed that the efficacy was not as pronounced as for other Lyme borreliosis vaccine candidates, e.g. OspA [ 23- 25]. In conclusion, the results suggest that certain adhesive properties by these decorin-binding proteins are important for the colonization of the human host.

Another characterized adhesin is BBK32 a fibronectin-binding protein that promotes B. burgdorferi attachment to glycosaminoglycans (GAG) [ 26]. Similarly to DbpA and DbpB, the expression of BBK32 is also up-regulated at 35 °C versus 23 °C indicating its importance in the mammalian host. Likewise, an inactivation of BBK32 leads to a significantly attenuated infectious phenotype of B. burgdorferi, indicating that the fibronectin-BBK32 interaction might be important for B. burgdorferi pathogenesis [ 25, 27].

Of all the different adhesins described in Borrelia spirochetes, perhaps P66 is one of the most interesting, in part because of its dual function. Hence, P66 has been shown to both act as a porin and as a mediator of interaction between B. burgdorferi and beta ₃-chain integrins, molecules that are found on various immune cells, blood platelets and endothelial cells. Although the function of P66 is not elucidated the integrin-binding activity of P66 is thought to aid in Borrelia escape from the site of the inoculum and in dissemination of the bacteria into tissues [ 28, 29]. The P66 adhesin is also the most studied porin in Borrelia and has a remarkably high single channel conductance indicating a possible unique role for these spirochetes ([ 30- 32]; Fig. ).

Also, many factors important for the dissemination of Borrelia spirochetes either hematogenously or directly through various tissues have been characterized. Most important in this process is the chemotaxis and motility apparatus that enables the spirochete to efficiently spread to various sites in the mammalian host [ 15]. An in-depth description of the molecular function of the motility and chemotaxis and its role during Lyme borreliosis has been summarized in [ 15, 33].

A functionally diverse and large group of virulence factors are the complement regulatory acquiring surface proteins (CRASPs). These proteins can bind complement inhibiting factor H (CFH), factor H-like protein 1 (CFHL-1) and several other factor H-related proteins (CFHRs). When binding to these complement-inhibiting proteins, the Borreliae can evade host-mediated complement killing [ 34- 38]. Until now, several CRASPs have been found in B. burgdorferi, including CspA, CspZ, ErpA, ErpC and ErpP.

The most prominent surface proteins of Lyme borreliosis Borrelia are the major Osps, i. e. OspA, OspB and OspC. These major Osps, exhibit a variable expression with high expression level of OspA and OspB in the tick and a simultaneous shift to OspC expression during feeding and after entering the mammalian host [ 39]. Thus, it appears that OspC expression is coupled to the spirochete transmission, as well as the presence within the mammalian host. Likewise, the OspA/B expression is associated with the arthropod host [ 39, 40]. Both OspA and OspC have adhesive properties that aid in the transmission of the spirochete from tick to mammal.

Another interesting surface located lipoprotein is the vlsE, which is a 35 kDa lipoprotein of Lyme borreliosis Borrelia that is analogous to the VMP's of RF Borreliae (see below) and can undergo antigenic variation. This antigenic variation mechanism is used by the bacteria to escape recognition and elimination by the host immune system. The expression site of vlsE together with a continuous site of 15 vls silent cassettes comprise the vls locus [ 41, 42]. The plasmid carrying the vlsE, lp28-1, is highly correlated with an infectious phenotype of B. burgdorferi, which is believed to be mainly due to the vls locus [ 43, 44].

Relapsing fever borreliosis

Many of the virulence factors described for Lyme borreliosis borreliae also occur in RF spirochetes, although not so thoroughly investigated. The infectious course during RF borreliosis, i. e. from the initial deposition of the spirochete in the skin to the subsequent colonization in various tissues, exposes the spirochetes to many defense mechanisms of the host. During the course of infection it occupies a variety of different environments or niches, including the skin, the circulation, and eventually peripheral organs with their own specific milieu. It means that the RF Borrelia will combat several components of the host immune system. To accomplish survival in all different environments the spirochete has developed mechanisms to adapt and survive in different surroundings of the animal host, which then defines its virulence characters.

An RF borreliosis infection initially starts with either a tick bite or by a louse blood meal. Usually, the tick bite is painless and therefore unnoticed. The anesthesia is caused by the inoculation of a variety of effectors secreted along with the tick saliva, including pain killers, anticoagulation factors and anti-inflammatory substances. All these factors are important for a successful infection and support the tick in obtaining a successful blood meal with a subsequent transmission of the disease [ 45- 47]. In addition, tick saliva inhibits the activity of polymorphonuclear leucocyte (PMN), which further reduces the killing of the spirochete [ 48]. Next, the RF Borrelia must penetrate both the extracellular matrix (ECM) and the endothelial lining of the blood vessels in order to maintain and spread the infection to adjacent tissues. As for Lyme borreliosis spirochetes the characteristic shape as well as the motility and chemotaxis mechanisms are important for the dissemination, as demonstrated by the loss of endothelial penetration ability of flagella mutants [ 49]. Also, the host protease activity is part of the spreading process as shown by studies in plasminogen-deficient mice ( plg), which demonstrated that dissemination of RF Borrelia to tissues is delayed when the plasminogen activator system (PAS) is missing, and with a simultaneous reduction of bacterial load in the brain and the heart of infected animals [ 50, 51]. Additionally, an activation of matrix metalloproteinases (MMP's) also follows plasminogen binding and activation on the spirochete surface [ 52]. Thus, the conclusion is that utilization of host proteases supports spreading of the RF Borrelia spirochete.

As mentioned, RF borreliosis is in the initial stages a blood-borne infection that subsequently multiplies in the circulation, where it reaches very high numbers. Several of the Old World RF Borrelia species, e. g. B. duttonii, B. crocidurae, and B. hispanica, frequently interact with cells in the blood, primarily the erythrocytes. Thus, these Old World RF species cause the red blood cells to aggregate, a phenomenon termed erythrocyte rosetting (Fig. ). The mechanism of red blood cell rosetting was first shown by Mooser in 1958 [ 53]. Moreover, in vitro models later suggested that the interaction is a way for the spirochete to cover itself with components that may inhibit detection by the immune response [ 54]. Another hypothesis, although not proven, is that the spirochete forages on the erythrocytes, i. e. collecting nutrients from the cells during its interaction with erythrocytes. Throughout an infection with RF Borrelia there are large amounts of spirochetes present, reaching up to 1 billion of spirochetes per milliliter of blood. Thus, it is evident that a considerable amount of nutrients are needed to support the growth of the RF borreliae population in the blood. The grazing theory was further backed-up by the findings that RF Borrelia species have specific genes that enable them to use purines from serum as metabolites for synthesis of various macromolecules [ 55]. These particular genes are not present in Lyme borreliosis spirochetes that interestingly never reach high densities in the blood displaying an important difference between these two genera. In addition, the purine hypoxhantine is abundant in human plasma and also produced by erythrocytes indicating a possible interaction with red blood cells as a means for nutrient acquisition as well as other metabolites needed for growth [ 55]. The RF Borrelia interactions with cells in the circulation might be a virulence strategy to increase and lengthen the time this pathogen can be retained within the host. Besides, erythrocytes and possibly other blood cells can provide nutrients or shelter from the host immune response and might augment penetration into distant tissues and organs.

Next and the most noticeable virulence factor of RF Borrelia is the mechanism of antigenic variation. This is the mechanism that drives the antigenic variation of the surface located outer surface variable membrane proteins (Vmp's) and as such prolongs the infection as long as possible. Numerous reviews and book chapters have been written about this process and are not discussed in detail. In short, the mechanism of RF was described by early researchers as a process in which every relapse of the infection was caused by a newly appearing variant of the original infecting strain. This indicated that the recurrent variant was a result of an antigenic variation of a dominant and immunogenic surface protein, the so called variable major proteins (Vmp) [ 56, 57]. Throughout every fever peak during RF, the majority of the spirochetes express one type of Vmp, eliciting the host to mount an antibody response towards this serotype that subsequently results in a clearance of this particular serotype from the circulation. In parallel, a second wave of spirochetes with a new serotype will emerge and multiply. These bacteria are expressing a Vmp that is no longer recognized by the immune system and that causes a new bout of spirochetemia with high fever. This process of multiphasic antigenic variation may be repeated several times. Only one Vmp is expressed at one time in a single expression locus, other variants are silent and located on several linear plasmids, but by recombination the expressed Vmp is replaced by one of these silent variants. These bacteria, the new serotype, will start to express the new Vmp and thus evade antibody-mediated killing [ 57- 60].

Finally, the progress of RF involves tissue invasion and colonization connected to an additional escape from the immune defense. A comprehensive and recently published review article by Talagrand-Reboul et al. summarizes the infection biology of RF borreliosis [ 61].

As summarized in this review the RF and Lyme borreliosis spirochetes have many characteristics in common, including the overall organization of the genome with a well-conserved synteny of the chromosome displayed as a very similar gene order. In contrast, a larger variability is seen in the various linear and circular plasmids of the different spirochetal types, i. e. RF and Lyme borreliosis borreliae. Although both types are transmitted by ticks and most are characterized as bacterial zoonoses there are several prominent differences between the two groups.

The RF group presents a more variable disease spectrum, including tick-borne and louse-borne RF as well as the separate and specific types of avian and bovine borreliosis. In contrast, Lyme borreliosis is more homogeneously characterized by the different stages of disease presented in various tissues. There is also a different disease progression that separates these two types of spirochetes as RF borreliosis is a blood-borne disease with a large amount of spirochetes in the blood whereas Lyme borreliosis is characterized by a very low amount of spirochetes in the blood but a predilection for various tissues, including skin, joints and the nervous system. Moreover, the Lyme borreliosis spirochetes are transmitted by prostriate ixodid ticks while RF spirochetes are more variable and can be transmitted by argasid ticks, prostriate ixodid ticks, metastriate ixodid ticks and human body lice. Another difference is the ability of most RF tick-borne species to undergo transovarial transmission in the ticks.

Conclusion

This review article does not have the intention to lead the reader to any conclusion or decision about the suggested split of the Borrelia genus into two new species, Borrelia and Borreliella. This suggested split has indeed initiated quite a turmoil within the Borrelia community. This article summarizes the basic differences and similarities of these two pathogenic groups of spirochetes, i. e. causing RF and Lyme disease (Lyme borreliosis). In parallel to this split within the genus there is also a recommendation to change the family name of various spirochetes, where Borrelia and Borreliella will be placed in the new family Borreliaceae. In connection to this we also acknowledge the problem of using the collective name Borrelia burgdorferi sensu lato for Lyme borreliosis spirochetes as well as the common mix-up between Lyme borreliosis and RF borreliae among lay people and in the healthcare setting. however, there are a number of important and practical caveats regarding the name change. The diagnostics geared to recognize Borrelia infections as well as the treatment regimens are anchored in the present nomenclature. It has been painstakingly built by many stakeholders and represents literally billions of dollars in investments from private and public funds. Changing the names would entail added costs with little or no de facto gain. Furthermore, the chances for confusion and misdiagnosis are increased and could in the end result in an increased risk for the patients suffering from the disease. While changes in nomenclature may be pertinent for genetic and taxonomic reasons, any modification has to be well thought through and the risks carefully considered.

We believe that the last word on this issue has not yet been said and we are eagerly waiting for the next step in this Borreliaceae drama. Either it might just fade away as happened to the renaming of the genus Chlamydia chlamydiophila. Alternatively, a decision similar to the ruling for keeping the name for Yersinia pestis instead of giving it the status of being a subspecies of Y. pseudotuberculosis as violation of the international code of nomenclature for bacteria [ 62].

Notes

Conflict of interest

S. Bergström and J. Normark declare that they have no competing interests.

References

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3. Barbour AG, Adeolu M, Gupta RS. Division of the genus Borrelia into two genera (corresponding to Lyme disease and relapsing fever groups) reflects their genetic and phenotypic distinctiveness and will lead to a better understanding of these two groups of microbes. Int. J. Syst. Evol. Microbiol. 2017 [[ PubMed] Google Scholar]

5. Felsenfeld O. Borrelia: strains, vectors, human and animal borreliosis. St. Louis: Warren H Green; 1971. [ Google Scholar]

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8. Afzelius A. Verhandlungen der Dermatologischen Gesellschaft zu Stockholm, 28 Oct 1909. Arch. Dermatol. Syph. 1910;101:404. [ Google Scholar]

47. Sonenshine DE, Roe MR, editors. Biology of ticks. Oxford: Oxford University Press; 2014. [ Google Scholar]

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# data[].attributes.attachment_link.title
Microbiological features distinguishing Lyme disease and relapsing fever spirochetes

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The recent proposal of splitting the genus Borrelia into two genera in the newly formed family of Borreliaceae, i. e. Borrelia and Borreliella has motivated us to reflect upon how these organisms has been characterized and differentiated. This article therefore aims to take a closer look on the biology and virulence attributes of the two suggested genera, i. e.

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A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity

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A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity

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Dianne K. Newman, Editor

Dianne K. Newman, California Institute of Technology/HHMI;

ABSTRACT

Plant-derived compounds and other natural substances are a rich potential source of compounds that kill or attenuate pathogens that are resistant to current antibiotics. Medieval societies used a range of these natural substances to treat conditions clearly recognizable to the modern eye as microbial infections, and there has been much debate over the likely efficacy of these treatments. Our interdisciplinary team, comprising researchers from both sciences and humanities, identified and reconstructed a potential remedy for Staphylococcus aureus infection from a 10th century Anglo-Saxon leechbook. The remedy repeatedly killed established S. aureus biofilms in an in vitro model of soft tissue infection and killed methicillin-resistant S. aureus (MRSA) in a mouse chronic wound model. While the remedy contained several ingredients that are individually known to have some antibacterial activity, full efficacy required the combined action of several ingredients, highlighting the scholarship of premodern doctors and the potential of ancient texts as a source of new antimicrobial agents.

IMPORTANCE

While the antibiotic potential of some materials used in historical medicine has been demonstrated, empirical tests of entire remedies are scarce. This is an important omission, because the efficacy of "ancientbiotics" could rely on the combined activity of their various ingredients. This would lead us to underestimate their efficacy and, by extension, the scholarship of premodern doctors. It could also help us to understand why some natural compounds that show antibacterial promise in the laboratory fail to yield positive results in clinical trials. We have reconstructed a 1,000-year-old remedy which kills the bacteria it was designed to treat and have shown that this activity relies on the combined activity of several antimicrobial ingredients. Our results highlight (i) the scholarship and rational methodology of premodern medical professionals and (ii) the untapped potential of premodern remedies for yielding novel therapeutics at a time when new antibiotics are desperately needed.

INTRODUCTION

Antibiotic resistance is a clear and present danger to human health, and there are worryingly few new antibiotics in the developmental pipeline ( 1). A rich source of new antimicrobials potentially resides in medieval and early modern medical texts: microbial infection has been a constant presence throughout human history, and manuscript evidence shows that early modern and premodern societies used a range of natural compounds to treat conditions that are clearly recognizable as microbial infections. Some substances used in premodern treatments for infection (e.g., extracts from garlic [ 2, -5]) show antimicrobial or virulence-reducing effects under certain conditions, but experiments that test the antibiotic activities of entire historical remedies are few and far between. This is an important omission, because the efficacy of these "ancientbiotics" could rely on the combined activity of their various ingredients. This would lead us to underestimate their efficacy and, by extension, the scholarship of premodern doctors. It could also help to explain why the in vitro antibacterial activity of individual compounds (e.g., garlic [ 6]) does not always reliably translate into in vivo antimicrobial potential.

Bald's Leechbook ( 7) (Materials and Methods) is an English medical text from the Anglo-Saxon period. The manuscript was written in the 10th century and contains remedies for various ailments, including clearly recognizable microbial infections. Medieval medicine has generally been dismissed as backwards or superstitious, but recent scholars have suggested that among the remedies there may be methods and recipes that indicate a more factual application ( 8, -10). One of Bald's remedies, a salve for a "wen" or lump in the eye (), is particularly interesting to the modern microbiologist. It aims to treat a condition caused by bacterial infection and contains ingredients with the potential for antimicrobial activity. The recipe instructs the reader to crush garlic and a second Allium species (whose translation into modern English is ambiguous), combine these with wine and oxgall (bovine bile), and leave the mixture to stand in a brass or bronze vessel for 9 days and nights.

The "wen" is most likely a sty: an infection of an eyelash follicle. Today, most styes are caused by the Gram-positive bacterium Staphylococcus aureus ( 11), which also causes other severe and persistent infections of various tissues. The spread of methicillin-resistant S. aureus (MRSA) strains has exacerbated this problem ( 11, 12). MRSA is particularly associated with chronic wound infections, which affect increasing numbers of people and are often highly resistant to treatment ( 11, 13).

The ingredients combined to treat this infection appear promising to the modern microbiologist. Allium species produce a range of antimicrobial compounds ( 3). Ajoene, which is found in garlic ( A. sativum), and allicin, found in garlic, ramsons ( A. ursinum), and spring onions ( A. fistulosum), may prevent biofilm formation by S. aureus and other bacterial species ( 4) and interfere with quorum sensing and virulence in Pseudomonas aeruginosa ( 5). Other antimicrobial compounds produced by Allium species include flavonoids, such as quercetin, kaempferol, and their derivatives, found in leek ( A. ampeloprasum/ A. porrum) and onion ( A. cepa) ( 3), and the antimicrobial peptide Ace-AMP1, so far only characterized in onion ( 14). Bile may also be antibacterial: it is generally thought to limit bacterial overgrowth in the host small intestine ( 15). Wine may act as a source of plant-derived antimicrobial small molecules or simply as a solvent for the extraction of compounds from the plant matter in the recipe. Finally, it is possible that copper salts may leach from the vessel in which the eyesalve is prepared. Copper surfaces prevent bacterial growth ( 16), and host-derived copper plays a role in immune defense ( 17). Interestingly, there is evidence that Allium-derived compounds may act synergistically with other antimicrobial agents, including copper ( 18) and tobramycin ( 2, 5).

We therefore sought to test the effect of Bald's eyesalve on S. aureus and to determine whether any antibacterial activity found could be attributed to a single ingredient or was reliant on combining the ingredients according to the instructions laid down by "Bald."

(Our work was put forward as a poster/oral presentation for the Society for General Microbiology Annual Conference 2015, Birmingham, United Kingdom, 30 March to 2 April 2015.)

RESULTS

Reconstruction of Bald's eyesalve.

We reconstructed four independent batches of the recipe (batches A to D; see Materials and Methods). For each batch, we made two variants of the recipe, one using onion (ES-O) and one using leek (ES-L) for the ambiguous Allium species. We chose these because they both produce antimicrobial flavonoids that are not found in garlic, and onion also produces an antimicrobial peptide ( 3, 19). We also made preparations of the individual ingredients at approximately the same concentrations as they are found in the recipe (batches A and B), versions of the recipe with single ingredients omitted (batch C), and a version of the recipe which was tested before and after the 9-day waiting period (batch D). All of these were prepared in glass bottles, to which we added squares of brass sheet to simulate the brass/bronze vessel. The complete recipes resulted in clear, slightly acidic (pH 4.6 to 4.7) brownish liquids. The full recipes were apparently self-sterilizing: we cultured bacteria from aliquots of the leek and oxgall preparations but not from aliquots of ES-O or ES-L (see Data Set S1 in the supplemental material).

Bald's eyesalve kills planktonic and biofilm cultures of S. aureus.

As shown by the results in , both ES-O and ES-L prevented growth of planktonic cultures of S. aureus growing aerobically at 37°C in synthetic wound fluid (SWF) ( 20). Aliquots of cultures were plated out at the end of the incubation period, and no colonies were observed (detection limit, 30 CFU). Furthermore, 5 µl of each culture was transferred to 1 ml of fresh SWF and incubated overnight at 37°C, but no growth was observed, showing that Bald's eyesalve was bactericidal, rather than simply bacteriostatic, under these conditions.

However, during infection, bacteria often exist as dense, antibiotic-resistant biofilms in a matrix of self-excreted polysaccharides and soft tissue from the host ( 11, 13, 21). We therefore grew S. aureus for 24 h at 37°C in a synthetic model of soft tissue infection comprised of SWF solidified with collagen ( 20). We exposed the resulting mature biofilms to batches A and B of ES-O or ES-L or preparations of the individual ingredients for a further 24 h (). A generalized linear model revealed a significant effect of treatment (χ ² ₇ 1.5e+10, P < 0.001); post hoc Tukey tests showed that only the results for ES-O and ES-L were significantly different from the results for the control treatment ( P < 0.001), causing marked reductions in the numbers of viable cells recovered from synthetic wounds. None of the individual ingredients alone had a significant effect on viable cell counts. There was also a significant effect of batch (χ ² ₁ 4.2e+9, P < 0.001) and a significant interaction between batch and treatment (χ ² ₇ 2.0e+9, P < 0.001); this was because the full recipes of batch B were slightly more bactericidal than those of batch A, and the effects of the individual ingredient preparations differed between batches. The full recipes retained bactericidal activity after 30 days of storage at 4°C (see Fig. S1 in the supplemental material).

Bald's eyesalve is only effective when the recipe is followed carefully.

We then made a third batch (batch C) of ES-O and ES-L, along with variants in which individual ingredients were systematically omitted. These were tested against S. aureus in the synthetic wound biofilm model. A generalized linear model revealed a significant effect of treatment (χ ² ₁₁ 3.1e+9, P < 0.001). We used Dunnett's test to test comparisons that were of interest. First, we contrasted the results of each recipe with those of the control. Both ES-O and ES-L caused a significant reduction in viable cell numbers ( t ₂₁ = 6.38, P < 0.001, and t ₂₁ = 6.25, P < 0.001, respectively); this was comparable to the results for batches A and B (). When onion/leek was dropped from the common recipe base, partial bactericidal activity was still observed ( t ₂₁ = 3.38, P = 0.015). Bactericidal activity was also retained when brass or oxgall was dropped from either ES-O ( t ₂₁ = 6.00, P < 0.001, and t ₂₁ = 3.80, P = 0.005, respectively) or ES-L ( t ₂₁ = 7.08, P < 0.001, and t ₂₁ = 5.38, P < 0.001, respectively). Dropping either garlic or wine from ES-O or ES-L completely removed the bactericidal activity: the viable cell counts were not significantly different from the results for the control ( P ≥ 0.166). We then compared the results for the modified recipes that retained bactericidal activity with the results for their parent recipes. The bactericidal power of the common recipe base when onion/leek was omitted was significantly impaired compared with that of either ES-O or ES-L ( t ₆ = 9.307, P = 0.005, and t ₆ = 18.2, P < 0.001, respectively). Omitting brass from either ES-O or ES-L had no effect on their bactericidal activities ( t ₁₀ = 0.684, P = 0.897, and t ₁₀ = 0.685, P = 0.896, respectively). Omitting oxgall decreased the bactericidal activity of ES-O ( t ₁₀ = 4.66, P = 0.001) but not that of ES-L ( t ₁₀ = 0.710, P = 0.885).

The combination of garlic, wine, and the other Allium species thus appears crucial for the full efficacy of Bald's eyesalve, and when onion is used in place of leek, oxgall is also necessary for full activity. Brass may be dispensable, but it is possible that its presence may facilitate activity against other bacterial species; additionally, using a brass pot may have allowed the Anglo-Saxon physician to start work with an uncontaminated vessel, as the copper content could prevent bacteria colonizing its surface.

Batch D of Bald's eyesalve was used to test whether the 9-day waiting period was important. ES-O and ES-L, with a water control, were tested in the synthetic wound biofilm model (i) immediately after preparation and (ii) after 9 days (). Analysis of variance (ANOVA) revealed significant effects of treatment ( F 2,24 = 110.9, P < 0.001), time (immediate/after 9 days) ( F 1,24 = 410.2, P < 0.001), and their interaction ( F 2,24 = 44.3, P < 0.001). Post hoc Tukey tests showed that while both fresh and 9-day-old ES-O and ES-L killed cells (comparisons versus the respective controls, P ≤ 0.037), the number of viable cells left after treatment with either version of the eyesalve was lower when the eyesalve had been left to stand for 9 days prior to use (both P < 0.001). ES-O and ES-L caused a 2-log drop in viable S. aureus cells if used immediately after preparation but a 4- to 5-log drop if used after 9 days (; consistent with data in and ).

Bald's eyesalve kills MRSA in chronically infected animal tissue.

All the ingredients in Bald's eyesalve are edible, and the likelihood of copper accumulating to levels toxic to mammalian cells seems low. Therefore, we tested its efficacy against S. aureus in a mouse model of chronic wound infection ( 22) using a thoroughly modern pathogen: methicillin-resistant S. aureus (MRSA) ( 12). Mice were administered wounds as previously described ( 22) and infected with MRSA. Four days postinfection, wounds were excised and submerged in ES-O, ES-L (Batch B), sterile saline, or the clinical last-line antibiotic vancomycin (100 µg·ml −1) for 4 h. As shown by the results in , vancomycin did not cause significant reductions in viable bacteria during this relatively very short exposure time (comparison of least-squares means adjusted for the experimental block) ( t ₁₆ = 1.45, P = 0.491). However, ES-O and ES-L caused statistically significant drops in the numbers of viable cells recovered from wounds (approximately 10-fold reductions) ( t ₁₆ = 3.94, P = 0.006, and t ₁₆ = 3.66, P = 0.010, respectively). Batches A and B of ES-O and ES-L were also tested in the mouse wound model after 3 months of storage at 4°C, but without the vancomycin comparison (see Fig. S2 in the supplemental material): in this experiment, ES-L but not ES-O caused a statistically significant drop in the numbers of viable cells recovered from wounds (an approximately 10-fold reduction). Thus, while there is some interbatch variability in activity, Bald's eyesalve shows potential as an antistaphylococcal agent in vivo.

DISCUSSION

In summary, we have reconstituted a 1,000-year-old remedy for bacterial infection and shown that it kills the most common cause of the infection it was designed to treat. Bald's eyesalve eliminates S. aureus in planktonic culture and reduces viable cell numbers by several orders of magnitude in a synthetic model of established biofilm infection. This effect depends upon the combination of several ingredients and upon the 9-day storage period specified in the recipe. Furthermore, a highly antibiotic-resistant strain of S. aureus is susceptible to Bald's eyesalve when infected tissue from a mouse chronic wound model is exposed to the recipe. In this model, significant bactericidal activity was observed after a brief period of exposure during which the current last-line clinical antibiotic (vancomycin) did not kill significant numbers of bacteria. Our findings contrast with those of a previous attempt to test Bald's eyesalve by Brennessel et al. ( 23), who found it ineffective against S. aureus in disk diffusion assays; however, these authors do not specify the methods of preparation and do not give quantitative results or details of replication, so we do not know exactly how their tests were conducted.

Future work will explore the mechanism(s) by which Bald's eyesalve kills S. aureus. We note that several of the ingredients may damage biological membranes. Bile salts act as surfactants ( 15), the Ace-AMP1 antimicrobial peptide present in onion is thought to have lipid-binding activity ( 19), and saponins, present in a wide variety of Allium spp., can bind the sterols present in biological membranes ( 3). Other antimicrobial compounds present in Allium spp. have been shown to act as enzyme inhibitors (allicin and flavonoids) ( 3). Wine may be a source of further plant-derived antimicrobial small molecules, or it may simply act as a solvent for the extraction of compounds from the other ingredients in the recipe, with the 9-day waiting period providing time for molecule extraction.

It is interesting that copper was entirely dispensable for antistaphylococcal activity in our synthetic wound model. Copper has been shown to have broad-spectrum antibacterial activity ( 16, 17), and we would expect copper salts to accumulate in our preparations: we observed copper compounds forming on the surface of the brass squares used to simulate the preparation vessel, and Brennessel et al. (who used an actual brass pot), found that copper salts accumulated to high levels in their version of Bald's eyesalve (M. Drout, personal communication). It is possible that we simply did not have a large enough copper surface to allow copper to accumulate to biologically active levels in our recipe. Alternatively, the copper vessel itself may have allowed the Anglo-Saxon physician to begin work with an uncontaminated pot due to the resistance of copper surfaces to bacterial colonization. A third possibility is that copper is entirely dispensable: copper alloy bowls of the Anglo-Saxon period can be highly decorated (even inlaid with silver) and appear as grave goods in high status burials as a marker of feasting power ( 24), and perhaps the more humble vessels may have been a marker of the physician's professional status.

The combined activity of molecules derived from the different ingredients might explain why the activity of Bald's eyesalve is greater than the sum of its parts: the mixture may attack the bacteria on several fronts. This is an important consideration for future work on natural antimicrobial compounds, as strong in vitro activity of individual compounds has not always translated into positive in vivo results ( 6). Alternatively, novel compounds may be formed as molecules from different ingredients react over the 9-day storage period specified by the recipe. Future research into antibiotics derived from natural materials would benefit from considering potential combinatorial activity of different ingredients, and premodern medical texts provide an excellent starting point for this strategy. Such work will require extensive collaborative research involving historians, linguists, microbiologists, and analytical/medicinal chemists.

When we describe Bald's eyesalve as being "designed" to treat eye infection, we do not use the term lightly. There has been considerable debate about the levels of scholarship and scientific method among early medieval medical practitioners ( 8, 9, 25). This may be partly because of an almost complete absence of theoretical material, and the fact that the surviving corpus of Anglo-Saxon medicine seems to be compilations of excerpts and treatises ( 26). Furthermore, many recipes-such as the one that requires having a virgin get water from an eastward-flowing spring for a cyst "which pains the heart," from the mid-11th century Lacnunga-look rather dubious as remedies. Medicine, as some scholars have claimed, was a craft and not a science in early medieval Europe ( 26), and there was a lack of explanatory frameworks. Texts like Bald's Leechbook were most likely composed in a monastic environment, and in many text sources, there is a conflation of spiritual and practical healing. More credence has been given to later medieval texts, associated with the School of Salerno and the growth of university-based training ( 27).

However, our finding that the combination of ingredients used is crucial for bactericidal activity supports the hypothesis that this "ancientbiotic" was systematically constructed based on empirical knowledge. The fact that Anglo-Saxon recipes do not state detailed amounts of each component requires the practitioners to have had some knowledge about how much of each ingredient to use. It is also notable that numerous "alternative" recipes are often given for a condition-indicating that a trained physician could adapt treatments when necessary. If medieval physicians really did use observation and experience to design effective antimicrobial medicines, then this predates the generally accepted date for the adoption of a rational scientific method (the formation of the Royal Society in the mid-17th century) and the modern age of antibacterial medicine (Lister's use of carbolic acid in the late 19th century) by several hundred years.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

We used a standard laboratory wild-type strain of S. aureus (Newman) for the initial in vitro tests of antibacterial activity. For the mouse chronic wound model, we used S. aureus Mu50 (ATCC 700699). Synthetic wound fluid (50% [wt/vol] fetal bovine serum, 50% peptone water) ( 20) was used as the growth medium for all experiments. All cultures were grown aerobically at 37°C.

Bald's eyesalve.

The recipe () calls for equal amounts (" begea emfela") of the Allium species and equal amounts of wine and oxgall, but it is not clear whether these two pairs of quantities are equal to each other, nor whether ingredients were measured by weight or volume. We decided to combine equal amounts by volume of all of the recipe ingredients. For the complete eyesalve recipes, 25 ml peeled, finely chopped garlic bulb was mixed with either 25 ml peeled, finely chopped yellow onion or 25 ml finely chopped leek (leaves), and these ingredients were crushed with a mortar and pestle for 2 min (the recipe asks for it to be pounded well: " gecnuwe wel tosomne"). Vegetables were purchased fresh from a greengrocer. Twenty-five milliliters of wine (Pennard Organic Wines) was added to the crushed alliums; we used an organic English white wine produced by a vineyard situated 7 miles from the village of Panborough in Somerset: a charter of ad 956 tells us that the Anglo-Saxon monastery of St. Mary's Abbey in Glastonbury held land rights to a vineyard at Panborough ( 28). We then added 25 ml bovine bile salts (Fluka/Sigma Aldrich) dissolved in water at a concentration of 87 mg·ml −1; this is the natural concentration of bile salts in the bovine gallbladder (source, Sigma Aldrich technical support). The mixture of alliums, wine, and oxgall was placed in a sterile 250-ml Duran bottle and, following the reasoning of Brennessel et al. ( 23), nine clean and sterile 15-mm squares of 0.51-mm 260 brass sheet (Hypertriton, Inc., Canada) were added to the bottle to simulate the copper alloy vessel (" arfæt "). The lid was closed and the bottle wrapped in foil (as the original vessel would not have been transparent and we did not know if any ingredients were light sensitive). The recipes, denoted ES-O for the version containing onion and ES-L for the version containing leek, were then left refrigerated at 4°C for 9 days. While the Anglo-Saxons did not have refrigerators, neither did they have centrally heated buildings: we reasoned that the daily fluctuations in ambient temperature in the laboratory caused by heating during working hours were probably less realistic than a constant cool temperature.

To test the antibacterial effects of the individual ingredients of the recipe, when we made batches A and B, we also combined 6 ml of each ingredient (the vegetables were finely chopped and crushed as described above) with 17 ml of distilled water in a foil-wrapped glass bottle containing two 15-mm squares of the brass sheet and left at 4°C for 9 days. This is an approximate scaling down of the recipe given the constraints imposed by measuring chopped vegetables accurately and by requiring a whole number of brass squares. To provide a control treatment, 23 ml distilled water and two 15-mm squares of the brass sheet were left at 4°C for 9 days.

We repeated the preparation of full recipes and individual ingredients twice, using fresh ingredients each time, to produce two independent batches of the eyesalve or individual ingredient preparations (batches A and B). We later repeated the preparation a third time (batch C), making complete ES-O and ES-L recipes plus versions of the recipes that were each missing one ingredient, using the scaled-down, 24-ml total volume that was used for the individual ingredient preparations for batches A and B.

The recipe calls for the mixture to be strained and "purified well" (" hlyttre wel ") before use or further storage. We debated how to simulate this and whether we should sterilize the mixtures. We decided that any microbes present in the ingredients are part of the "natural" state of the medicine and would not have been removed by filtering through cloth (probably linen). Therefore, we briefly centrifuged each recipe/ingredient to pellet the solid material and filtered the remaining liquid through a filter small enough to remove solid particles of vegetable matter but large enough to let bacterial cells through (5 µm). Because the oxgall does not fully dissolve in the mixture, the finished eyesalve contains a fine white precipitate (especially noticeable when taken directly from cold storage). All versions of the eyesalve or its ingredients were thus briefly vortexed before use to ensure a homogenous suspension.

Batch D of the complete recipes was made as described above but in two subbatches from the same ingredients. One subbatch was made without brass, shaken for 10 s, and then filtered and used in experiments immediately. Brass was added to the other subbatch, and this was stored for 9 days at 4°C, following the original recipe.

Growth and testing of planktonic cultures in synthetic wound fluid.

Cultures of S. aureus were grown for 6 h (mid-log phase) in SWF at 37°C on an orbital shaker, and 200-µl aliquots of this culture (containing 10 ⁴ to 10 ⁵ cells) were transferred to 12 wells of a 96-well microtiter plate. One hundred microliters of sterile distilled water, ES-O, or ES-L (Batch A only) was added to four wells each. Cultures were incubated at 37°C in a microplate spectrophotometer (Tecan Infinite) for 18 h with periodic shaking; every 15 min, the cultures were shaken for a few seconds and the optical density at 600 nm (OD ₆₀₀) of each well was measured. Because the eyesalve recipes were colored, we standardized the OD readings by subtracting the OD for each well at time zero.

Culturing and treatment of synthetic wound infections.

S. aureus was streaked onto LB agar and incubated aerobically at 37°C for 24 h. Multiple colonies were pooled to inoculate starter cultures in SWF. These were incubated aerobically at 37°C on an orbital shaker for 6 h. The cultures were then diluted to an OD ₆₀₀ of 0.05 in SWF, and 100-µl amounts added to wells of 24-well cell culture plates containing synthetic wound medium. Synthetic wounds comprised 400 µl polymerized collagen (2 mg·ml −1 collagen, 0.01% acetic acid, 60% [vol/vol] SWF, 0.01 M sodium hydroxide). For experiments on batches A and B of the eyesalve, additional synthetic wounds were mock inoculated with 100 µl sterile distilled water instead of with S. aureus to reveal any growth of bacteria resident in the eyesalve or its component ingredients. Synthetic wounds were incubated aerobically at 37°C for 24 h to allow the establishment of bacterial biofilms within the collagen matrix.

Batches A and B of the full recipes and individual ingredient preparations were tested as follows. For S. aureus-infected wounds and the no-infection control, five replica synthetic wounds were overlaid with 200 µl of each complete recipe or individual ingredients. A further five infected and noninfected synthetic wounds were overlaid with 200 µl sterile distilled water to provide mock-treated controls. Treated synthetic wounds were then incubated aerobically at 37°C for 24 h. The collagen was then depolymerized by treating each wound with 500 µl 0.5 mg·ml −1 collagenase. An aliquot of each wound was serially diluted and plated on LB agar to enumerate the number of viable cells present.

For batch C (full recipes and recipes missing a single ingredient), this experiment was repeated but each preparation was tested in triplicate. For batch D (full recipes before and after 9-day storage), each preparation was tested in five replica wounds. For batches C and D, only a single no-infection control wound was used for each experiment; this was visually inspected to confirm the absence of contaminating bacterial growth and not analyzed further.

Mouse chronic wound infections.

Adult female Swiss-Webster mice were administered wounds as previously described ( 22) and infected with approximately 10 ⁵ CFU S. aureus Mu50. At 4 days postinfection, mice were euthanized and their wound tissue was extracted and cut into 3 equal pieces, which were weighed. Wound sections were then submerged in 300 µl sterile saline, ES-O, or ES-L for 4 h, after which they were rinsed in sterile saline and homogenized. Wound homogenates were then serially diluted and plated on Mueller-Hinton agar with 4% NaCl and 6 µg/ml oxacillin (MRSA screen agar) to determine CFU/g tissue. This experiment was carried out twice on separate days, and data from the two blocks combined in one analysis. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center (protocol number 07044), and an NC3Rs ARRIVE checklist ( 29; see also www.nc3rs.org.uk) is available for this work.

Statistical analyses.

All data were analyzed with R (version 2.14.0; R Foundation for Statistical Computing, http://www.R-project.org). Different models and their residuals were explored to find the most appropriate analysis for each data set. The data sets for S. aureus growth in synthetic wounds treated with batches A to C of the recipes/ingredients were highly skewed; therefore, these data sets were analyzed using generalized linear models with Poisson error. ANOVAs of log-transformed data were used to analyze (i) the 30-day retest of batches A and B in synthetic wound infections and (ii) the test for an effect of the 9-day waiting period using batch D. In all analyses where both batches A and B were used, we tested for effects of treatment, batch, and their interaction. Post hoc comparisons were conducted using the multcomp package ( 30). For the S. aureus synthetic wound data for batch C, one replica wound treated with eyesalve minus onion/leek had to be dropped from the data set, as it completely dried out during incubation. Data on MRSA viable cell counts in mouse chronic wounds were not suitable for analysis using general linear models and so were analyzed by fitting least-squares means for each treatment adjusted for the effect of the experimental block; Tukey tests were then used to test pairwise differences between treatments, with P values adjusted for multiple comparisons. The growth curves of the S. aureus reporter strains were compared using the compare GrowthCurves function of the statmod package (version 1.4.21; http://CRAN.R-project.org/package=statmod). For each strain, growth curves from different treatments were compared using 1,000 permutations of the data and the Holm-Bonferroni correction for multiple comparisons.

Experimental data and R code have been deposited with Dryad.org (doi: 10.5061/dryad.mn17p).

SUPPLEMENTAL MATERIAL

Data Set S1

Bacterial species cultured from recipes and ingredient preparations. Download

Figure S1

Bald's eyesalve retains antistaphylococcal activity for 30 days of storage. The onion (ES-O) and leek (ES-L) variants of the recipe (batches A and B) retained bactericidal activity against S. aureus biofilms in synthetic wounds after 30 days of storage (ANOVA, treatment, F 2,24 = 217, P < 0.01; batch, F 1,24 = 0.16, P = 0.693; and interaction, F 2,24 = 0.196, P = 0.823). Download

Figure S2

Bald's eyesalve kills MRSA in a mouse chronic wound infection. Adult female Swiss-Webster mice were administered wounds and infected with ca. 10 ⁵ CFU S. aureus Mu50. Four days postinfection, mice were euthanized. Wound tissue was extracted and cut into three equal pieces, which were weighed, submerged in 300 µl sterile saline, ES-O, or ES-L (batches A and B, after 3 months of storage at 4°C) for 4 h, and then rinsed in sterile saline and homogenized. Viable bacteria were enumerated by plating. ES-L but not ES-O killed MRSA in wounds (ANOVA, F 2,12 = 6.17, P = 0.014; post hoc Tukey test versus the control, ES-O, P = 0.069, and ES-L, P = 0.014). There was no effect of eyesalve batch (ANOVA, batch, F 1,12 = 0.443, P = 0.518; interaction, F 2,12 = 0.583, P = 0.573). The graph shows the means of data from six mice per treatment (three for each batch of the eyesalve). Download

ACKNOWLEDGMENTS

S.P.D., F.H., C.L., and A.E.L.R. conceived the study; C.L. and F.H. identified the recipe; A.E.L.R., F.H., K.P.R., and R.G. conducted the experimental work; F.H. analyzed the data; and all authors contributed to manuscript preparation.

This work was funded by the University of Nottingham Interdisciplinary Centre for Analytical Science (sandpit grant to F.H., A.E.L.R., C.L., and S.P.D.), the Natural Environment Research Council (NE/J007064/1 to S.P.D.), the Human Science Frontier Programme (RGY0081/2012 to S.P.D.), and the University of Nottingham Institute for Medieval Research and School of English Research Committee (C.L.).

We thank the two anonymous reviewers. The development of the work benefitted from discussions with Conor Kostick, Steve Atkinson, Erin Connelly, Laura Piddock, James Gurney, Jason Millington, Mike Drout, and Marvin Whiteley. We also thank Emma Rayner for sourcing the manuscript facsimile used in , and the British Library for permission to reproduce it.

The authors declare no conflicts of interest.

Footnotes

Citation Harrison F, Roberts AEL, Gabrilska R, Rumbaugh KP, Lee C, Diggle SP. 2015. A 1,000-year-old antimicrobial remedy with antistaphylococcal activity. mBio 6(4):e01129-15. doi:10.1128/mBio.01129-15.

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A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity

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Dianne K. Newman, Editor Dianne K. Newman, California Institute of Technology/HHMI; Plant-derived compounds and other natural substances are a rich potential source of compounds that kill or attenuate pathogens that are resistant to current antibiotics.