Introduction

Members of the genus Xenorhabdus are Gram-negative, asporogenic, fermentative, facultative anaerobic rods with a respiratory and fermentative metabolism, and belong to the family Enterobacteriaceae. They grow optimally at 28 °C, are oxidase and catalase negative and do not reduce nitrate to nitrite (Thomas and Poinar 1997; Akhurst and Boemare 2005). Acid is produced from the fermentation of glucose, but without the release of CO2. N-acetyl-glucosamine, glycerol, fructose and mannose are usually fermented (Akhurst and Boemare 2005). Xenorhabdus species are known to produce two phenotypic variants that differ in morphology and physiology. Phase I cells are larger than phase II cells, absorb certain dyes and produce proteases, lipases and antibiotics (Akhurst 1980; Boemare and Akhurst 1988).

Xenorhabdus spp. are closely associated with entomopathogenic nematodes of the family Steinernematidae that infects insects such as Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera (Laumond et al. 1979; Poinar 1979). Although the association between Xenorhabdus spp. and nematodes is species-specific, a single species may infect different Steinernema spp. (Fischer-Le Saux et al. 1997; Lee and Stock 2010). More than 20 Xenorhabdus species have been characterised, with approximately 11 of these species being associated with more than one Steinernema nematode species (Tailliez et al. 2006; Stock 2015).

Steinernema dauer larvae or non-feeding infective juvenile (IJ) transport Xenorhabdus to the haemocoel by entering natural openings on the body of the insect. Once in the haemocoel, the IJ nematodes release an immunesuppressive agent that suppresses the activity of antimicrobial peptides produced by the insect (Götz et al. 1981). IJ develop into amphimictic females and males that feeds on the proliferating bacteria. Endo- and exotoxins (e.g., DNases, lipases and lecithinases) are released by the bacteria, killing the insect within 24–48 h. Colonization of the cadaver by other microorganisms is prevented by the release of antimicrobial compounds from the respective Xenorhabdus species. The bacteria and bio-converted host tissue supports the sexual reproduction of the nematodes as long as nutrients are available. The nematodes, colonized with a few cells of Xenorhabdus are then released into the environment to repeat the cycle of infection (Adams and Nguyen 2002).

In this study, the bacterial strain WS9 was isolated from an unknown entomopathogenic nematode Steinernema sp. WS9. The bacteria was identified using phenotypic and genotypic characteristics.

Materials and methods

Isolation of Xenorhabdus sp. and maintenance of cultures

Galleria mellonella L. (Lepidoptera: Pyralidae) larvae were exposed to IJs of Steinernema sp. WS9 (KP325086; MF443108) collected from a litchi orchard in Friedenheim (25°30.927′S 30°58.681′E), Mpumalanga, South Africa. After 18 h, the live Galleria larvae were dipped in 98% alcohol, bent and pricked close to a proleg, using a sterile syringe. A drop of haemolymph, was spread-plated onto Nutrient Agar (NA, Biolab, Biolab Diagnostics, Midrand, South Africa), supplemented with 0.025% (w/v) bromothymol blue and 0.004% (w/v) triphenyltetrazolium chloride (TTC), known as NBTA (Kaya and Stock 1997; Ferreira et al. 2013). The plates were incubated at 30 °C for 48 h. Colonies that absorbed bromothymol blue were regarded as Xenorhabdus spp. and streaked onto NBTA to obtain pure cultures.

Strain WS9, isolated from one colony, was cultured in Tryptone Soy Broth (TSB, Biolab) and stored at − 80 °C in cryotubes with 40% (v/v) glycerol. All experiments were performed with phase I cells, stored in glycerol, to prevent phase shifting. Phase I cells were differentiated from phase II cells by the absorption of bromothymol blue from NBTA plates. All assays were performed with cultures from glycerol to prevent phase shifting. Escherichia coli Xen 14 was cultured in Luria broth (LB, Biolab) and E. coli DH5α transformants in LB, supplemented with 100 µg ampicillin/ml. Bacillus subtilis subsp. subtilis BD170, Listeria monocytogenes EDGE, Staphylococcus aureus Xen 5, Pseudomonas aeruginosa Xen 5 and Salmonella typhimurium Xen 26 were cultured in brain heart infusion (BHI, Biolab). Stock cultures were prepared in 40% (v/v) glycerol and stored at − 80 °C.

Cell morphology and phenotypic characteristics

Strain WS9 was gram stained and the size of a single cell determined using a Leica DM2000 research microscope (Leica Microsystems), equipped with a camera, computer and digital image software [Leica Application Suite (LAS), version 3.5.0]. Oxidase activity was determined using Kovács oxidase reagent. Catalase activity was recorded by suspending a colony in a drop of 5% (v/v) H2O2. Optimal growth temperature was determined by growing cultures at 26, 30, 37 and 42 °C for 24 h in TSB. Changes in growth were monitored by recording optical density readings at 600 nm. Lecithinase activity was determined as described by Ferreira et al. (2013). Lipase activity was determined by streaking 48-h-old cultures onto media containing 10% (w/v) peptone, 5% (w/v) sodium chloride, 0.1% (w/v) CaCl2∙2H2O and either 10% (v/v) Tween 40, Tween 60 or Tween 80. All experiments were done in triplicate.

Carbohydrate fermentation and assimilation profiles were determined using API 50 CHE and 20 NE test strips, respectively (BioMérieux, Marcy I’Etoile, France), according to the instructions of the manufacturer. Results were recorded after 10 days of incubation at 30 °C.

Genotypic characterization and phylogenetic analysis

Genomic DNA was extracted using a modification of the method described by Crouse and Amorese (1987). The 16S rDNA and housekeeping genes glutamyl-tRNA synthetase catalytic subunit (gltX), recombinase A (recA) and DNA polymerase III beta chain (dnaN) were amplified using the primers listed in Table 1. 16S rDNA primers were from Inqaba Biotechnology (Pretoria, South Africa) and all other primers from Integrated DNA Technologies (Coralville, Iowa, USA). Amplifications were performed using Q5® High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) or GoTaq® HotStart DNA Polymerase (Promega Corporation, Madison, Wisconsin, USA) and a SwiftMinipro thermal cycler (Esco Healthcare, Malaysia). Amplified fragments were ligated into plasmids pGEM®T Easy (Promega Corporation) or pJET1.2 (Thermo Fisher Scientific), according to instructions of the manufacturer. The plasmids were transformed to E. coli DH5α and cloned cells selected from colonies on LB agar supplemented with 100 µg/ml ampicillin. Positive clones were confirmed by sequencing. The sequencing facilities at the DNA sequencing unit, Central Analytical Facility, University of Stellenbosch, were used.

Table 1 Primers used for sequencing
Full size table

Whole genome sequencing (WGS) was used to determine the precise species of isolate WS9. Genomic DNA was sequenced on the Illumina Miniseq (Illumina, San Diego, California, USA) instrument. Before sequencing, genomic DNA was fragmented using the M220 Focused-Ultrasonicator (Thermo Fisher, Waltham, MA, USA). The NEBNext® Ultra™ DNA Library Prep Kit and Illumina® NEBNext® Multiplex Oligos (New England Biolabs Inc., Ipswich, MA, USA) were used for preparing sequencing libraries. This service was delivered by the Centre for Proteomic and Genomic Research (CPGR). The accession number for the WGS data of strain WS9 is SAMN07327420.

De novo assemblies of Illumina PE reads were constructed using SPAdes v3.10.1. Contigs containing the 16S rDNA and housekeeping genes, gltX, recA, dnaN, DNA gyrase B (gyrB) and initiation factor B (infB) were identified using BLASTn v2.6.0 + analysis and compared to respective gene reference sequences. MEGA6.0 (Tamura et al. 2013) was used to align sequences. The maximum likelihood method was used to construct phylogenetic trees from single gene sequences and concatenated sequences. The Kimura two-parameter model (Kimura 1980) was used to calculate the distance metrics for aligned sequences. The robustness of individual branches was determined by bootstrapping with 1000 replicates. Parameters were kept constant for the construction of all phylogenetic trees. Protein-coding sequences were compared to previously published sequences by performing translated BLAST searches (BLASTx, National Center for Biotechnology Information). Gene sequences of closely related species and outgroups were obtained from the National Center for Biotechnology Information (NCBI).

Testing for antibacterial activity

Antibacterial activity was determined by spotting 5 µl of a 12-h-old culture of WS9 onto TSB agar (TSA). The plates were incubated at 30 °C for 24, 48, 72 and 96 h, respectively, and then overlaid with 12-h-old cells of B. subtilis subsp. subtilis BD170 suspended in BHI agar (1%, w/v). Spot inoculated plates that were incubated for 96 h were overlaid with L. monocytogenes EDGE, S. aureus Xen 5, P. aeruginosa Xen 5 and S. typhimurium Xen 26, each suspended in BHI agar (1%, w/v), and E. coli suspended in LB agar (1%, w/v). Results were recorded after 24 h of incubation at 37 °C.

Results and discussion

Phenotypic and biochemical characteristics

Strain WS9 is Gram-negative, rod-shaped and 0.8–1.1 by 3.1–6.2 µm in size. Catalase and oxidase were not produced. All colonies absorbed bromothymol blue from NBTA plates, which is characteristic for Xenorhabdus spp. (Kaya and Stock 1997). Optimal growth was recorded at 26 and 30 °C, with slightly better growth at 30 °C. Strain WS9 does not produce lecithinase, and is negative for the hydrolysis of Tweens 60 and 80, but positive for Tween 40.

Strain WS9 produced acid from the fermentation of ribose, xylose, glucose, fructose, mannose, dulcitol, N-acetylglucosamine, maltose, melibiose, gentiobiose and fucose. Acid production from glycerol, arabinose, galactose, inositol, mannitol, esculin ferric citrate, trehalose, luxose, arabitol and potassium gluconate was weak. Strain WS9 assimilated glucose, mannose, N-acetyl-glucosamine, maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate and phenylacetic acid. No other tested carbohydrates were metabolized. Nitrate was not reduced to nitrite.

Antibacterial activity

The antibacterial activity of strain WS9 reached maximum level after 72 h of growth. Colonies of strain WS9 inhibited the growth of B. subtilis subsp. subtilis BD170, L. monocytogenes EDGE, S. aureus Xen 5, E. coli Xen 5 and S. typhimurium Xen 26. No antibacterial activity was recorded against P. aeruginosa Xen 5, which is most likely due to a low secondary metabolite concentration. Most Xenorhabdus spp. produce one or more antimicrobial compounds (Webster et al. 2002). Further research is required to identify the antibacterial compounds produced by strain WS9.

Phylogenetic analysis of strain WS9

Strain WS9, grouped with the type strain of X. griffiniae when 16S rDNA, gltX, recA, dnaN, gyrB and infB sequences were concatenated (Fig. 1). Comparison of 16S rDNA, gltX, dnaN and gyrB sequences (Online Resources 1–4), separately, grouped strain WS9 with X. griffiniae, but with X. ehlersii and Xenorhabdus sp. TZ01 (JQ687358; JQ687369–JQ687373) when sequences of recA and infB (Online Resources 5, 6) were compared. This suggests that strain WS9 is phylogenetically related to X. griffiniae, and to a lesser extent to X. ehlersii and Xenorhabdus sp. TZ01. Nucleotide similarity searches produced lower identity percentages for X. ehlersii and Xenorhabdus sp. TZ01 compared to X. griffiniae, despite the fact that query coverages were similar, with exception of the recA gene (Online Resource 7). Protein-coding sequences of gltX, dnaN and gyrB obtained for isolate WS9 were similar, but not identical, to protein-coding sequences listed for X. griffiniae (Online Resource 7). However, corresponding to phylogenetic trees, protein-coding sequences for recA and infB were similar to those of X. ehlersii and Xenorhabdus sp. TZ01, respectively. Taking into consideration all the above mentioned, the bacterial isolate WS9 has been identified as a strain belonging to the species X. griffiniae. Closely related X. ehlersii has been isolated from Steinernema longicaudum Shen & Wang (syn. Steinernema serratum) (Lengyel et al. 2005; Tailliez et al. 2006), and Xenorhabdus sp. TZ01 from Steinernema pwaniensis (Půža et al. 2016). The Steinernema sp. strain WS9 is closely related to S. pwaniensis from Tanzania, and both belongs to the Karii-clade, while S. longicaudum belongs to the Longicaudum-clade (Spiridonov and Subbotin 2017).

Fig. 1
figure 1

Phylogenetic relationship of strain WS9 with known Xenorhabdus spp. The maximum likelihood tree was constructed using concatenated sequences of six genes (16S rDNA, gltX, recA, dnaN, gyrB and infB). Three Photorhabdus spp. were used as outgroups. Type strains are indicated by a superscript T. Bootstrap values of more than 70% are shown at branch points. The bar indicates a sequence divergence of 2%

Full size image

Xenorhabdus griffiniae has previously been isolated from the nematode Steinernema hermaphroditum Stock, Griffin & Chaerani (Nguyen and Hunt 2007) and more recently from the nematode Steinernema khoisanae strain BMMCB (Mothupi et al. 2015). Based on the internal transcribed spacer (ITS) region (KT027382), S. khoisanae BMMCB is 91% similar to the type species, S. khoisanae SF80 (DQ314287), and 92% to Steinernema sp. strain WS9 (KP325086). According to Nguyen and Hunt (2007), a sequence similarity in the ITS region of 95% or less to the most closely related species indicate a possible new Steinernema species. This suggests that strain BMMCB could be neither S. khoisanae nor Steinernema sp. WS9, but appears to be a new Steinernema species.

Previously, Spiridonov et al. (2004) group all three nematode species associated with Xenorhabdus griffiniae in one clade (clade V). According to recent molecular information (Spiridonov and Subbotin 2017), the three nematode species associated with X. griffiniae belong to different clades; S. khoisanae SF80 belongs to the Khoisanae-clade, Steinernema sp. WS9 to the Karii-clade and S. hermaphroditum to the Longicaudum-clade. Host switches of the bacterial symbionts are generally observed between nematodes belonging to the same clade, however, increasing reports have shown that host switches can occur between nematodes from different clades, which is also the case for this study (Lee and Stock 2010; Çimen et al. 2016; Dreyer et al. 2017).

This study reports, for the first time, an association between X. griffiniae and an unknown locally isolated Steinernema sp. WS9. Accession numbers for the PCR obtained sequences 16S rDNA, gltX, recA and dnaN are KT954035, KT954028, KT954030 and KT954033, respectively.