Streptomyces isolates from the soil of an ancient irish cure site, capable of inhibiting multi-resistant bacteria and yeasts

isolates the of an ancient irish cure site, capable inhibiting multi-resistant Abstract: Traditional Irish medicines are often intertwined with ritual and spirituality, making it difﬁcult to substantiate the validity of their claims. In this manuscript, we use molecular and microscopic techniques to investigate some microorganisms that might be responsible for the reputed healing properties of an ancient Irish soil cure known as the Blessed clay from a site in Boho in the West Fermanagh Scarplands. We previously reported the isolation of an antibiotic producing bacteria from this soil. In this report, we characterize the antibiotic activity of a further six isolates of Streptomyces from this source. Two of these isolates inhibit the growth of multi-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa , two inhibit the growth of the yeast Starmerella bombicola , and two have as yet undetermined activity. Genetic analysis of these Streptomyces reveals the potential to synthesize varieties of antibiotics similar to cypemycin, griseochelin, macrolactams, and candicidin. From these observations, we suggest that part of the medicinal reputation of the Blessed clay may lie in the diversity of antimicrobial producing Streptomyces isolated from this soil. These ﬁndings highlight the potential for antibiotic discovery in this area.


Introduction
The record of traditional Irish folk medicine, essentially passed on by word of mouth, is fading from the living landscape. The principal reasons for this are the advancing age of the population, the sharp decline in the use of traditional medicine, and of course, the reliability of modern medicines. However, in a reversal of fortune, researchers are now looking towards traditional medicines as a potential source of new pharmaceutical compounds [1][2][3][4][5].This necessity is partly due to a reduction in the supply of new antibiotics caused by years of under-investment [6,7]. Indeed, the World Health Organization (WHO), in their Global Pathogen Priority (GPP) catalogue, identified a group of multiresistant pathogens, including methicillin resistant Staphylococcus aureus and multi resistant Pseudomonas aeruginosa, as priority targets for the research and development of new antibiotics [8]. These challenges have prompted researchers to pursue more innovative methods of drug discovery. One of these approaches, pioneered by Geoffrey Cordell, was the analysis of traditional folk-medicine. Geoffrey discovered that traditional medicines yielded far more successful lead compounds in the development of anticancer drugs than random plant screenings [9]. These ideas were quickly adopted by other researchers. Pioneering work by the group of Julian Davies in British Columbia discovered that the sacred clay, used for millennia by the Heiltsuk peoples of Kisameet Bay, was able to combat many multi-resistant hospital pathogens [3]. In another part of the globe, researchers in Jordan attributed the therapeutic activity of 'Red clay' to a high proportion of antibiotic producing bacteria found in the soil [10]. Researchers have discovered that antibiotic producing organisms could also have a close relationship with plants. This was the case for Streptomyces sp. strain Y3111, which lives in association with the traditional Chinese medicinal plant Heracleum souliei and produces anti-tubercular (anti-BCG) compounds named heraclemycins [1]. Some researchers have even reconstituted ancient medicines from old texts that have been proven to be effective against antibiotic resistant organisms such as methicillin resistant Staphylococcus aureus (MRSA) [2]. The common theme running through many of these discoveries has been the presence of Streptomyces. Together with other closely related genera, these organisms produce as much as 50% of the world's current antibiotics, as well as anticancer, antiparasitic, and antifungal compounds [11].
The West Fermanagh Scarplands are a remote range of limestone hills on the border between N. Ireland and Eire. This area has a long tradition of folk cures associated with rocks, soils, and wells, some of which are named after the diseases they cure, such as jaundice, scurvy, and ague. One of the traditional medicines from this area concerns the soil that covers the grave of a cleric known as Fr McGirr. Fr McGirr belonged to a group of priests who emerged at the end of the penal laws in Ireland. Many of these people were familiar with both spirituality and local folk healing practices [12]. In his later years' Fr McGirr stated that the soil that covered him would be able to cure the same diseases as it did when he was alive. This soil or 'Blessed clay', as it is described by locals, is wrapped in cloth and is given for ailments as diverse as toothache or tuberculosis. In our previous research, we isolated one species of Streptomyces from this soil that inhibited several antibiotic resistant pathogens [13]. Our current investigation involves the isolation and characterization of other Streptomycetes isolated from this soil. We also used the antibiotic prediction software antiSMASH [14,15], in combination with in vitro antimicrobial analysis, to optimize the potential antimicrobial identification from the genomes of these isolates.
Streptomyces isolates McG2 up to McG7 were also isolated from the same soil sample, as described above. One gram of this soil sample was diluted in 1 mL sterile water, vortexed, and cultured on International Streptomyces Project (ISP) 2 agar (1/5th strength) and starch agar (Oxoid, Hampshire, UK), as previously described [13]. Streptomyces isolates were characterized by observing their distinctive colony morphology (small, embedded colonies with powdery surfaces) on ISP2 media. These species were further identified using whole genome sequencing. Stocks of these Streptomyces were frozen at -80 • C in 18% glycerol after their initial isolation.

Antimicrobial Tests
Antimicrobial tests followed a modified agar overlay method [16]. Briefly, an agar plug/core of Streptomyces that had grown on soy flour mannitol (SFM) agar for 9-15 days was placed within wells made in 15 mL of 1.5% agar. This was overlaid with Muller-Hinton agar containing pre-diluted test organism at 42 • C in the case of P. aeruginosa, tryptic soy agar (TSB) in the case of S. aureus, and Sabouraud dextrose agar in the case of S. bombicola. In cases where the overlay methods were not consistent, a sterile swab was used to apply a dilution of 1 × 10 6 cfu/mL of test organism. Zones of inhibition in the confluent growth of test pathogens was deemed to indicate antimicrobial activity. Inhibition zones less than 1mm from agar core were recorded as resistant.
Antibiograms followed the Kirby-Bauer protocol [17]. Organisms were grown on Muller-Hinton agar and tested against a variety of antibiotic discs, which were placed on top of the agar. Agar plates were incubated overnight at 37 • C. The presence of a zone of inhibition greater than 1 mm from the edge of the disk indicated inhibition. The suspension was centrifuged, and the supernatant was transferred to 2 mL Eppendorf tubes. Next, 500 µL of ATL lysis buffer and 80 µL of proteinase were added and incubated for 10 min at 70 • C. A further 16 µL RNase and 500 µL were then added to the buffer and incubated at 70 • C for 7 min. A further 600 µL of ethanol was then added. The suspension was transferred to the mini spin column and centrifuged at 6010× g for 1 min. This step was repeated until the whole suspension had passed through. The column was washed twice, and the DNA was eluted in 50 µL of elution buffer. Extracted DNA was quantified with a Nano Drop spectrophotometer.
Genomic DNA libraries were prepared using Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA). The manufacturers protocol was changed slightly by adding two nanograms of DNA instead of one as an input, and the PCR elongation time was changed from 30 s to one minute. DNA quantification and library preparation were performed on a Hamilton Microlab STAR automated liquid handling system. Pooled libraries were quantified using the Kapa Biosystems Library Quantification Kit for Illumina on a Roche light cycler 96 qPCR machine. Libraries were sequenced on the Illumina HiSeq using a 250 bp paired end protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cut off of Q15 [18]. De novo assembly was performed on samples using SPAdes version 3.7 [19], and contigs were annotated using Prokka 1.11 [20].

Taxonomic Position of Streptomyces Isolates Using a Maximum-Likelihood Phylogeny
A multi-locus phylogeny was reconstructed by the method reported previously [13]. Briefly, whole genome sequences (WGS) for all Streptomyces were retrieved autonomously from GenBank. Genomes were annotated in PROKKA [20], and the resulting translated coding domain sequences were used to generate a multi-locus, concatenated alignment of 400 proteins in PhyloPhlan [21]. Proteins used for the alignment were those shown to be conserved among bacterial genera [21]. A maximum-likelihood phylogeny was reconstructed in FastTree and rendered in iTOL [22,23]. The robustness of the phylogeny was assessed using 1000 bootstrap replications. The phylogeny was reconstructed with 921 Streptomyces genomes (including McG designated isolates). The phylogeny was outgroup rooted along the lineage leading to Frankia (five species). The full list of species in the phylogeny can be found in Supplementary Data (File S1). Genome-to-genome distance calculations were performed in GGDC web server (http://ggdc.dsmz.de/ server accessed on 22 May 2021) using formula 3 to account for the use of draft genomes.

Secondary Metabolite Analysis
Gene clusters known to be involved in secondary metabolite biosynthesis, selfimmunity, or resistance were identified using Antibiotics and Secondary Metabolite Analysis Shell (anti-SMASH) version 4.0.0 [14]. The relaxed metabolite matching parameter in antiSMASH was chosen for comparison of the Streptomyces genomes. This detects welldefined clusters containing all required parts of a biosynthetic gene cluster, as well as partial clusters missing one or more functional parts. The GenBank sequence files (from Prokka annotation) were submitted to the web interface selecting all extra features of annotation.

Microbial Characterization
We isolated six Streptomyces from the 'Blessed clay' of Fr McGirr, which we labelled as Streptomyces spp. McG2 to McG8 (herein referred to as McG2 to McG8). These were identified from colony morphology, microscopy, and whole genome sequencing. We have previously characterized Streptomyces sp. myrophorea isolate McG1 (NCTC 14177) [13].
Streptomyces spp. McG2 to McG8 grew well on ISP2, PDA, and starch agar. All isolates were characterized by small, embedded colonies with powdery surfaces that emitted earthy odors. All of these Streptomyces isolates had a high salt (NaCl) tolerance (Table 1). Microscopically, all Streptomyces isolates were Gram-positive, filamentous, with branching and aerial hyphae that produced spores.

Antimicrobial Activities of the Isolated Streptomyces Strains
The antimicrobial activity of the Streptomyces isolates was tested against methicillin resistant Staphylococcus aureus (MRSA) (ATCC 43300), Pseudomonas aeruginosa (PA 01), and the yeast Starmerella bombicola on agar plates using a modified version of the Lehrer assay ( Figure 1 and Table 2). The antibiotic resistance profiles of the test organisms are shown in Table 3.

Genome Assembly of Streptomyces Isolates from the Boho Clay
Streptomyces genomes were assembled de-novo by MicrobesNG (Birmingham University, Birmingham, UK). The k-mer spectrums of contigs larger than 2000 bp were calculated based on an approach described previously [24]. For four of the isolates, sets of low coverage contigs with k-mer spectrums significantly different from the five largest contigs in the assembly were identified as probable contaminants. All small contigs of less than 500 bp were removed from the assemblies (Table 4).

Prediction of Antibiotic Gene Cluster Similarities
The potential of the Streptomyces isolates to produce antibiotics and other secondary metabolites was predicted using antiSMASH (version 4), which compared our unknown gene sequences with previously documented antibiotic/secondary metabolite biosynthetic gene clusters (BGC). These BGCs contain the genes necessary to produce secondary metabolites, including enzymes and pathway-specific regulatory genes.

Streptomyces Isolate Phylogeny
The relationships between the Streptomyces isolates from the Boho clay were calculated using the maximum-likelihood of relatedness. This placed isolates McG2, McG5, and McG6 alongside the S. albidoflavus clade, suggesting that these were likely variants (Figure 3a).  The closest species to Streptomyces sp. McG7 and McG8 was Streptomyces sp. FxanaD5 (Figure 3c). These new isolates were added to the data set of [5,13]. The full phylogeny is available from the authors on request.

Discussion
There has been an increasing interest in the potential of traditional medicines to provide new medicines over the past few decades [3]. However, many of the older tradition medicines are hard to decipher because of their complicated relationship with rituals and mythology. Using a series of microbiological methods, we isolated a group of Streptomyces from the Blessed clay of Fr McGirr in the West Fermanagh Scarplands. Several of these isolates inhibited the growth of multi-resistant pathogens and yeasts. These add to the growing list of Streptomyces sp. isolated from traditional medicines that are able to produce antibiotics against fungi and bacteria [39]. We were also able to predict the potential synthesis of a variety of antimicrobial substances from these isolates. We think that these discoveries may provide a possible explanation as to why this soil has historically been used as a folk medicine. There is also the possibility that other types of bacteria could also be associated with the reputed antimicrobial activity of this soil; however, in this study we concentrated solely on Streptomyces. Additionally, the activity of the Blessed clay could equally be based on some type of clay mineral, such as Montmorillonite [40,41] or sulfurand iron-reducing bacteria [42]. However, the Blessed clay in this study has a silty-loam consistency rather than clay-like, similar to the rest of the soil found in this area.
Given the continuous spiritual significance of this site, we assume many of the folk medicines found in this area are derived from much older cultures. Since this particular traditional soil medicine was passed down by word of mouth, we are still uncertain of the conditions under which it could yield its full therapeutic potential. Therefore, even though we managed to induce the production of antibiotics from Streptomyces isolated from this soil under laboratory conditions, this might still not be the complete repertoire of its activity.
We are aware that limestone areas similar to Boho have long been regarded as a rich source of antibiotic producing Streptomyces [43][44][45] In vitro testing showed that only Streptomyces isolates McG1, McG2, and McG3 had any inhibitory effect on multi-resistant bacteria. The identities of the compounds responsible for this activity were difficult to discern from the antiSMASH predictions since there were several biosynthetic gene clusters identified for each isolate. Streptomyces isolates McG1 and McG3 encoded antibiotic synthesis clusters similar to Streptomyces griseus polytetramic macrolactam (SGR PTM), an antifungal and antioxidant [36]. Streptomyces isolates McG1 and McG3 also encoded gene synthesis clusters similar to those of griseochelin (zincophorin), a carboxylic acid antibiotic that is active against Gram-positive bacteria and viruses. Streptomyces isolates McG1 and McG5 also have gene synthesis clusters for cypemycin, a Gram-positive antibiotic that also has activity against leukemia [34]. Finally, a gene cluster from Streptomyces isolates McG7 and McG8 showed similarity to those previously identified for the synthesis of cyslabdan, an antimicrobial potentiator [33]. Several researchers have also shown that some species of Streptomyces that only produce low levels of antibiotic when cultivated alone are able to induce other species to express new antibiotics [47]. This may be an interesting direction to follow in future research.
Altogether, the Streptomyces species isolated here are effective against multi-resistant bacteria and fungi. It may be possible that this spectrum might extend to anticancer and antiviral activity, but this is still the subject of ongoing research. This new source of Streptomyces strains may be a useful resource in helping to replenishing the ever-decreasing range of antibiotics capable of combating multi-resistant microbial pathogens.

Conclusions
We identified a group of Streptomyces from the Blessed clay in the Boho region of West Fermanagh. We have shown through genome analysis and by in vitro tests that some of these Streptomyces have the ability to produce antimicrobial compounds that inhibit the growth of clinically significant multi-resistant pathogens in vitro. We do not know how to activate this soil in situ, but we feel that the abundance of Streptomyces discovered in the Boho clay might be responsible for its reputed healing abilities. We think that these findings might provide a basis for future antibiotic discovery.