Antimicrobial Activities of Symbiotic and Non-symbiotic Bacteria Associated with the Entomopathogenic Nematode, Steinernema feltiae

The use of antibiotics against a range of pathogenic bacteria has increased in recent years, leading to the development of drug resistance, which makes disease control challenging. Thus, the need for the development of new antibacterial medications is critical. Natural resources, such as entomopathogenic bacteria (EPB), provide a rich source of metabolites with well-known antibacterial properties. The present study aimed to investigate the antibacterial activity of symbiotic ( n = 1) and non-symbiotic ( n = 8) entomopathogenic bacterial species associated with the entomopathogenic nematode (EPN) Steinernema feltiae against four multidrug-resistant bacterial species. Bacterial cells and filtrates from Xenorhabdus bovienii strongly inhibited the growth of Staphylococcus aureus (33.3 and 28.9 mm) and Escherichia coli (24.6 and 21.6 mm) in disk diffusion, minimum inhibitory concentration (MIC) (2 and 8 µl/ml) and minimal bactericidal concentration (MBC) (4 and 12.5 µl/ml) assays. In conclusion, the direct application of endogenous S. feltiae -associated EPB, especially X. bovienii , appears promising as an antibacterial agent against multidrug-resistant bacteria (MRBs).


INTRODUCTION
Antibiotic multidrug resistance has adverse implications for public health worldwide.In terms of public health, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus cereus, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa are important multidrug-resistant bacteria. 1ntibiotics are commonly used due to their effectiveness against a wide range of pathogenic bacteria.3][4] Multidrugresistant bacterial infections, although difficult to measure accurately, are expected to result in nearly 10 million deaths annually by 2050. 5herefore, treatments other than antibiotics are required to target bacteria that are resistant to multiple drugs.
One treatment strategy is the use of biological compounds derived from bacterial or natural sources.These compounds (secondary metabolites or natural products) are a key starting point when developing new pharmaceuticals.Novel compounds with unique structures, high activity, and high selectivity have been discovered via screening natural products. 6Fungi, 7,8 plants, 9 and bacteria 6,10,11 are the three major producers of natural products in nature.An alternative that seems preferable is using antimicrobial peptide (AMP) molecules, which are synthesized by soil-dwelling organisms.Many bacterial species produce antimicrobial toxins in the form of secondary metabolites.In entomopathogenic nematode/bacterium symbiotic relationships, the prokaryotic symbiont's antimicrobials have preserve the monoxenic environment for EPB in the intestine of infective dauer EPN juveniles. 12enorhabdus and Photorhabdus, members of the Enterobacteriaceae family of gram-negative bacteria, which form symbiotic associations with infected EPN juveniles of the Steinernema and Heterorhabditis genera, respectively, provide new bioactive pharmaceuticals for treating microorganisms. 13EPNs deliver cooperative bacteria into the insect hemolymph when they infect a target insect.These bacteria release toxins and enzymes, which kill the insect host within 48 hours. 14,157][18] These bacteria are motile rods, oxidase-negative, non-spore forming, chemoorganotrophic heterotrophs, with respiratory and fermentative metabolism and facultative anaerobes. 19n the past, the association between EPNs and their mutualistic bacteria was thought to be monoxenic.][22] Xenorhabdus, the primary symbiont, and a dozen other regularly occurring microbiota (e.g.Deftia, Stenotrophomonas, Pseudomonas, Achromobacter, Alcaligenes, Pseudochrobactrum, Brevundimonas, and Ochrobactrum) constituted the bacterial group associated with laboratoryreared dauers from Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema weiseri. 23The general hypothesis is that non-symbiotic bacteria 'hitchhike' in infected juvenile vectors at random through the cuticle or inter-cuticular region and enter the insect haemocoel during infected juvenile penetration. 24enorhabdus strains are thought to have commercial potential in the production of novel antibiotics, which could be used to manage therapeutically significant pathogens, as well as oral insecticidal toxins for use in biocontrol. 25revious research demonstrated that bacterial antimicrobial resources, including symbiotic or non-symbiotic bacterial cells and filtrates, successfully prevented the growth of K. pneumonia, E. coli, and Enterobacter cloacae, 26 Bacillus subtilis and Botrytis cinerea, 27 Streptococcus pyogenes and S. aureus, 28,29 Rhizoctonia solani, Phytophthora capsici, and Bacillus anthracis 30 and Fusicladium effusum. 31These bacteria produce numerous secondary metabolites with antibiotic activity. 29hese metabolites include chaiyaphumine, 32 1-carbapen-2-em-3-carboxylic acid, 26 3-hydroxy-2-isopropyl-5-phenethylphenyl carbamate, 33 3,5-dihydroxy-4-isopropystilbene, 27 2-isopropyl-5-(3-phenyl-2-oxiranyl0-benzene-1,3diol, 34 and benzaldehyde. 30Data on the antibacterial potential of EPB linked to EPNs in South western part of Saudi Arabia are lacking.Thus, this study aimed to assess the antibacterial effectiveness of EPB associated with S. feltiae against four multidrugresistant bacterial species (B.cereus, E. cloacae, E. coli, and S. aureus) using disk diffusion, minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays.

S. feltiae-associated bacteria
6][37] Nine isolates of EPB (Xenorhabdus bovienii, Stenotrophomonas maltophilia, Stenotrophomonas tumulicola, Pseudomonas mosselii, Pseudochrobactrum saccharolyticum, Serratia liquefaciens, Lysinibacillus xylanilyticus, Advenella kashmirensis and Aeromonas hydrophila) were maintained on NBTA medium (nutrient agar with 0.025% bromothymol blue and 0.004% triphenyl tetrazolium chloride) and incubated 2 days at 28°C.To produce cell suspensions or cell-free conditioned filtrates, Five millilitres of Luria-Bertani (LB) broth were inoculated with one colony of each bacterial isolate, and they were stirred constantly (220 rpm) overnight at 28°C.Then, 400 ml of LB medium with 100 ml aliquots of the culture that had been shaken for 24 hours at room temperature were transferred to flasks and agitated at 200 rpm for five days.The multiplied bacterial culture was centrifuged (13,000 rpm for 30 minutes) at 4°C to obtain the supernatant and bacterial pellets.After that, a 0.22 m Millipore filter was used to separate a filtrate free of cells, and the pellet was resuspended in sterile distilled water.Further dilution of the filtrate with sterilized-distilled water was done to obtain concentrations of 300, 150, 75, 50, 25, 12.5, 8, 4, 2 and 1 μl/ml and then stored at 4°C.A spectrophotometer was used to adjust the bacterial cell culture from OD600 to 1.0.A spread plate with a 10-fold serial dilution generated a bacterial culture containing 1 × 10 6 (CFU/ml).

Multidrug-resistant bacteria preparation
Four isolates of MRBs (E.coli, S. aureus, B. cereus and E. cloacae) were employed.The bacteria were plated onto Mueller-Hinton agar (MHA), which was then incubated at 37°C for 24 hours.In order to adjust the 0.5 McFarland standard for turbidity, one colony was dissolved in 0.85% sodium chloride.A sample of the bacterial culture (100 μl) was then spread on the MHA for a disk diffusion test. 38

Antibacterial activity
An MHA plate containing the MRBs was coated with 20 μl of each isolate's cell suspension (10 6 CFU/ml) or filtrate (150 μl/ml) to test the antibacterial potential of the nine entomopathogenic bacterial species.Following that, the plates were left to incubate for 24 hours at 37°C.A clean area from the edge of a bacterial colony that was expanding read positive results.A disc sensitivity test was conducted on EPB isolates that suppressed at least one multidrug-resistant bacterium.

Disk diffusion method
Twenty microliters of each filtrate (150 μl/ml) or cell suspension (10 6 CFU/ml) from the nine bacterial isolates were loaded onto sterile 6 mm paper discs, which were then placed on MHA plates with the isolates that were resistant to multiple drugs.Positive controls included antibiotic discs with ampicillin and penicillin, and negative controls included discs with distilled water.Eight replicates of each entomopathogenic bacterial isolate were tested against various MRBs.The plates were then stored at 37°C for 24 hours.Using a ruler, the clear zone's diameter (mm, representing the zone of inhibition) was estimated.

MIC and MBC assays
To determine the MIC, the broth microdilution technique was employed, utilizing the bacterial filtrates that give the best results for disk diffusion.Ten serial dilutions of the bacterial filtrate (300, 150, 75, 50, 25, 12.5, 8, 4, 2 and 1 μl/ml) were undertaken in a 96-well micro-titre plate.In a control treatment, multidrug-resistant bacteria were incubated in sterile Mueller-Hinton broth.After that, the plates were kept for 24 hours at 37°C.The MIC was the minimal filtrate amount that caused the well-located bacteria were not proliferating.To determine the MBCs, a subculture of 10 µl from each well of the 96-well MIC microtitre plate was placed onto MHA plates.The plates were then incubated at 37°C for 24 hours.The lowest concentration of each filtrate at which bacteria did not multiply was the MBC.All MIC and MBC assays were performed in triplicate.

Statistical analysis
Results are displayed as mean±standard error (M±SE).A one-way analysis of variance was conducted to statistically analyse the data, using the CoStat program, followed by multiple comparisons.P-values that were 0.05 or less were regarded as significant.

Screening of EPB isolates against multidrugresistant bacteria
The filtrates and the cell suspension of bacteria associated with S. feltiae inhibited most of the tested multidrug-resistant bacteria, with the level of inhibition varying from weak to strong (Table 1).E. coli was sensitive to eight of the EPB isolates, while seven of them were effective against S. aureus.B. cereus and E. cloacae were susceptible to six of the bacterial isolates.Among all the EPBs examined, X. bovienii filtrates and cells showed the strongest inhibition activity (+++) against all the tested multidrug-resistant bacteria.Cells of S. maltophilia strongly inhabited E. coli, E. cloacae, and S. aureus.Filtrates of this bacterium showed strong inhibition potential against E. cloacae and S. aureus and moderate inhibition activity (++) on B. cereus and E. coli.Although S. tumulicola and P. mosselii cells exhibited strong inhibition activity against E. coli and S. aureus, their filtrates exhibited moderate or weak (+) inhibition activity against these bacteria.No inhibition activity was detected when B. cereus, E. coli and E. cloacae bacteria were treated with P. saccharolyticum cells or filtrates.Except for E. coli, which was susceptible only to L. xylanilyticus cells, all the MRBs were resistant to its cells or filtrates (Table 1).

Disk diffusion method
The method of disk diffusion was used to verify the antimicrobial effect of the nine symbiotic and non-symbiotic bacteria associated with S. feltiae (Table 2

MICs
The values of MIC for the nine symbiotic and non-symbiotic bacteria linked to the EPN S. feltiae against four bacterial species were also assessed, as indicated in Table 3. EPB cell-free conditioned media with MICs between 2 and 300 µl/ml prevented the multidrug-resistant bacteria from growing.Among the bacteria tested, S. aureus (2 µL/ml) exhibited the highest estimated susceptibility to the X. bovienii filtrate, the next is E. coli (8 µl/ml), E. cloacae and B. cereus (12.5 µl/ml).All the tested bacteria were also sensitive to the S. maltophilia filtrate, which showed the strongest inhibitory activity (12.5 µl/ml) on S. aureus and E. cloacae, followed by E. coli (25 µL/ml) and B. cereus (50 µl/ml).S. tumulicola exhibited strong activity against S. aureus and E. coli (50 µl/ml) and moderate activity against E. cloacae and B. cereus (75 µl/ml).The MIC of S. liquefaciens was 75 µl/ml for all the tested bacteria.At a high MIC value (300 µl/ml), the L. xylanilyticus filtrate was only effective against E. coli. A. kashmirensis, A. hydrophila, penicillin and ampicillin also exhibited low activity against S. aureus, with an average MIC value (300 µl/ml).

MBCs
Except for EPB L. xylanilyticus, the multidrug-resistant bacterium S. aureus was most vulnerable to all filtrates, with MBCs ranging from 4 to 300 µl/ml (Table 4).With an MBC of 4 µl/ml, the X. bovienii filtrate had higher activity against S. aureus than any of the other tested bacteria, but a MIC value of 12.5 µl/ml was reported for E. coli, E. cloacae, and B. cereus.S. aureus was the least susceptible bacterium that was suppressed by A. kashmirensis and A. hydrophila.E. coli had less sensitivity to L. xylanilyticus and B. cereus to A. kashmirensis, with these having the highest MBC values (300 µl/ml).

DISCUSSION
The present study investigated nine symbiotic and non-symbiotic entomopathogenic bacterial species previously isolated from the EPN S. feltiae against four multidrug-resistant bacteria.The findings support those reported in a previous study, in which the five EPBs associated with S. feltiae were identified, and their efficiency against the two aphid species Aphis illinoisensis and A. punicae, was evaluated. 37The results of the current study also confirm those of earlier work, in which the authors isolated Photorhabdus and Xenorhabdus spp.from Heterorhabditis and Steinernema spp., respectively, in the same province and revealed their complex behaviours against Meloidogyne incognita infecting pomegranate under greenhouse conditions. 35he current research clearly demonstrates that entomopathogenic bacterial isolates provide a novel approach for preventing the spread of several multidrug-resistant bacteria.
Based on their antibacterial activity, the majority of the nine species of S. feltiaeassociated bacteria have been shown to prevent the proliferation of pathogenic bacteria, with variation in their antibacterial activity range.This might be as a result of each bacterium's ability to synthesize therapeutic molecules or the multidrugresistant bacteria's sensitivity.In addition, it was clear that the bacterial cells of the nine species were more potent than were the filtrates at eliminating multidrug-resistant bacteria, despite having low toxicity.Among the evaluated bacterial isolates, the strongest inhibitory impact was observed in cells and filtrates from the symbiont X. bovienii, and L. xylanilyticus displayed the weakest inhibition activity against all the tested multidrug-resistant bacteria.6][27][28][29][30][31] Similarly, these results agree with those of other research on the pathogenic soybean fungus Sclerotinia sclerotiorum and the larvae of the fall webworm (Hyphantria cunea). 39,40hus far, 29 species of Xenorhabdus and 20 species of Photorhabdus that produce different types of natural products have been isolated worldwide, including Europe, Australia, America, Africa and Asia. 19,41Xenorhabdus strains of EPB have strong commercial potential in the area of new antibiotic production, with such antibiotics targeting clinically relevant bacteria. 25ith the exception of P. saccharolyticum, the data obtained showed that E. coli was susceptible to all of the tested bacterial filtrates and cells.S. aureus was susceptible to all isolates, except L. xylanilyticus and A. kashmirensis.E. cloacae showed resistance to P. saccharolyticum, L. xylanilyticus and A. hydrophila.Furthermore, B. cereus appeared resistant to P. saccharolyticum, L. xylanilyticus and A. kashmirensis.Accordingly, based on values of the inhibition zone, MIC and MBC, the toxicity of EPBs toward the tested MRBs could be arranged as follows, in descending order: X. bovienii > S. maltophilia > S. tumulicola > S. liquefaciens > P. mosselii > A. kashmirensis > A. hydrophila > P. saccharolyticum > L. xylanilyticus.In descending order, the susceptibility of the MRBs was as follows: E. coli > S. aureus > E. cloacae > B. cereus.These findings are in accordance with those of earlier studies, which showed that X. budapestensis, X. szentirmaii, X. innexi, X. ehlersii, X. nematophila, X. bovienii and X. cabanillassii, as well as P. luminescens, inhibited the growth of clinical and multidrug-resistant isolates of S. aureus, E. coli, K. pneumoniae, and B. subtilis. 26,42t is evident that Xenorhabdus strains possess a wide variety of antibacterial components, making them potential sources of novel antibiotics against S. aureus, E. coli, E. cloacae and B. cereus that resist conventional antibiotics. 43These results were in agreement with those of previous research, which reported that Xenorhabdus produced derivatives of xenocoumacin and amicoumacin, 44,45 each of which has been demonstrated to be an effective antibiotic against S. aureus. 29All Photorhabdus spp.produce isopropylstilbene, 46,47 which has a wide range of biological properties, including antibiotic activity against E. coli and S. aureus. 33It has been proven that the S. aureus strains ATCC20475, PB36, and PB57 are sensitive to the inhibitory action of Photorhabdus extracts. 48Interestingly, the antibacterial activity of complete cell-free media was significantly higher than that of any isolated, recognized or patented molecules (e.g.nematophin). 49Xenorhabdus strains' antibiotically active, unpurified, cell-free liquid cultures are effective against a wide range of pathogens, including bacteria, fungus and protozoa.In earlier research, the protease inhibitor protein-encoding gene from the symbiotic bacterium X. bovienii strains BJFS526 and Xbpi-1 was discovered, produced, and evaluated for its impact on the pea aphid Acyrthosiphon pisum. 50,51ccording to Fuchs et al., 52 X. szentirmaii is a uniquely important source of peptides with excellent antibacterial properties that inhibit almost all known phytopathogens.In the present study, S. maltophilia and S. tumulicola isolates, either cells or filtrates, ranked second to X. bovienii in inhibiting the growth of the tested multidrugresistant bacteria.The results agree with those of a previous study, which revealed the termiticidal activity associated with S. maltophilia's synthesis of bacterial chitinases. 53In addition, they are in accordance with prior research that discovered S. maltophilia might have antagonistic effect against a spectrum of fungi and bacteria that are multidrug resistant. 54Berg 55 stated that S. maltophilia suppressed the phytopathogen Rhizoctonia solani growth, probably because of antibiosis and the production of certain lytic enzymes that destroy pathogenic fungi.Further studies showed that the metabolic complexity of S. maltophilia is responsible for the creation of novel bioactive compounds, including metabolites that could be used in the biocontrol of bacteria, in addition to the existence of enzymes that can be employed in therapeutic applications. 56In the present study, S. liquefaciens and P. mosselii exhibited moderate inhibition activity against the tested bacteria.The other EPB isolates, L. xylanilyticus, P. saccharolyticum and A. hydrophila, exhibited less bactericidal activity.In contrast, a previous study reported that crude chitinase from A. hydrophila may act as a potent biocontrol agent against insects and that it may be a good alternative to chemical pesticides. 57The present study confirmed the potential of one symbiotic and eight nonsymbiotic entomopathogenic bacterial species associated with the EPN S. feltiae in Saudi Arabia as antibacterial agents.[37]

CONCLUSION
In the current study, the antibacterial efficacy of one symbiotic and eight non-symbiotic entomopathogenic bacterial species associated with S. feltiae against four important multidrugresistant bacterial species was investigated.Overall, the results indicated that the EPB isolates had varying effects on several multidrug-resistant bacterial species.This might be due to the capacity of each bacterial species to produce beneficial compounds or the sensitivity of the MRBs to the specific metabolites produced by each symbiont or non-symbiont.Among the species tested, the symbiont X. bovienii exhibited excellent antibacterial activity against the four tested pathogenic bacterial species.The findings might serve as a starting point for the discovery of new bioactive substances.

Table 1 .
, Figure).At a concentration of 150 Antibacterial activity of filtrates or cells of nine EPB species against multidrug-resistant bacteria /ml filtrates and 10 6 CFU/ml cells for all nine EPBs, inhibitory zones (means±SE) ranging from 0.0 to 33.3 mm (P < 0.05) were observed.The nine isolates' bacterial cells were more efficient than their bacterial filtrates against all of the studied MRBs, causing a mean inhibition zone of 8.86 mm for the cells and 6.53 mm for the filtrates.The largest clear zones (33.3 and 28.9 mm) were obtained when S. aureus bacterium was exposed to X. bovienii cells and filtrates, a No inhibition (-): 0-5 mm; weak inhibition (+): 6-10 mm; moderate inhibition (++): 11-15 mm; strong inhibition (+++): > 15 mm μl

Table 2 .
Antibacterial potential of filtrates or cells of nine EPBs against multidrug-resistant bacteria according to disk diffusion analysis aIn this experiment, each treatment was represented by four repeats with two plates.The inhibition zone diameters ± standard errors are depicted by numbers in each column

Table 3 .
MICs of EPB filtrates (µl/ml) against multidrug-resistant bacteria a There were three repetitions of each treatment in this test.The numbers in each column indicate the MIC of each symbiotic or non-symbiotic bacterium.ND = not detected respectively.Moreover, E. coli, B. cereus, and E. cloacae growth was inhibited, with average zones of (24.6 and 21.6 mm), (21.6 and 17.0 mm), and (19.5 and 17.9 mm), respectively.S. maltophilia cells and filtrates also inhibited the growth of S. aureus, E. cloacae, E. coli and B. cereus, with clear zones of 20.4 and 19.3 mm, 19.4 and 18.0 mm, 17.3 and 14.5 mm, and 14.0 and 12.0 mm, respectively.With inhibition zones of 17.9 and 13.6 mm, 12.9 and 9.4 mm, 17.1 and 13.3 mm, and 11.8 and 7.8 mm for S. aureus, E. cloacae, E. coli, and B. cereus, respectively, the cells and filtrates of S. tumulicola were the third most lethal to the examined bacteria.S. liquefaciens ranked fourth in terms of inhibition zone activity against S. aureus (11.8 and 7.6 mm, 11.5 and 7.4 mm, 18.9 and 8.8 mm and 13.1 and 7.9 mm, respectively).Contrarily, S. aureus treated with filtrates of A. kashmirensis and A. hydrophila exhibited the smallest inhibitory zone (0.38 mm).L. xylanilyticus cells and filtrates showed no inhibition activity on B. cereus and E. cloacae (Table2).

Table 4 .
MBCs of EPB filtrates (µl/ml) against multidrug-resistant bacteria All experiments were conducted in triplicate.The numbers in each column indicate the MBC of each symbiotic or non-symbiotic bacterium.ND = not detected a