Effect of Lipopolysaccharide Extracted from Enterobacter cloacae on some Pathogenic Bacteria

A 106 different specimen were collected to isolate and diagnose some gram negative and gram-positive bacteria, as it was performed for the period from 15 August to 30 November 2019 from AL-Salam and Al-Khansaa educational hospitals in Mosul. Lipopolysaccharide (LPS) was extracted from Enterobacter cloacae by using the Methanol Chloroform method. extract was diagnosed, using some biochemical tests such as the Molisch test to detect carbohydrate content and Folin reagent in order to determine proteins concentration, lipid solubility in organic solvent and Bio drop technique as well as to detect nucleic acid concentration. The extraction methods showed its efficiency with low protein and nucleic acids concentration in the extract. Lipopolysaccharide demonstrated its efficiency inhibiting microbial growth on all test isolates at concentrations 1100, 1000 μg\ ml while its lipopolysaccharide showed its inhibition of Proteus mirabilis at all concentrations used. LPS showed possibility used as inhibitory, antimicrobial growth.


INTRODUCTION
Outer membrane (OM) of Gram negative (G-) bacteria are a round membrane enclosed entities of internal component cell (Jan, 2017). LPS anchored in the (OM) of all (G-) bacteria can pose risk to human health that ranges from irreversible shock to death (Su and Ding, 2015). LPS have many benefits to bacterial cell as it contributes to the structural integrity and protection barrier against harsh environments (Reyes et al., 2015). Also, facilliting biofilm formation, antibiotic resistance and protecting bacteria from threats, LPS consist of three domains that include lipid A, core oligosaccharide and O-antigen (Huszczynski et al., 2020). LPS potent endotoxin causes chronic immune responses associated with inflammation (Salguero et al., 2019). Immune system responses to a low doses LPS lead to rapid clearing of pathogens but a higher doses LPS lead to uncontrolled over production cytokine that lead to endotoxic shock that is often lethal (Williams, 2019). In other word, less immunogenic LPS could serve as therapeutics to antagonize an overreacting innate immune system (Steimle et al., 2016). Enterobacter cloacae belonging to order Enterobacteriales, family Enterobacteriaceae gram negative bacteria bacilli shaped (De-Lamaza et al., 2020). Facultative anaerobic it is related with opportunistic infections that is specific in immunocompromised patients (Hong et al, 2018).and nosocomial infections (Jin et al., 2018). Also, these bacteria have a broad-spectrum antibiotic resistance (Annavajhala et al., 2019). The extracted LPS by Methanol-Chloroform method was much easy less costly and safer but lack purity (Kalambhe et al., 2017).

MATERIALS AND METHODS
A 106 specimen were collected that include urine, stool, blood, pus, sputum, nipple swab, vaginalis, nipple discharge and ear swab. The specimen was directly cultured on media then identification was completed depending on gram stain, morphological and biochemical tests using IMVIC to ensure diagnosis used a VITEK 2 technique was utilized. (Koneman et al., 2017;Cappuccino and Welsh, 2020;De-Lamaza et al., 2020).

LPS extraction:
LPS was extracted according to (Mirzae et al., 2011). The dried LPS was dissolved in deionized water and placed in the deep freezer -80˚C temperature then lyophilized placed in the lyophilizer (Christ lyophilizer made in Germany).

Biochemical test to detect LPS:
Folin to determine protein concentration, Molisch to detect carbohydrate, Solubility in organic solvent tests used to detect lipid also use Bio drop technique to detect nucleic acids concentration. (U and Sampath, 2014;Awasthi et al., 2013) Usage of crude of LPS as antibacterial against some pathogenic bacteria: The antibacterial activity of LPS was extracted from pathogenic Enterobacter cloacae done against Proteus mirabilis as gram negative bacteria and Staphylococcus aureus as gram positive bacteria, using agar well diffusion method according to (Chittepu, 2019). Experimental bacteria were cultured on petri Dish containing 25 ml Muller Hinton agar after comparing bacterial growth with 0.5 McFarland standards tube that equals 1.5×10 8 . A (6mm) cut in agar diameter was performed by a sterile Pasteur pipette and then removed the agar cut by forceps filled 0.1 ml of each well in the different concentration of LPS (1100, 1000, 900, 800, 700) µg \ ml after being cultured with bacteria by swab (three replications were made for each concentration of LPS extract for each bacteria). Also, preparation control sample without of LPS using normal saline to fill in the well. The plats were incubated in 37˚C for 24 hours in upright position to keep LPS extract in the well, then the effects LPS on microbial growth were evaluated by measuring the diameters of growth inhibition zones on pathogenic bacteria (Al-Azawy and Abdullah, 2017).

Bacterial isolate:
The following isolates were diagnosed: Enterobacter cloacae, Staphylococcus aureus and Proteus mirabilis. The antimicrobial activity assay of LPS extract of Enterobacter cloacae were done against experimental bacterial isolate in different concentrations of (1100, 1000, 900, 800, 700) µg\ml that were evaluated by measuring the diameters of growth inhibition zones on bacterial strain and the results are shown in (Tables 2, 3).     Fig. (1), and LPS has also demonstrated its efficacy in inhibiting Proteus mirabilis Fig. (2) at all concentrations (1100, 1000, 900, 800, 700) μg\ml. which lowest inhibitor concentration was only 700 µg\ml for Proteus mirabilis among the isolates under study . And this inhibitory effect is compatible with the researcher (AL-Azawy and Abdullah, 2017). LPS effect on gram negative bacteria was more than gram positive bacteria. The LPS mechanisms inhibitory effect on microorganisms its effect on is due to its capability of having anti-adhesive agent that inhabits bacterial adhesion on the surface (Tanek et al., 2018) However, the mechanism of LPS antimicrobial remains indistinct but certain hypotheses were proposed that sustained specific evidence showing loss of membrane integrity: antimicrobial LPS is as a result of the adhesion property of the surface of active agents for the cell membrane integrity that leads to subsequent collapse of nutrition cycle or fatty acid moieties of LPS inserted in to the cell membrane. Instigates a proliferation of membrane size and ultra-structural changes, other hypothesis assumes that lps able to form pores and disrupt the plasma membrane (Ines and Dhouha, 2015). Also, LPS can increase cell membrane sponginess, occurs due to disarrangement of plasma membrane phospholipids (Sotirova et al., 2009), increasing permeability of plasma membrane cells as interact lipids with fatty acids content in the cell membrane leads to inhibition of the membrane -restricted enzyme and efflux of intracellular cytoplasmic components (Cameotra and Makkar, 2004). LPS interacting with membrane protein leads to increase initiating conformational changes of lipids and proteins Sanchez et al., 2006;Sotirova et al., 2008).