Molecular Characterization and Antibacterial Activities of Photorhabdus and Xenorhabdus from Mizoram, North-East India

Photorhabdus and Xenorhabdus are the bacterial symbionts of insect pathogenic nematodes, Heterorhabditis and Steinernema , respectively. This study aims to characterize the bacterial symbionts from Mizoram, North-east India and to evaluate their antibacterial potential. The bacterial isolates were characterized using recA and gyrB gene regions. The ethyl acetate extract of bacterial isolates was tested against pathogenic bacterial strains, viz. Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 10145), and Bacillus subtilis (ATCC 11774) using disk diffusion method. Analysis of recA and gyrB genes revealed that the Photorhabdus isolates were P. hindustanensis , and P. namnaonensis . This study constitutes the first documentation of P. namnaonensis from India. The two isolated Xenorhabdus belong to X. vietnamensis and X. stockiae . The ethyl acetate extracts of the studied bacteria suppressed the development of all the microorganisms tested. Based on MIC and MBC values, the highest activity was exhibited by TS ( P. hindustanensis ) and TD ( P. namnaonensis ) isolates against P. aeruginosa and K. pneumoniae respectively. The lowest inhibitory activity was observed on both Xenorhabdus isolates (RF and PTS) against B. subtilis. This study focuses on the existence and identification of symbiotic bacteria from Mizoram, an Indo-Burma biodiversity hotspot region, and details their activity against different pathogenic bacteria. Since these metabolites could be potent antibiotics, further research is required to better understand the genetic information, chemical composition, and method of action against other microorganisms.


INTRODUCTION
Microbial resistance to classical antibiotics has long been a serious health concern worldwide.Though resistance occurs naturally, the overuse or misuse of the existing antibiotic drugs in a variety of formats exerted selective pressure on certain microorganisms.This, in turn, develops resistance against antibiotics resulting in less or no effectiveness to treat various diseases. 1][4] The widespread antibiotic resistance was reported globally and declared a pandemic. 5Recently, the World Health Organization (WHO) has listed a group of pathogen strains, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.These strains are becoming more virulent and resist multidrug; therefore, effective antibiotics are urgently required for treatment. 2,6Also, these organisms can avoid the effects of the current antimicrobial medications and are also responsible for the majority of nosocomial infections ., 2,7 thereby consequently linked to the highest risk of mortality and morbidity, which raises healthcare expenses. 8Thus, an increase in the development of resistance in clinically important bacterial strains has led to the demand for the discovery and development of effective antibiotics.
Photorhabdus and Xenorhabdus are gram-negative bacteria under the family Morganellaceae.They formed a mutual relation with insect pathogenic nematodes viz Heterorhabditis and Steinernema, respectively. 9hey have a cosmopolitan distribution (except Antarctica) and are highly effective at controlling a variety of insect pests. 10Altogether, Photorhabdus has 28 recognized taxa, including 22 species, six of which are further classified into subspecies. 11o far, 28 taxa of Xenorhabdus associated with the nematodes have been identified including two recently identified subspecies. 12When the mutualistic association infects the insect larvae, the symbiotic bacteria produce broad-spectrum compounds which are lethal to the infected larvae, including activity against bacteria, fungi and parasites. 135][16] The bacteria multiplied within the insect host, the nematodes ingest the bacteria and consume the cadaver for growth and reproduction. 17When the food resources are exhausted, the emergence of the infective juveniles (IJs) from the host insect occurred and IJs eventually seek a new host. 18 h e s e c o n d a r y m e t a b o l i t e s o f Photorhabdus spp.and Xenorhabdus spp.0][21] and fungi 14,[22][23][24] to a great extent.In addition to antibacterial and antifungal, the bioactive compounds have been claimed to be effective against insects. 13,25,26Therefore, these bioactive compounds might be the solution to the novel antimicrobial compounds and could be used to overcome the limitations of effective antibiotics in combating certain disease-causing microbes.
The present study aims to characterize the insect pathogenic nematode-associated bacteria from Mizoram, North-east India using two housekeeping genes, viz.recA and gyrB gene regions, and to further assess the antibacterial activity of the bacterial symbionts against four pathogenic bacteria viz.E. coli (ATCC 10536), K. pneumoniae (ATCC 10031), P. aeruginosa (ATCC 10145), and B. subtilis (ATCC 11774) using the disk diffusion method.

Isolation and identification of bacterial symbionts
The symbiotic bacteria were isolated from entomopathogenic nematodes (EPNs) viz.The freshly emerged infective juveniles (IJs) of insect pathogenic nematodes were macerated for isolation of bacterial symbionts. 27Prior to the maceration process, the IJs were washed with 10% sodium hypochlorite (w/v) to prevent external tegument contamination.The IJs were further crushed in sterile PBS buffer using a micro-pestle.A volume of 100 µl was inoculated on nutrient agar with 0.0025% bromothymol blue and 0.004% triphenyl tetrazolium chloride (NBTA). 27,28followed by incubation for 48 h at 28R"C.
Bacterial cells from overnight nutrient broth culture were harvested and Phenol Chloroform Isoamyl-alcohol (PCI) method was used for genomic DNA extraction. 29For phylogenetic analysis, a set of primer-recA1 F (5'-GCTATTGATGAAAATAAACA-3') and recA2 R (5'-RATTTTRTCWCCRTTRTAGCT-3') was used for the amplification of recombinase A gene. 30 In addition, another set of primer-1200FgyrB (5'-GATAACTCTTATAAAGTTTCCG-3') and 1200RgyrB (5'-CGGGTTGTATTCGTCACGGCC-3') was used for amplification of gyrase B gene. 30 The PCR conditions applied for recA were 5 min at 94°C for denaturation followed by 30 cycles for 1 min at 94°C, 1 min at 55°C for annealing and 1 min at 72°C for extension followed by 5 min at 72°C.For gyrB, the PCR conditions were set as follows: 5 min at 94°C for denaturation followed by 30 cycles for 1 min at 94°C, 45 s at 58°C for annealing and 2 min at 72°C for extension followed by 7 min at 72°C.The PCR products were directly sequenced in a forward direction.

Sequence alignment and analysis
Sequence editing was performed using FinchTV 1.4.0 (http://www.geospiza.com)and alignment was done using clustalW (MEGA X). 31 Sequences were compared with the nearest matches available species from GenBank using the BLASTN algorithm.For phylogenetic analysis, 29 and 30 available taxa of Xenorhabdus and Photorhabdus respectively including an outgroup species, E. coli K-12 was recovered from NCBI GenBank.
To calculate genetic distance, pairwise sequence comparisons using the Kimura 2-Parameter approach under the Gamma distribution were used.The maximum likelihood tree (ML) was generated following the lowest BIC score (K2P with G + I sites) and branches statistically supported by a replicate of 1000 bootstraps.The generated sequences were deposited to NCBI GenBank (Accession number ON314147-ON314170).
In addition, the nucleotide sequences of recA and gyrB genes were concatenated, aligned and the ML tree was constructed separately for Photorhabdus and Xenorhabdus spp with the available type strain retrieved from NCBI GenBank.

Preparation of pathogenic bacteria.
Four strains of pathogenic bacteria, viz.E. coli (ATCC 10536), K. pneumoniae (ATCC 10031), P. aeruginosa (ATCC 10145), and B. subtilis (ATCC 11774) were maintained in Research and Instrumentation Centre, Pachhunga University College, Aizawl.A bacterial colony was transferred in nutrient broth followed by incubation at 30°C for a duration of 24 h.The overnight grown culture was adjusted to 0.5 McFarland standard for further antibacterial assay.

Preparation of bacterial extract
The extraction of bacterial metabolites and preparation of stock concentrations were prepared according to Muangpat et al. 21with a slight modification.The whole-cell suspension of the bacterial isolates was used for preliminary screening of antibacterial activity.A 50 µl of the cell suspension was incorporated into the agar well containing the spread pathogenic strain and kept at 30°C for 24 h.A void zone around the well was read as an inhibition zone.For all the bacterial isolates, a colony was shifted to a 1000 ml sterile nutrient broth, stored at 28°C in a shaker incubator for 48 h and subsequently transferred to 2000 ml of separating funnel.To extract the crude compound, the same volume of whole-cell suspension and ethyl acetate were mixed by inverting the funnel and laid at room temperature for 24 h.Further, ethyl acetate layer was collected, then evaporated using Rotavapor ® R-100 System-Buchi, Switzerland.To obtain the maximum amount of crude extract, the extraction procedure was repeated twice.
A stock solution was made by solvating 500 mg of the condensed bacterial extract in 1 ml of DMSO.A unit of 10 µl from the stock solution was pipetted out and impregnated into 6 mm paper disks.The paper disks were then placed on Mueller Hinton Agar (MHA) previously plated with the selected pathogenic strain followed by incubation at 30°C for a duration of 24 h.A caliper was used to measure the diameter of a clear zone (in mm).A drop of DMSO served as a negative control, whereas a standard ampicillin disk served as a positive control.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
The MIC of the bacterial extracts was performed following the microdilution method in a 96-well microtiter plate.The same volume of bacterial extracts and sterile Mueller Hinton Broth (MHB) was mixed in a well followed by two-fold serial dilutions.Then, each well was inoculated with 10 µl of microbial inoculum initially adjusted to 0.5 Mc Farland standard.After thorough mixing, plate incubation was performed at 30 °C for 24 h.For control, a DMSO mixture and nutrient broth with inoculum and the same mixture without inoculum were used.The MIC was determined as minimum bacterial extract concentration along with a clear well as detected by the unaided eye.
For MBC, 10 µl of the extract dilution representing the MIC along with two more concentrated diluted wells were streaked on MHA to observe visible growth.The plates incubation was done at 30 °C for 24 h and growth was observed corresponding to different concentrations.The MBC value was determined as minimum bacterial extract concentration without observable growth on MHA.

Statistical data analysis
The zone of inhibition size (in diameter) is given as Mean ± Standard Error of Mean (SEM).One-way ANOVA was carried out to determine a variation of inhibition exhibited by the bacterial isolates (at the level of P ≤ 0.05) against the selected pathogenic bacteria.

Characterization of the bacteria
The two Photorhabdus isolates TS and TD were isolated from H. indica and H. baujardi, respectively, while the two Xenorhabdus isolates, RF and PTS were isolated from S. sangi and S. surkhetense, respectively.
In addition, the ML tree constructed from the concatenation of the two nucleotide sequences (recA and gyrB genes) showed that the Photorhabdus isolates, TS and TD clustered cohesively with the type strain of P. hindustanensis H1 T (0.6% K2P distance) and P. namnaonensis PB45.5 T (2%) respectively.Also, the Xenorhabdus isolates, RF and PTS are closely related with the type strain of X. vietnamensis VN01 T (0.1% K2P distance) and X. stockiae TH01 T (3% K2P distance) respectively.
The maximum likelihood tree of bacterial symbionts and database sequences from NCBI GenBank are given in Table 1 and Figure 1-6.

Antibacterial activity
The extract of all bacterial isolates inhibited the growth of E. coli (ATCC 10536), K. pneumoniae (ATCC 10031), P. aeruginosa (ATCC 10145), and B. subtilis (ATCC 11774) within 24 h of incubation.(Figure 7).Additionally, from our study, the extracts of the bacterial isolates including standard ampicillin showed significant variations of growth inhibition against the selected pathogenic bacteria (df=4,43; F=31.96; p<0.05).However, no significant difference was observed in the activity of TS isolates and ampicillin(p>0.05)while the other extracts showed a significant difference in  Maximum likelihood tree of Photorhabdus isolates inferred from recA gene.The numbers at the nodes correspond to the bootstrap support (1000 replicates, 50% or more).GenBank accession numbers and strain codes were given along with each species activity when compared with standard ampicillin (p>0.05).Among the bacterial isolates, the extracts of TS and TD were found to be most potent against the pathogenic bacteria with a growth inhibition zone of 13.67-16.33mm diameter.The highest inhibition on the pathogenic bacteria was recorded with K. pneumoniae (ATCC 10031) where the bacterial extract of Photorhabdus isolates (TS) provided a clear inhibition zone of 16.33 ± 0.33 mm in diameter.In addition, the bacterial extract of X. vietnamensis inhibited the growth of all four tested bacterial strains, with a clear inhibition zone of 13-13.67 mm diameter.The extract of X. stockiae showed comparatively lower activity against the pathogenic bacteria as compared to the other isolates, exhibiting a clear inhibition zone of 10-11.67 mm diameter against the studied bacterial strains.The Mean ± SEM of the inhibitory activity of the bacterial isolates against pathogenic bacteria was given in Table 2.
The MIC and MBC values of Photorhabdus and Xenorhabdus extracts against the morbific subtilis at the MIC and MBC values of 3.90 mg/ml and 7.81 mg/ml, respectively.
In the case of Xenorhabdus isolates, both X. vietnamensis and X. stockiae show MIC Figure 4. Maximum likelihood tree of Xenorhabdus isolates inferred from gyrB gene.The numbers at the nodes represent bootstrap proportion value (1000 replicates, 50% or more).GenBank accession numbers and strain codes were given along with each species and MBC values ranging from 7.81-3.90mg/ml and 15.62-3.90mg/ml, respectively.The highest activity of Xenorhabdus isolates was observed at the same value of MIC and MBC (3.90 mg/ml) with X. vietnamensis and X. stockiae against E. coli (ATCC 10536) and K. pneumoniae (ATCC 10031), respectively.In addition, both the Xenorhabdus isolates show the lowest activity against B. subtilis (ATCC 11774) with a MIC value of 7.81 mg/ml and an MBC value of 15. 62 mg/ml.Therefore, as per the observed MIC and MBC values, the overall activities of Photorhabdus extracts were Figure 5. Maximum likelihood tree of Photorhabdus derived from two concatenated protein coding genes (recA and gyrB).The numbers at the nodes represent bootstrap proportion value (1000 replicates, 50% or more) and strain codes were given along with each species Figure 6.Maximum likelihood tree of Xenorhabdus from two concatenated protein coding genes (recA and gyrB).The numbers at the nodes represent bootstrap proportion value (1000 replicates, 50% or more) and strain codes were given along with each species higher against the test pathogenic organisms as compared to Xenorhabdus isolates.

DISCUSSION
The subsequent analysis of the ML tree inferred from recA and gyrB revealed that the two Xenorhabdus, RF and PTS isolates, clustered cohesively with the previously identified X. vietnamensis and X. stockiae respectively, and thus belong to it.In contrast, some complications occurred to resolve the exact identity of the two Photorhabdus isolates (TS and TD).Lalramchuani et al., 32 though forming a deep split between the two isolates, identified both the isolates as P. luminescens subsp.akhurstii using 16S rRNA gene.However, further analysis and reconstruction of the phylogenetic tree using the two housekeeping genes (recA and gyrB) revealed that TD isolates belong to P. namnaonensis.Analysis of the two genes further revealed that a consistent gap still exists between the two isolates by splitting into  when analyzed with recA gene.However, analysis using another coding gyrB revealed that TD isolates clustered closely with P. hainanensis (RCWD01).Therefore, the closer relationship of TD isolates with P. hainanensis (RCWD01) when analyzed with gyrB gene leads to confusion while there is a considerable separation between the two using another gene.The occurrence of this type of discrepancy might be the result of evolutionary pressure within different genes. 30t is thus clear that phylogenetic analysis using a single gene causes incongruence regarding species identification, particularly in closely related species.Moreover, the concatenated ML tree indicated that the TD isolates and P. namnaonensis PB45.5 T clustered together cohesively, thereby suggesting them to be similar species.Variations of activities including the degree of inhibition against the tested pathogenic organisms were observed among the isolates.This may be attributed to several factors such as the production of secondary active substances by Photorhabdus and Xenorhabdus, including media used for culture, medium pH, temperature, inoculation volume, fermentation time, rotary speed 33 and bacterium-nematode affected insect cadaver conditions. 34These conditions may result in variability in the activity outcomes of bacterial extracts among various workers worldwide.
0][21] The compound, lumicin from P. luminescens subsp.akhurstii showed satisfying activity against E. coli. 35The high potency of Photorhabdus isolates against targeted microbes might be due to the production of several bioactive compounds including isopropylstilbenes and ethylstilbenes derivatives. 14,15 urthermore, Photorhabdus spp.are also known to produce broad-spectrum antibiotic properties including carbapenem, which is a prominent class of b-lactam antibiotics, responsible for activity against E. coli, K. pneumoniae, and E. cloacae. 36enorhabdus spp.are recognized for producing a wide range of chemicals having considerable antibacterial, antifungal, insecticidal and nematicidal activities. 33X. stockiae extracellular metabolites show bactericidal activity against mastitis associated pathogens such as E. coli and B. subtilis and S. aureus. 37,38and further could inhibit the growth of P. aeruginosa, but failed to inhibit E. coli and K. pneumoniae. 20We have observed that the extract of X. stockiae suppressed the growth of all the tested pathogenic strains, including E. coli and K. pneumoniae, contradicting Muangpat et al. 20 However, the inhibition is comparatively lower compared to other studied isolates.Moreover, comparing the two Xenorhabdus isolates, the extract of X. vietnamensis showed higher activity than X. stockiae against all the tested pathogenic microorganisms.The potency of the genus Xenorhabdus against several microbes is due to the presence of compounds; fabclavine, 39 dithiolopyrrolone derivative 40 , indole derivative compounds, 41 xenocin, 42 etc.The present report on the potential antibiotic activity possessed by X. vietnamensis adds another important data on antibiotic activity existing among the genus Xenorhabdus.

CONCLUSION
The current study focuses on the occurrence and identification of symbiotic bacteria in Mizoram, North-east India, which is part of an Indo-Burma biodiversity hotspot region.This study provides information on the symbiont's ability to function against specific harmful microorganisms.Among the isolates, we observed that Photorhabdus isolates were found to be more active compared with Xenorhabdus isolates.However, detailed analysis and studies need to be carried on to increase the knowledge of the exact genetic information, the composition of chemical compounds, and the mode of action against other microbes since these extracted metabolites could be a promising antibiotic in the future.This finding will pave the way for the identification

Figure 1 .
Figure 1.Maximum likelihood tree of Photorhabdus isolates inferred from recA gene.The numbers at the nodes correspond to the bootstrap support (1000 replicates, 50% or more).GenBank accession numbers and strain codes were given along with each species

Figure 2 .
Figure 2. Maximum likelihood tree of Xenorhabdus isolates inferred from recA gene.The numbers at the nodes correspond to the bootstrap support (1000 replicates, 50% or more).GenBank accession numbers and strain codes were given along with each species

Table 1 .
Sequences analysed in this study

Table 2 .
Activity of bacterial extracts against pathogenic bacteria using disk diffusion method (Mean±SEM)

Table . 3
MIC and MBC of bacterial extracts against pathogenic bacteria (mg ml -1 )