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Predatory Profile of Myxobacteria Against Clinically Relevant OrganismsPaul Livingstone, Russell Morphew, David Whitworth - IBERS Aberystwyth University

Introduction

Abuse and overt use of antibiotics have resulted in multidrug resistance among pathogens which has troubled the medical world today. A renewed interest in novel antimicrobial agents has emerged within the research community which manifests from chemically modifying existing antibiotics to isolating natural compounds from potential producer organisms. Myxobacteria are predatory soil microbes studied extensively for their potential to produce natural products which can kill bacteria, fungi, viruses and parasites. Pharmaceutical researchers have isolated hundreds of myxobacterial secondary metabolites with potent antimicrobial properties, however very few enter even into pre-clinical trials. This is attributed to the technical difficulties in propagation of these organisms, the unstable nature of the isolated compounds and more importantly, lack of knowledge on their mechanisms of action. Therefore we employed a holistic approach of studying the predatory activity of 113 myxobacteria isolated from soil samples, against 10 clinically relevant organisms, to analyse their prey range.

MethodsSoil Sampling:Soil samples from various habitats including woodlands, gardens, farmlands, streams and open fields were collected from the Aberystwyth and Carmarthen areas in West Wales. E.coli bait and filter paper methods of isolation were employed. Suspicious growth of myxobacteria typically seen as swarming growth or fruiting bodies (Fig.1) was purified for further identification and predation assays.

16SrRNA Sequencing and Analysis:16S rRNA sequencing was performed using F27 and R1389 primers and the PCR products were sequenced. The assembled contigs were identified using the EzTaxon database and phylogenetic trees were constructed using MEGA 7 (Fig.5).

Predation Assay:A lawn culture method was employed in this assay. 10 prey organisms (Fig. 3) were grown in Luria Bertani (LB) broth and centrifuged. The washed pellet was spread onto a WAT (non nutrient water agar) agar plate to form a uniform lawn. Myxobacterialisolates were grown in AMB broth and centrifuged. 10μl of the cell pellet was spotted onto the prey lawn and incubated. The diameter of the zone of swarming was recorded on day 4 as a measure of predatory activity (Fig2). Predatory activity data for the 10 prey organisms were clustered using the hierarchal clustering method in R (Fig.6).

CA046D AB054A CA038 AB038B AB031 AB032C AB052 CA039B AB033 CA043A CA049B AB059A AB055A AB019 CA047A CA048 AB045 AB051 AB002 AB046 AB038C AB030 AB058 AB016 CA054B AB053A CA031A CA044C AB047A AB047C AB035B CA049A AB039B AB059B AB039A CA037B CA041A AB017 AB037 CA037A AB032A AB012 AB038A CA044A CA043C AB032B AB036B AB009 AB015 AB018 Corallococcus_coralloides_strain_DSM_2259 AB050B AB004 AB049A CA046A AB043B CA046C AB043A CA043D AB049B AB035A AB050A CA046B AB044 CA041B AB028 AB007 Corallococcus_exiguus_strain_91 CA047B CA031B CA031C CA054A AB047B AB048 CA052A CA051B CA040B CA034 CA059A CA043B CA053C

Corallococcus spp

AB022 Myxococcus_fulvus_strain_NBRC_100333 M. fulvus Myxococcus_stipitatus_DSM_14675 Pyxidicoccus_fallax_strain_DSM_14698 CA053A CA060B CA059B CA060A CA032A

Pyxidicoccus spp

CA040A CA056 CA051A CA033 CA039A CA053B AB053B AB055B

M.macrosporus

Myxococcus_macrosporus_strain_125-10-1 CA023 CA029 CA024 CA006 CA026 CA028 AB056 AB023 CA021 CA025 AB036A AB024B M.xanthus_DK1622 Myxococcus_virescens_strain_DSM_2260 CA027 AB024A AB025A CA005 AB025B CA010 CA018

M. xanthus / virescens

AB050C Sorangium_cellulosum_strain_0087-7-1 Sorangium spp Desulfovibrio_desulfuricans_(NR_036778)

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Fig.5. 16S rRNA based Phylogenetic Tree showing 6 clusters Fig.6. Predation Tree exhibiting good, moderate and poor groups

Fig.1. Colonies (swarming growth and fruiting bodies) of Myxobacteria

Fig.2. Myxobacteria showing a zone of predation on a prey lawn

Fig.3. Box and whisker plots illustrating the predatory activity of Myxobacteria for the 10 prey organisms

Results and Discussion

Novel Welsh Isolates113 myxobacterial strains were isolated from 77 soil samples from Carmarthen and

Aberystwyth areas. Corallococcus spp. were predominant (70%) followed by Myxococcus spp. (24%), Pixicoccus spp. (5%) and Sorangium spp. (1%).

Phylogenetic ClustersMyxobacterial strains were grouped into 6 distinct phylogenetic clusters (Fig. 5)

which were in accordance with EZTaxon assignments.

Broad Range of Predatory ActivitiesCluster analysis (Fig. 6) grouped the isolates into 3 broad groups – good, moderate

and poor predators. K. pneumoniae, E. coli and P. mirabilis were the best prey organisms while P. aeruginosa, S. aureus, S. epidermidis and S. saprophyticus were poor preys (Fig. 4).

Relationship Between Phylogeny and PredationThere was only partial congruence between phylogeny and predation from both the

predators’ and prey’s perspective. This suggests that predation is mechanistically influenced by transferable genetic factors than a constitutive trait.

Conclusion

The novel isolates exhibited a broad predatory activity against clinically relevant organisms

which can be explored for antibiotic discovery. Studying the predatory range of these

novel organisms will also open doors in exploiting alternative therapeutic options against

pathogenic organisms using the live organisms. Also, this study paves the way for a better

understanding of predatory mechanisms, through genome wides studies which we are

pursuing at the moment.

References1. Dawid W (2000) Biology and global distribution of myxobacteria in soils. FEMSMicrobiol Rev. 24: 403-27.2. Evans AG, Davey HM, Cookson A, Currinn H, Cooke-Fox G, Stanczyk PJ, Whitworth DE(2012) Predatory activity of Myxococcus xanthusouter-membrane vesicles and properties of their hydrolase cargo. Microbiology. 158: 2742-52. doi: 10.1099/mic.0.060343-0.3. Everitt, B. (1974). Cluster Analysis. London: Heinemann Educ. Books.4. Garcia R, Gerth K, Stadler M, Dogma IJ Jr, Müller R. (2010) Expanded phylogeny of myxobacteria and evidence for cultivation of the'unculturables'. Mol Phylogenet Evol. 57: 878-87. doi: 10.1016/j.ympev.2010.08.028.5. Korp J, Vela Gurovic MS, Nett M. (2016) Antibiotics from predatory bacteria. Beilstein J Org Chem. 12: 594-607. doi:10.3762/bjoc.12.58.6. Morgan AD, MacLean RC, Hillesland KL, Velicer GJ. (2010) Comparative analysis of myxococcus predation on soil bacteria. Appl EnvironMicrobiol. 76: 6920-7. doi: 10.1128/AEM.00414-10.

S. aureus

S. saprophyticus

S. epidermidis

Ps. aeruginosa

C. albicans

B. subtilis

Ent. faecalis

K. pneumoniae

Pr. mirabilis

Es. coliFig.4. Cluster tree of the prey organisms according their

susceptibility profile