Isolation and Ecology:
This strain was identified in a chronically infected endovascular graft in a 55-year-old shepherd, with bacterial cultures performed from the explanted graft at Guy’s and St Thomas’ Hospital (London, UK) confirming polymicrobial growth of Nocardia nova and Variovorax sp. To our knowledge, this is the first case of Variovorax sp. identified as a cause of human infection and the first description of this new species of Variovorax durovernum nov. sp. This Variovorax durovernum nov. sp. was cultured from three of five operative samples from the explanted graft after extended incubation, as well as from subcultures of brain heart infusion broth, after seven days of incubation. All positive cultures were also growing Nocardia nova, suggesting a symbiotic relationship between the two organisms.
The genus Variovorax was originally described in 1969 as the organism Alcaligenes paradoxus[1] which was reclassified to Variovorax paradoxus, due to its ability to devour a ‘variety of substrates’, ‘voraciously’. V. paradoxus is a betaproteobacterium best known as a plant-promoting rhizobacterium and has important biodegradative processes in nature [2]. All members of the genus are benign soil rhizospheres; there are twelve recognised members of the genus Variovorax described, V. dokdonensis [3], V. soli [4], V. boronicumulans [5, 6], V. ginsengisoli [7], V. defluvii [5] and V. guangxiensis [8], V. gossypii [9], V. humicola [10], V. ureilyticus,V. rhizosphaerae and V. robiniae [11]. Members of the genus Variovorax are noted to have diverse metabolic processes; a property has been utilised as a biotechnological agent to degrade pollutants from contaminated sites [12]. Genomic studies placed the Variovorax genus within the Comamonadaceae family [13].
Like Pseudomonas aeruginosa, Variovorax sp. can display swarming motility or form biofilms depending on environmental pressures. Variovorax sp. can be engaged in mutually beneficial interactions with other plant rhizospheres such as V. boronicumulans [6] that accumulates boron and V. ginsengisoli [7] that has denitrifying properties. These bacterial species in various ecosystems display synergistic relationships which could explain the environmental niche for the Variovorax sp.and Nocardia nova to proliferate on the microclimate of our patients endovascular graft.
16S RNA phylogeny:
Variovorax durovernum nov. sp. DNA was extracted from blood agar culture by bead beating for 4m/s for 40s using Matrix E beads (MP Biomedicals™) on FastPrep-24™ 5G Instrument (MP Biomedicals™). Extracted DNA was washed with 0.5X of Agencourt AMPure XP beads (Beckman Coulter-A63881) and prepared for nanopore sequencing using the Genomic DNA (SQK-LSK109 - Oxford Nanopore Technologies (ONT)) followed by sequencing for 24 hrs on a GridION (ONT), following manufacturer’s instructions.
Data after 24 hours sequencing were used for downstream analysis. Data was filtered with Filtlong v0.2.0 to retain the best 1 Gb of sequencing data. De novo assembly was p erformed using Flye v.2.9 [14] and further polished using medaka v.1.5.0 [15]. Genome was annotated using prokka v1.14.6 [16]. The consensus sequence is available on the European Nucleotide Archive (ENA) under the accession number: ERS6367071.
Nucleotide sequences representing the 16S rRNA gene of the isolate were identified in 3 contigs, with fragment size of up to 1538bp which all shared 100% sequence identity. The 16S rRNA gene sequence was confirmed by targeted 16S sequencing performed by the Public Health England reference laboratory in Colindale, UK. 36 closely related species to the genus Variovorax were identified from literature and 16s rRNA sequences were taken from NCBI RefSeq database [10]. SNP analysis was performed using snp-dists v0.8.2 [17]. Comparing the 16S rRNA gene across the reference species, the 16S rRNA gene of Variovorax isolates differed by a minimum of 6 SNPs (99.6% identity) for V. paradoxus and 7 SNPs for V. boronicumulans and V. guangxiensis (99.5%) (Table 1). Multiple sequence alignment was performed using MUSCLE v5.1 [18] and 16S rRNA-based maximum-likelihood phylogenetic tree produced using IQ-TREE v2.2.0 [19] with SH-like approximate likelihood ratio test and bootstrap replication both set to 1000. Tree visualised using iTOL v6.5.2 [20] (Figure 1). Despite having closer SNP distance to V. paradoxus the 16S rRNA gene of the GSTT-20 isolate segregates with V. boronicumulans with high confidence. To further characterise genomic difference, other measures of overall genome relatedness index (OGRI) are presented below.
Genome Features:
The GSTT-20 isolate was compared to 7 Variovorax species with publicly available genomes. Digital DNA-DNA hybridisation (dDDH) was performed using Type genome strain server (TYGS) [21] and Average nucleotide identity (ANI) was performed using FastANI v1.33 [22]. Whilst the 16s rRNA analysis positions the isolate closest to V. boronicumulans and V. paradoxus, the dDDH and ANI analysis delineates the isolate as the separate species (Table 2). The dDDH and ANI score for V. boronicumulans was 32.3% and 88.57% respectively. Similarly, the dDDH and ANI score for V. paradoxus was 31.1% and 87.50% respectively. Both of these values fall below intra-species thresholds of 70% dDDH and 95% ANI.
Next, the 7 species and the GSTT-20 isolate underwent phylogenomic analysis. Pan-genome analysis of the 8 genomes was investigated using Roary v3.13.0 [23] at 70% BLASTP identity threshold set for inference of core genes. A total of 1530 core genes were identified, along with a further 16605 accessory genes. Core genome alignment was performed within Roary. 16S rRNA-based maximum-likelihood phylogenetic tree produced using IQ-TREE v2.2.0 with SH-like approximate likelihood ratio test and bootstrap replication both set to 1000. Tree visualised using iTOL v6.5.2 (Figures 2). The GSTT-20 isolate clusters closely to V. guangxiensis, V. gossypii and V. boronicumulans, but exists on its branch. A SNP analysis was performed using snp-dists v0.8.2 revealing a total of 461887 SNPs across the 8 species, with a core genome length of 1.63 Mb. Compared with the GSTT-20 isolate, V. guangxiensis, V. gossypii and V. boronicumulans contain 136367, 130335 and 146897 SNPs respectively.
Despite the similarity in 16S rRNA similarity, the difference in dDDH, ANI and core-genome SNP analysis, support the classification of Variovorax nov sp. as a novel species [24].
Physiology and Chemotaxonomy:
Variovorax durovernum nov. sp. was isolated and grew on blood, MacConkey, nutrient, trypticase soy agar and Brain heart Infusion broth (BHI) after 48 hrs of incubation at 37°C in aerobic and on chocolate agar at 37°C after 48 hrs of incubation in CO2 incubation. GSTT-20T isolate grew on blood agar at 30°C and 20 °C. The isolate did not grow in a NaCl concentration higher than 1% when incubated aerobically at 37°C for 5 days, either at 4°C or 40°C and no growth was observed in a pH=8 dilution. Colonies on blood agar appeared as yellow, mucoid colonies, catalase positive, oxidase negative and on the Gram stain, Gram negative rods were seen. Motility test following wet mount method [25], showed that GSTT-20T is a motile bacterium.
API 20NE and API 20E were performed following the manufacturer's instructions, to study the assimilation of carbon sources and determine the presence of phenotypic enzymes. GSTT-20T did not assimilate any of the carbon sources tested, only urease and nitrate reduction and esculin hydrolysis was observed. (Table 3)
To examine the cellular fatty acid profile, the isolate was cultured at 28°C for 48 hrs on Trypticase Soy Broth Agar and fatty acid profiled using the Sherlock V6.4 system supplied by Midi Inc. The key acids in the isolate were 10:0 3OH (9.90%), Sum In Feature 3 16:1 ω7c/16:1 ω6c (16.36%), 16:0 (24.23%), 17:0 cyclo (13,35%), and Sum in Feature 8 18:1 ω7c, 18:1 ω6c. The minor acids are 10:0 (3.16%), 12:0 (2.92%) 14:0 (1.10%), 14: 2OH (2.10%), 16:1 2OH (3.10%), 16:0 2OH (1.43%), and 18:0 (0.74%). Compared to the closest match in the RTSBA6 6.21 library, the GSTT-20 isolate has a similarity index of 0.184 (18.4%) i.e. not very similar to V. paradoxus. The key difference between GSTT-20 isolate and the library entry for V. paradoxus and V. boronicumulans was that the GSTT-20 isolate had about 2-3 times the expected 10:0 3OH. (Table 4)
Ethics and consent:
All experimental protocols were approved by North West - Preston Research Ethics Committee (18/NW/0584). All methods were carried out in accordance with relevant guidelines and regulations. The patient provided informed consent for the publication of this article.
Protologue:
Variovorax durovernum nov. sp. is a short, curved gram-negative rod. Round, yellow, mucoid colonies grow in aerobic conditions on chocolate and 5% sheeps blood agar at 37°C. This organism is oxidase positive and catalase negative.
The name Variovorax durovernum nov.sp. was suggested by our patient, who had the organism isolated from their infected prosthetic aortic graft. The name 'Durovernum' is the Latin name for Canterbury; an ancient town in the United Kingdom where they work in the local area as a shepherd. It is likely Variovorax nov. sp. was an opportunistic pathogen, as all other members of the genus are soil organisms with no previous documented reports of human infection, with agricultural exposure facilitating the infected prosthesis. The proposed name for this novel species is Variovorax durovernum. nov.sp.