Xenorhabdus genus was first described by Thomas and Poinar. In their study, nine isolates were taken from the lumen of nematodes of the genera Neoaplectana and Heterorhabditis.¹ Xenorhabdus spp. exist in a symbiotic relationship that can be pathogenic or mutually beneficial. The life cycle of the bacteria starts and ends with the colonisation in the intestines Xenorhabdus spp. start their life cycle by colonising the nematodes (Figure 1). The nematodes upon entering the insect, release the bacteria into the haemolymph. Xenorhabdus spp. multiplies and produces toxins that kill the insects. The nematodes multiply by consuming nutrients from the insect cadaver, which is converted by the bacteria. The Xenorhabdus spp. exhibits mutualism with the nematodes by providing the specific nutrients for their growth and exhibits pathogenicity towards insects.²

Entomopathogenic bacteria
Entomopathogenic nematodes are a group of insect-pathogenic that mostly belong to the genera Steinernema and Heterorhabditis. Entomopathogenic nematodes exist in a mutualistic relationship with bacterial genera Xenorhabdus and Photorhabdus.³ One species of nematodes associates with only one species of bacteria, whereas one species of bacteria can associate with many species of nematodes. The Entomopathogenic nematodes enter the insect host by cuticle penetration through the openings of the host, reaching the gut they convert the host tissue into nutrients for their reproduction.⁴ Entomopathogenic nematodes are mostly free-living soil organisms, so isolated from the soil using the Galleria mellonella,⁵ Galleria mellonella Linnaeus, 1758 and Achroia grisella Fabricius, 1794 are the key pests of honeybee colonies worldwide. They cause wax comb damages which lead to colony perishing and absconding. To control these pests, their genetic background and evolution should be considered. In this study, genetic diversity and demographic history of 2 species of wax moth were explored based on mitochondrial genes (COI) as a bait. The larvae that are infected by Steinernema appear black in colour, whereas those infected with Heterorhabditis appear brick red in colour. This pigment is associated with the symbiotic bacteria. The nematodes are collected using the white trap method followed by surface sterilization and molecular characterization.⁶

Galleria mellonella
Galleria mellonella is commonly known as the Greater Wax Moth (GWM). This GWM refers to a collection of moths that infiltrate and overrun the bee hives and their products. Egg, larva, pupa, and adult moth are the different life stages of the G. mellonella (Figure 2). The life cycle takes around weeks to months based on the biotic and abiotic factors. The GWM is nocturnal and is active at the onset of the scotophase, that is, between 18:00-24:00 hours. During this phase, the mating of the male and female moth occurs; the female flies towards the male when it is attracted by the acoustic sound from the male. After mating, the female flies back to the hive and lays around 50 to 100 eggs in the cleft and cranny to increase the survival rate of the egg and larvae. The larvae feed on the honey, pollen, and brood resulting in the destruction of the hive structure. Prior to pupation, the larval stage lasts from 28 days to 6 months. The pupa stage takes around 1-9 weeks, and the life cycle continues.⁷

Xenorhabdus isolation
The G. mellonella larvae are reared artificially and are used as bait to isolate the Xenorhabdus. Soil samples from different locations were collected, and the large debris was removed. The soil samples are moistened, to this the G. mellonella is placed and the container is closed with the cloth to provide a dark condition with proper aeration. The temperature of this setup is maintained at around 25-28 °C. This setup is checked regularly for 7 days to identify any infection in the larvae.⁸

The growth temperature of Xenorhabdus lies in the range between 32 °C and 42 °C. The bacteria can be isolated through an indirect or direct method. The direct method involves the isolation of the bacteria from the infective nematodes, whereas the indirect method, involves the isolation from the G. mellonella haemocoel⁹ isolated from at least 27 species of Steinernema nematodes and collected in 32 countries, using three complementary approaches: 16S rRNA gene sequencing, molecular typing and phenotypic characterization. The 16S rRNA gene sequences of the Xenorhabdus strains were highly conserved (similarity coefficient >95%. The G. mellonella is infected by the Steinernema nematode, and the dead cadaver appears black in colour. The cadaver is washed with ethanol, and it is placed in a sterile petri plate. The haemolymph is collected from the 3^rd segment of the insect cadaver, and it is streaked in NBTA after 4 days of incubation in the dark. The morphological identification provides a primary confirmation of the species. The Xenorhabdus species appears as dark blue colonies.¹⁰ These colonies are further cultured in the LB media under shaking conditions to obtain the secondary metabolites for 78 hours at 30 °C. The supernatant is collected for further analysis of the secondary metabolites.¹¹

Secondary metabolites
The secondary metabolites produced by Xenorhabdus bacteria have multiple activities, like anti-bacterial, anti-fungal, insecticide, protozoal, and also against cancer cells (Figure 3). The antibiotic production in the X. nematophila is regulated by the Lrp gene, but this gene activity is not the same in all other species of Xenorhabdus. The isolated secondary metabolite includes depsipeptides, xenocoumacins, rhabdopeptides, bicornitun, etc (Table 1).¹² Xenorhabdus khoisanae J194 with South African origin was cultured in non-aerated and aerated broth; it was observed that there was a rapid increase in biomass in non-aerated broth compared with to aerated. The sequence encoding novel peptides rhabdin and xenopep was confirmed using whole-genome sequencing. Rhabdin is effective against Gram-positive bacteria such as S. aureus ATCC 25923, S. aureus Xen 31, L. monocytogenes and Gram-negative bacteria such as K. pneumoniae, P. aeruginosa, and E. coli. Xenopep was effective only against Gram-positive bacteria such as S. aureus ATCC 25923, L. monocytogenes, and S. epidermidis.¹³

Table (1):
Secondary metabolites produced by various Xenorhabdus spp. against a range of pathogens and their mode of action

Applications
Anti-bacterial activity of secondary metabolites from Xenorhabdus spp.
EPNs from 230 soil samples were isolated and it was identified as the genus Steinernema and Heterorhabditis. From these isolates 6 Xenorhabdus and 8 Photorhabdus was isolated, and the antibacterial activity was evaluated against the 2 MRSA (Methicillin-resistant Staphylococcus aureus) strains, 2 MDR (Multidrug-resistant) strains, 1 XDR (Extensively drug-resistant) strain, and E. faecalis ATCC 51299 by the disk diffusion method. The results stated that the Xenorhabdus had no inhibitory effect against any antibiotic-resistant strain. In Photorhabdus, the P. luminescens subspecies was able to inhibit a broad range of antibiotic-resistant strains. 1 Xenorhabdus and 3 Photorhabdus strains were tested against 5 antibiotic-resistant strains, and the result indicated the MIC and MBC range between 7.81-0.98 mg/ml, exhibiting moderate to strong activity. Time-kill study was performed against S. aureus strain PB36 (MRSA), and it expressed that Photorhabdus reduced the S. aureus strain PB36 (MRSA) 10⁵ to 10³ CFU/ml within 30 minutes; also, there was no bacterial regrowth, exhibiting the strong bactericidal activity (Figure 4). On the contrary, the S. aureus strain PB36 treated with Xenorhabdus continued to grow, referring to no bactericidal activity.¹⁴ 10 EPN were isolated from different agricultural sites, and it was found that 2 symbiotic bacteria evinced antibacterial activity. The primary characterisation was performed based on the triphenyl tetrazolium and bromothymol blue dye absorption from NBTA plates. It revealed that the colonies, which was blue in colour, were Xenorhabdus and brown were Photorhabdus species. Further molecular characterisation was performed using 16S rRNA sequencing. The antibacterial compound was isolated by the solvent extraction method using ethyl acetate, followed by HPLC. The extract from the Xenorhabdus and Photorhabdus exhibited antibacterial activity against 6 bacterial pathogens (E. coli, S. aureus, B. subtilus, P. mirabilis, E. faecalis, and P. stutzeri). At increasing concentration of the Xenorhabdus and Photorhabdus strain, displayed increasing zone of inhibition. Compared to Photorhabdus, Xenorhabdus evinced strong antibacterial activity.¹⁵

Anti-fungal activity of Xenorhabdus spp.
Two Photorhabdus and three Xenorhabdus species were used to study the antifungal activity against Cryphonectria parasitica¹⁶ Fusarium oxysporum¹⁷ Rhizoctonia solani¹⁸ and Sclerotinia sclerotiorum.¹⁹ The Xenorhabdus strain X. szentirmaii has strong anti-fungal activity against C. parasitica compared to other strains (Table 2). In the case of F. oxysporum all the bacterial strains had an anti-fungal activity; the X. szentirmaii at a concentration of 5% had the highest inhibition even after 15 days of incubation, which was absent in the case of other strains. Whereas the other Xenorhabdus strains had no effect against F. oxysporum. The R. solani and S. sclerotiorum were inhibited only by the X. szentirmaii at the end of 5^th and 10^th day of inhibition and the other strains had no inhibitory effect. A dose of 40% of X. szentirmaii supernatant inhibited the R. solani and 20% dose inhibited the S. sclerotiorum. The antifungal activity of X. szentirmaii is due to the fabclavine compounds.²⁰

Table (2): Antifungal compounds derived from various bacterial strains

The Xenorhabdus indica strain AB releases a volatile organic compound that has an anti-fungal activity against F. oxysporum. The VOCs inhibit the growth by 60% through spore germination inhibition. A total of six VOCs was isolated, among which four had anti-fungal activity against F. oxysporum. The four VOCs are methyl anthranilate, decanenitrile, aniline, and furfuryl alcohol. The methyl anthranilate and decanenitrile inhibit the growth by hindering the mycelial growth. A 50 µL/plate quantity of methyl anthranilate has the property of inhibiting the growth and reproduction of F. oxysporum through a membrane-breaking mechanism. Aniline reduces the spore germination rate by 60% with a dose of 50 µL.²¹

The Xenorhabdus bovienii supernatant had a strong inhibition against the Fusarium solani NK-NH1 strain with just 2 days of inhibition. Ven diagram analysis was made between the control and the treatment group to identify the unique metabolite responsible for the inhibitory action. Around 52.75% is the lipids and lipid like compounds. The VOCs (2-pentanone, 3-(phenylmethyl), and 1-hexen-3-one, 5-) produced by the Xenorhabdus bovienii contributed to the anti-fungal activity.²²

10 μg/mL of Xcn1 isolated from X. nematophila YL001 exhibited a strong inhibition against Sclerotinia sclerotiorum, R. solani, B. cinerea, and E. turcicum. The samples treated with Xcn1 underwent morphological changes like coiled and disfigured hyphae with minimal distribution. S. sclerotiorum makes the medium acidic by producing oxalic acid during growth. This acidification process occurred in the Xcn1-treated group, but with some time delay. This process did not render with the inhibitory effect of the Xcn1.²³

Xenorhabdus nematophila was cultured in different media such as LB, TSB, and NB. There was a significant difference in the inhibitory effect of the X. nematophila. The metabolites extracted after 3 days from LB media had a strong inhibition against Botrytis cinerea²⁴ compared to the other TSB and NB.²⁵

Insecticidal and larvicidal activity of Xenorhabdus spp.
Xenorhabdus nematophila and Xenorhabdus innexi insecticidal activity against the Manduca sexta²⁷ was evaluated. Xenorhabdus nematophila exhibited insecticidal activity by producing several metabolites like rhabduscin that reduces the activation of the phenoloxidase (PO) system by 30%. This PO system is a crucial part of the insect’s innate immune system. Xenorhabdus innexi has no activity against M. sexta. Xenorhabdus innexi is non-toxic against most of the insects that were tested, as it does not possess virulent genes to secrete the toxins. The X. innexi, along with its nematode S. scapterisci, possesses some toxicity, but when it is tested along there was no insecticidal activity. This is due to the absence of genes for Tc toxins and other virulent characteristics.²⁸

Xenorhabdus hominickii supernatant was extracted using 4 different organic solvents, such as hexane, ethyl acetate, chloroform, and butanol. The butanol extract exhibited strong inhibition against haemocyte PLA₂ of Spodoptera exigua,²⁹ which has a crucial role in the insect’s immune system (Figure 5). From the butanol extract, fifteen fractions were evaluated, of which two fractions and eight subfractions expressed strong inhibition. From the leaf-dipping method for evaluating the feed toxicity, it was inferred that 3-ethoxy-4-methoxyphenol (EMP) and phthalimide (PM) from the eight compounds indicated strong insecticidal activity.³⁰ Steinernema carpocapsae mutualist with Xenorhabdus nematophila expressed high toxicity against larvae of P. brassicae³¹ Pieris brassicae Linnaeus (Lepidoptera: Pieridae. Xenorhabdus nematophila HB310 strain releases protein toxin complex (Tc), which has insecticide properties.³²

Xenorhabdus sp. isolated from Corcyra cephalonica,³³ which was infected by an entomopathogenic bacterium. The Xenorhabdus sp. occurs in two forms or two phases. Phase I is referred to as the primary form, which absorbs bromophenol blue and appears as dark blue rod-shaped colonies. Phase II is also referred to as the secondary form. MacConkey agar medium appears in red, confirming the presence of this form after 1 week of time. These two forms have the ability to produce lecithinase, but the primary form produces a higher concentration compared to the secondary form. The larvicidal activity of the lecithinase was studied against Culex quinquefasciatus³⁴ larvae. The lecithinase from the Xenorhabdus sp. exhibited high activity at 24 °C, with a pH of 7. The activity of the lecithinase was inversely correlated with the pH. The haemolytic property of the lecithinase was studied through a blood agar plate. The results confirmed that lecithinase concentration is positively correlated with the larvicidal activity against the larvae.³⁵

Two and four isolated Xenorhabdus and Photorhabdus species were chosen for larvicidal activity testing against Aedes aegypti³⁶ and C. quinquefasciatus larvae (Figure 6). Photorhabdus exhibited strong larvicidal activity against them after 24 hours of exposure.³⁷

The cell suspension and cell-free supernatants of the Photorhabdus and Xenorhabdus were tested for the larvicidal activity against Culex pipiens. It was concluded that the cell-free supernatant showed a high mortality rate compared to the cell suspension against the development stages of Culex pipiens.³⁸ Xenorhabdus bovienii KCS-4S cell-free suspension exhibited an increasing mortality rate of 95% with a gradual increase in the exposure time of 72 hours against the first two larval development stages. This is the only strain that also expressed a mortality rate over 50% against the pupae stage. The time taken to kill 50% of the population (LT50) was higher in the case of cell suspension than in the cell-free supernatant.³⁹

Anti-tumour
HN_xs01 strain isolated from nematodes obtained from the foot of Yuelu Mountain using Galleria mellonella as bait was identified as 99% similar to X. stockiae. The anti-tumour activity was verified using B16 melanoma cells; the cells that were treated with the HN_xs01 exhibited signs of apoptosis that include morphological changes like shrinkage and detachment, unlike the control cells, which were elongated and attached. Three compounds were purified from the crude extract of the strain, and the molecular formula was determined using NMR spectra, and these structures belonged to rhabdopeptides. The difference between the three compounds (1,2,3) is due to the number of residues of the amino acid N-methyl-valine. The cytotoxicity assay on the A431 cells and K562 cells revealed that compound 3 has strong cytotoxic activity compared to the other two compounds. The anti-tumour targeting activity of the strain was tested using C57BL/C mice bearing the B16 tumour. Different concentrations of HN_xs01 were administered intravenously. At high concentrations, the mice died immediately; lowering the concentration lowered the mortality of the mice, and the safe concentration was identified in the range of 1 × 10⁶ CFU/ 100 μL and 5 × 10⁷ CFU/100 μL. Comparison of HN_xs01 with the probiotic E. coli Nissle 1917 was performed to study the tumour-specific colonisation, and it was observed that the HN_xs01 strain colonised only in the tumour-specific area, whereas in the case of Nissle 1917 was detected in the other organs of the mice, like the liver, kidney, and spleen, than in the tumour region. It was also observed that after the injection of the mice with the HN_xs01 strain, the weight decreased, and it regained its normal weight after 3 days. It was observed that the HN_xs01 mimicked the pulmonary metastasis of B16 tumours and controlled them more effectively.⁴⁰ The purified supernatant fractions obtained from the UHPLC peak E and F was analysed for the anti-tumour activity on the HUVEC, B16, 4T1, and HeLa cells. It was observed that the peak E fraction had a strong inhibitory effect and was less toxic to the non-tumour cells, and it was further termed as XNAE (Figure 7). This XNAE was used in the CCK-8 reduction assay to assess the in vitro inhibitory effect on the HUVEC, B16, and HeLa cells. There was no effect on the HUVEC, whereas notable cell migration inhibition in B16 and HeLa cells. The ability of the XNAE to induce apoptosis and arrest the cell cycle was tested on the B16 cell. The result suggested that there was some extent of apoptotic activity on the B16 cells, and the cell cycle was arrested in the G1 phase, inhibiting the proliferation.¹¹