Research Article | Open Access
Mary Lalramchuani1,2, Lalramliana1 , Hrang Chal Lalramnghaki1,3,Vanramliana1 and Esther Lalhmingliani2
1Department of Zoology, Pachhunga University College, Mizoram University, Aizawl, Mizoram, India.
2Systematics and Toxicology Laboratory, Department of Zoology, Mizoram University, Aizawl, Mizoram, India.
3Developmental Biology and Herpetology Laboratory, Department of Zoology, Mizoram University, Aizawl, Mizoram, India.
Article Number: 8619 | © The Author(s). 2023
J Pure Appl Microbiol. 2023;17(3):1560-1577. https://doi.org/10.22207/JPAM.17.3.18
Received: 13 April 2023 | Accepted: 10 July 2023 | Published online: 11 August 2023
Issue online: September 2023
Abstract

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.

Keywords

Antibiotic, Photorhabdus, Xenorhabdus, gyrB, recA

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 Furthermore, widespread antibiotic use and self-medication by farmers and patients, respectively, as well as hospital infection exposure, have accelerated the growth of multidrug-resistant (MDR) bacteria globally.2–4 The widespread antibiotic resistance was reported globally and declared a pandemic.5 Recently, 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.2,6 Also, 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.8 Thus, 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.9 They have a cosmopolitan distribution (except Antarctica) and are highly effective at controlling a variety of insect pests.10 Altogether, Photorhabdus has 28 recognized taxa, including 22 species, six of which are further classified into subspecies.11 So far, 28 taxa of Xenorhabdus associated with the nematodes have been identified including two recently identified subspecies.12 When 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.13 Meanwhile, several compounds produced by bacterial symbionts are known to protect the insect cadaver microenvironment due to their antimicrobial, nematicidal, acaricidal and insecticidal activity.14-16 The bacteria multiplied within the insect host, the nematodes ingest the bacteria and consume the cadaver for growth and reproduction.17 When 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

The secondary metabolites of Photorhabdus spp. and Xenorhabdus spp. including methanol and ethyl acetate extracted bioactive compounds from the fermented culture media were found to be effective in controlling various pathogenic bacteria.19-21 and fungi 14, 22-24 to a great extent. In addition to antibacterial and antifungal, the bioactive compounds have been claimed to be effective against insects.13,25,26 Therefore, 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.

Materials and Methods

Isolation and identification of bacterial symbionts
The symbiotic bacteria were isolated from entomopathogenic nematodes (EPNs) viz. Heterorhabditis indica (location: 23.740N 92.952E), H. baujardi (location: 22.350N 93.060E), Steinernema sangi (location: 23.370N 93.161E) and S. surkhetense (location: 22.960N 92.612E) which were randomly collected from four different localities of Mizoram, North-East India. The freshly emerged infective juveniles (IJs) of insect pathogenic nematodes were macerated for isolation of bacterial symbionts.27 Prior 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).27,28 followed 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.29 For 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.21 with 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.

RESULTS

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.

The total length of the recA sequence developed is 860 bp. The two isolates, TS and TD, consistently exhibited 2.2% K2P distance gap (98.1% similarity) between them. Further, based on the analysis of the developed gene region with closely related type species from the GenBank, the Photorhabdus isolates (TS) exhibited a closer relationship with P. hindustanensis (PUWT01) (99.53% similarity with 0.8% K2P distance). Another Photorhabdus isolate (TD) showed 98.59% (1.6% K2P distance) similarity with P. namnaonensis (LOIC01). Further analysis showed that the Photorhabdus (TS) isolate exhibited 99.41% (0.9% K2P distance), 98.24% (2% K2P distance) and 98.13% (2% K2P distance) similarity with P. akhurstii subsp. akhurstii (RCWE01), P. hainanensis (RCWD01) and P. akhurstii subsp. bharatensis (RCWU01) respectively, whereas, the Photorhabdus isolate (TD) exhibited 98% (2.2% K2P distance), 97.83% (2.3% K2P distance), and 96.9% (3.0% K2P distance) similarity with P. hindustanensis (PUWT01), P. akhurstii subsp. akhurstii (RCWE01) and P. hainanensis (RCWD01) respectively. Simultaneously, the two Xenorhabdus isolates, RF and PTS exhibited highest similarity with X. vietnamensis (FJ823401) (99.69% similarity with 0.3% K2P distance) and X. stockiae (KX826948) (99.4% similarity with 0.6% K2P distance) respectively. Further, the isolates, RF showed 95.5% similarity with X. japonica (FJ823400) and PTS isolates showed 93.21% similarity with X. innexi (FJ823423).

The total length of the gyrB sequence developed is 1050 bp. The two Photorhabdus isolates, TS and TD showed 96.88% similarity (3.2% K2P distance) between them. Further analysis of the isolates and comparison with the type species on NCBI GenBank showed that the Photorhabdus isolates (TS) exhibited 99.43% (0.5% K2P distance) similarity with P. hindustanensis (PUWT01), whereas, Photorhabdus (TD) isolates exhibited the highest similarity with P. hainanensis (RCWD01) (98.30% similarity with 1.7% K2P distance. Comparisons with other closely related species show that Photorhabdus isolates (TS) exhibited 97.83% (2.2% K2P distance), 96.60% (3.5% K2P distance), 97.17% (2.9% K2P distance) similarity with P. akhurstii subsp. akhurstii, P. akhurstii subsp. bharatensis (RCWU01) and P. hainanensis (RCWD01), respectively, whereas, the Photorhabdus (TD) isolates showed 97.26% (2.8% K2P distance), 96.22% (2.8% K2P distance) and 96.88% (3.2% K2P distance) similarity with P. namnaonensis (LOIC01) and P. akhurstii subsp. akhurstii (RCWE01) and P. hindustanensis (PUWT01) respectively. Simultaneously, the Xenorhabdus isolates, RF and PTS, showed 99.88% and 99.15% (0.0-0.8% K2P distance) similarity with the database sequence of X. vietnamensis (EU934514) and X. stockiae (KX826949) respectively. Further, among the Xenorhabdus isolates, RF showed 95.48% similarity with X. japonica (EU934513) and PTS showed 93.97% similarity with X. innexi.

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 H1T (0.6% K2P distance) and P. namnaonensis PB45.5T (2%) respectively. Also, the Xenorhabdus isolates, RF and PTS are closely related with the type strain of X. vietnamensis VN01T (0.1% K2P distance) and X. stockiae TH01T (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.

Table (1):
Sequences analysed in this study.

Strain
Species
Nematode Host
NCBI Accession No. (recA)
NCBI Accession No. (gyrB)
Country
References
TS1
Photorhabdus. hindustanensis
H.  indica
ON314156
ON314147
Mizoram, India
This study
TS2
Photorhabdus. hindustanensis
H. indica
ON314157
ON314148
Mizoram, India
This study
TS3
Photorhabdus hindustanensis
H. indica
ON314158
ON314149
Mizoram, India
This study
TD1
Photorhabdus namnaonensis
H. baujardi
ON314159
ON314150
Mizoram, India
This study
TD2
Photorhabdus namnaonensis
H. baujardi
ON314160
ON314151
Mizoram, India
This study
TD3
Photorhabdus namnaonensis
H. baujardi
ON314161
ON314152
Mizoram, India
This study
RF1
X.  vietnamensis
S.  sangi
ON314162
ON314168
Mizoram, India
This study
RF2
X. vietnamensis
S. sangi
ON314163
ON314169
Mizoram, India
This study
RF3
X. vietnamensis
S. sangi
ON314164
ON314170
Mizoram, India
This study
PTS1
X.  stockiae
S. surkhetense
ON314165
ON314153
Mizoram, India
This study
PTS2
X.  stockiae
S. surkhetense
ON314166
ON314154
Mizoram, India
This study
PTS3
X.  stockiae
S. surkhetense
ON314167
ON314155
Mizoram, India
This study
MEX47-22T
Photorhabdus luminescens subsp. mexicana
H. mexicana
PUJX01
PUJX01
Mexico
43
Hb T
P. luminescens subsp. luminescens
H. bacteriophora
FMWJ01
FMWJ01
Australia
43
JART
P. luminescens subsp. venezuelensis
H. amazonensis
JAPFFZ01
JAPFFZ01
Venezuela
11
C1 T
P. khanii subsp. khanii
H. bacteriophora
AYSJ01
AYSJ01
USA
43
MEX20-17T
P. khanii subsp. guanajuatensis
H. atacamensis
PUJY01
PUJY01
Mexico
43
TTO1T
P. laumondii subsp. laumondi
H. bacteriophora
WSFH01
WSFH01
Australia
44
BOJ47T
P. laumondii subsp. clarkei
H. bacteriophora
NSCI01
NSCI01
Iran
44
FRG04T
P. akhurstii subsp. akhurstii
H. indica
RCWE01
RCWE01
Australia
45
H3T
P. akhurstii subsp. bharatensis
Heterorhabditis sp.
PUWU01
PUWU01
India
45
SF41T
P. heterorhabditis subsp. heterorhabditis
H. zealandica
RCWA01
RCWA01
South Africa
46
Q614T
P. heterorhabditis subsp. aluminescens
Heterorhabditis sp.
JABBCS01
JABBCS01
Australia
46
9802892T
P. australis subsp. australis
Clinical specimen
JONO01
JONO01
Australia
46
PB68.1T
P. australis subsp. thailandensis
H. indica
LOMY01
LOMY01
Thailand
46
AM7T
P. noenieputensis
H. noenieputensis
JQ424881
JQ424884
South Africa
44
C8404T
P. hainanensis
Heterorhabditis sp.
RCWD01
RCWD01
China
44
PB45.5 T
P. namnaonensis
H. baujardi
LOIC01
LOIC01
Thailand
47
HG29 T
P. caribbeanensis
H. bacteriophora
RCWB01
RCWB01
Guadeloupe
44
H1T
P. hindustanensis
Heterorhabditis sp.
PUWT01
PUWT01
India
45
DSM 23513T
P. kleinii
H. georgiana
JAJAFY01
JAJAFY01
North America
44
DSM 15194 T
P. kayaii
H. bacteriophora
JAJAFZ01
JAJAFZ01
Turkey
44
LJ24-63T
P. bodei
H. beicherriana
NSCM01
NSCM01
China
44
BA1T
P. aegyptia
H. indica
JFGV01
JFGV01
Egypt
44
39-8T

PT1.1

P. thracensis
H. bacteriophora
CP011104
JAGJJU01
Turkey
48, 44
XINach T

M1021

P. temperata
H. megidis
JAJAFX01
AUXQ01
Russia
44, 49
DSM 23271 T
P. stackebrandtii
H. bacteriophora
PUJV01
PUJV01
North America
44
T327 T
P. tasmaniensis
H. zealandica
PUJU01
PUJU01
Australia
44
UCH-936T
P. antumapuensis
H. atacamensis
JAHZMK01
JAHZMK01
Chile
50
3107 T
P. cinerea
H. downesi
PUJW01
PUJW01
Hungary
44
3265-86T
P. asymbiotica
Clinical specimen
RBLJ01
RBLJ01
USA
44
APURET
P. aballayi
H. amazonensis
JAPFCD01
JAPFCD01
Switzerland
11
VN01 T
X. vietnamensis
S. sangi
FJ823401
EU934514
Vietnam
30
DSM16522 T
X. japonica
S. kushidai
FJ823400
EU934513
Japan
30
ID10 T
X. griffiniae
S. hermaphroditum
FJ823399
EU934525
Indonesia
30
DSM16337 T
X. ehlersii
S. serratum
FJ823398
EU934524
China
30
FRM16 T
X.  doucetiae
S. diaprepesi
FJ823402
EU934526
Martinique
30
PR06-A T
X.  romanii
S. puertoricense
FJ823403
EU934515
Puerto Rico
30
SaV T
X.  kozodoii
S. arenarium
FJ823404
EU934522
Russia
30
G6 T
X. poinarii
S. glaseri
FJ823409
EU934543
USA
30
SF87 T
X. khoisanae
S. khoisanae
AB685736
AB685735
South Africa
52
Q1 T
X. miraniensis
Steinernema sp.
FJ823414
EU934520
Australia
30
Q58 T
X. beddingii
Steinernema sp.
FJ823415
EU934516
Australia
30
KE01 T
X. hominickii
S. karii
FJ823410
EU934517
Kenya
30
USNJ01 T
X.  koppenhoeferi
S. scarabaei
FJ823413
EU934532
USA
30
ATCC19061 T
X. nematophila
S. carpocapsae
FN667742
FN667742
USA
53
DSM16338 T
X. szentirmaii
S. rarum
FJ823416
EU934534
Argentina
30
VC01 T
X. mauleonii
Unknown
FJ823417
EU934533
St. Vincent
30
VP-2016b
X. stockiae
S. surkhetense
KX826948
KX826949
India
54
TH01 T
X.  stockiae
S. siamkayai
FJ823425
EU934542
Thailand
30
DSM16336 T
X.  innexi
S. scapterisci
FJ823423
EU934540
Uruguay
30
DSM16342 T
X. budapestensis
S. bicornitum
FJ823418
EU934535
Serbia
30
IMI397775 T
X.  magdalenensis
S. australe
FJ798401
JF798402
Chile
51
DSM17382 T
X. indica
S. thermophilum
FJ823421
EU934538
India
30
USTX62 T
X. cabanillasii
S. riobrave
FJ823422
EU934537
USA
30
T228 T
X. bovienii subsp. bovienii
S. feltiae
JANAIF01
JANAIF01
Australia
12
XENO-1T
X. bovienii subsp. africana
S. africanum
JAMGSK01
JAMGSK01
Africa
12
VLST
X. lircayensis
S. unicornum
JACOII01
JACOII01
Chile
55
30TX1T
X. thuongxuanensis
S. sangi
KX602194
KY451961
Vietnam
56
DL20T
X. eapokensis
S. eapokense
KX602188
KY451960
Vietnam
56
GDh7T
X. ishibashii
S. aciari
AB630947
AB630948
Japan
57
K-12
Escherichia coli
 
NC_000913.3
NC_000913.3
Gen Bank

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. 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

Figure 3. Maximum likelihood tree of Photorhabdus isolates inferred from gyrB 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 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

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 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 7. Antibacterial activities of bacterial isolates against pathogenic bacteria using disk diffusion method. (PA) P. aeruginosa (ATCC 10145, (BS) Bacillus subtilis (ATCC 11774), (KP) K. pneumoniae (ATCC 10031, (EC) E. coli (ATCC 10536), (1) TS, (2) TD, (3) RF, (4) PTS, (AMP) Ampicillin standard disk (10 µg) and (N) Negative control (DMSO)

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 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– 16.33 mm 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.

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

Bacterial strain
E. coli
(ATCC 10536)
K. pneumoniae
(ATCC 10031)
P. aeruginosa
(ATCC 10145)
B. subtilis
(ATCC 11774)
Standard
(Ampicillin)
TS
15.330.33
16.330.33
14.330.33
13.670.33
15.670.33
TD
140.57
14.330.33
13.670.67
13.670.33
15.670.33
RF
13.330.33
13.670.33
130.57
13.330.33
15.330.33
PTS
100.57
11.670.3
100.57
10.670.33
16.670.33

The MIC and MBC values of Photorhabdus and Xenorhabdus extracts against the morbific bacteria were given in Table 3. The MIC value of both Photorhabdus isolates ranges from 3.90–1.95 mg/ml and the MBC value range from 7.81–1.95 mg/ml. Furthermore, the Photorhabdus isolates, TS and TD exhibited the highest activity at the same MIC and MBC value (1.95 mg/ml) against P. aeruginosa (ATCC 10145) and K. pneumoniae (ATCC 10031), respectively. In addition, the lowest inhibitory activity was observed with Photorhabdus isolates (TS and TD) against B. subtilis at the MIC and MBC values of 3.90 mg/ml and 7.81 mg/ml, respectively.

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

Bacterial strain E. coli
(ATCC 10536)
K. pneumoniae (ATCC 10031) P. aeruginosa (ATCC 10145) B. subtilis
(ATCC 11774)
MIC MBC MIC MBC MIC MBC MIC MBC
TS 3.90 3.90 1.95 3.90 1.95 1.95 3.90 7.81
TD 1.95 3.90 1.95 1.95 3.90 3.90 3.90 7.81
RF 3.90 3.90 3.90 7.81 3.90 7.81 7.81 15.62
PTS 7.81 7.81 3.90 3.90 3.90 7.81 7.81 15.62

In the case of Xenorhabdus isolates, both X. vietnamensis and X. stockiae show MIC and MBC values ranging from 7.81–3.90 mg/ml and 15.62–3.90 mg/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 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 two sub-clades. The Photorhabdus (TS) isolate undoubtedly belongs to P. hindustanensis as it clustered cohesively with the type species of P. hindustanensis H1T (PUWT01). Meanwhile, the Photorhabdus (TD) isolate showed a deep split from Photorhabdus (TS) isolate clade and clustered closely with P. namnaonensis PB45.5T (LOIC01) when analyzed with recA gene. However, analysis using another coding gene, 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.30 It 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 PB45.5T 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 speed33 and bacterium–nematode affected insect cadaver conditions.34 These conditions may result in variability in the activity outcomes of bacterial extracts among various workers worldwide.

Consistent with our study, the more efficient antibacterial activities of Photorhabdus, compared to Xenorhabdus isolates, against several disease-causing bacteria i.e., E. coli, P. aeruginosa, and K. pneumoniae were also reported in previous studies.19-21 The compound, lumicin from P. luminescens subsp. akhurstii showed satisfying activity against E. coli.35 The 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 Furthermore, 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.36

Xenorhabdus spp. are recognized for producing a wide range of chemicals having considerable antibacterial, antifungal, insecticidal and nematicidal activities.33 X. stockiae extracellular metabolites show bactericidal activity against mastitis associated pathogens such as E. coli and B. subtilis and S. aureus.37,38 and further could inhibit the growth of P. aeruginosa, but failed to inhibit E. coli and K. pneumoniae.20 We 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 derivative40, 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 and investigation of certain symbiotic bacteria including their metabolites for the treatment of various diseases.

Declarations

ACKNOWLEDGMENTS
The authors would like to thank Principal, Pachhunga University College; Head, Department of Life Sciences, Pachhunga University College for providing the necessary research facilities to carry out this work. The authors acknowledge the support from DBT, Government of India for Advance Level Institutional Biotech Hub and DST, Government of Mizoram.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
This work was funded by National Mission on Himalayan Studies (NMHS) under the Himalayan Fellowship (U/I ID: HSF 2018-19/I-25/03; No. GBPNI/NMHS-2018-19/HSF 25-03/154, Dt. 17.12.2018). Research facilities were provided by DBT-BUILDER (BT/INF/22/SP41398/2021) of the Department of Biotechnology, Government of India.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.

ETHICS STATEMENT
Not applicable.

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