Synergistic effect of mannan extracted from Saccharomyces cerevisiae with antibiotics against multidrug resistant Escherichia coli

: Multidrug-resistant Escherichia coli (MDR-E. coli ) presents a formidable healthcare challenge, continuously evolving resistance to conventional antibiotics. The aim of this study focuses on assessing the synergistic effect and enhance the effectiveness of traditional antibiotics of mannan extracted from Saccharomyces cerevisiae on MDR-E. coli . Clinical samples (n=150) were collected from several hospitals in Baghdad for both sexes between August and December 2022, including 73 urine, 10 catheter, 13 wound, and 54 stool samples. Among the total collected samples, 112 E. coli isolates were obtained, with 54 derived from urine, 9 from catheter samples, 6 from wound samples, and 43 from stool samples. Antimicrobial susceptibility test (AST) revealed resistance to imipenem (0%) and amikacin (2%), with sensitivity observed for cefotaxime (25%), gentamicin (29%), ceftriaxone (28%), ciprofloxacin (41%), norfloxacin (34%), trimethoprim-sulfamethoxazole (48%), and tetracycline (35%). Mannan purified from S. cerevisiae demonstrated inhibitory effects on E. coli growth, with Minimum Inhibitory Concentrations (MIC) ranging from 100 to 12.5 mg/mL and Minimum Bactericidal Concentrations (MBC) from 25 to 200 mg/mL. Furthermore, mannan exhibited a synergistic effect with conventional antibiotics, pointedly boosting the efficacy of gentamicin (250%), cefotaxime (160%), ceftriaxone (340%), ciprofloxacin (344.4%), norfloxacin (337.5%), and trimethoprim-sulfamethoxazole (237.5%). This research addresses the public health threat posed by MDR-E. coli , highlighting the potential of S. cerevisiae mannan as a promising solution for inhibiting E. coli and improving antibiotic efficacy.


2.1.Samples collection and diagnosis
Samples were collected from August to December 2022 from Al-Elwiya Maternity Hospital, Ibn Al-Nafis Hospital, and Sheikh Zayed Hospital in Baghdad.These samples included 73 urine, 10 catheter, 13 wound, and 53 stool samples, totaling 150 from both male and female patients.E. coli isolates were cultured on MacConkey agar and EMB agar, and colonies were examined based on various characteristics after 24 hours of incubation at 37°C.Further, Gram staining and microscopic examination of isolates were performed.The VITEK®2 compact system, using GN Card ID, rapidly diagnosed the E. coli isolates from different clinical sources.The bacterial suspensions' turbidity was determined by the VITEK®2 DENSICHEK device, and results were recorded after approximately 4-6 hours in accordance with the instructions provided by BIOMÉRIEUX-France's.

2.3.Mannan extraction
To extract mannan from S. cerevisiae, 5 grams of yeast were mixed with a 1% sodium hydroxide solution (50 mL) and heated at 100°C for two hours.Subsequently, the soluble mannan oligosaccharide was cooled, neutralized to a pH of 7 using a diluted HCl (10%) solution, and then precipitated by adding 200 mL of absolute ethanol.The mixture was filtered following the protocol established by Huang et al. (2010) and the resulting precipitate was washed with both diethyl ether and absolute ethanol.The filtrate was discarded and the precipitate taken to be dissolved in an appropriate amount of distilled water.At this stage was obtained crude mannan.To obtain deproteinized mannan, Trichloroacetic acid (TCA) at a 10% concentration was used to adjust the pH of crude mannan to 3, initiating a 24-hour incubation at 4°C.Subsequent steps involved centrifugation, ethanol precipitation, further centrifugation, and sediment dissolution in distilled water, followed by a heating process at 121°C for three hours, based on the methodology outlined by Razan and Odongo (2020).More details about mannan extraction and purification can be consulted in (Fadhil Abbas Al-Helli and Abdul Sattar Salman, 2023).

2.4.Antibacterial activity of mannan against MDR-E. coli isolates
Mannan from S. cerevisiae was assessed for inhibitory activity in a 96-well microtiter plate.To find the MIC, each well was filled with sterile Mueller-Hinton broth (125 µL per well).Mannan solutions (concentration range: 200 to 3.125 mg/µL) were initially added to the first column and then serially transferred to the next wells, except the last column, maintaining 125 µL in each well.The positive control contained only Mueller-Hinton broth, and the negative control included a bacterial suspension.An E. coli bacterial suspension was created by diluting it 1:100 with Mueller-Hinton broth and added to wells with mannan and the positive control.The suspension's turbidity was adjusted to 0.5 McFarland, and the microtiter plate was covered and incubated at 37°C overnight.Following incubation, 30 µL of 0.015 Resazurin dye was added to all wells.The plate was further incubated for 1/2 to 3 hours at 37°C to check for a colour change, indicating dye reduction.The MIC was identified as the lowest mannan concentration preventing bacterial growth, with no dye reduction and colour change from dark blue to bright pink.A Mueller-Hinton agar plate was inoculated with a bacterial suspension exceeding the identified MIC.The Minimum Bactericidal Concentration (MBC) was defined as the lowest concentration where no bacterial colonies were observed to grow on the agar plate (Wu et al., 2023).

2.5.Evaluation of the combined effect of antibiotics and mannan on MDR-E. coli isolated from different sources.
The antibiotics to which the E. coli showed resistance were selected for this assay.The synergistic impact of six antibiotics only (gentamycin, cefotaxime, ceftriaxone, ciprofloxacin, norfloxacin, and trimethoprim-sulfamethoxazole) in combination with mannan was investigated using an adapted approach based on a modified method by Roy et al. (2010).Mannan-submerged paper discs were placed on Mueller-Hinton agar plates with MDR-E.coli.After 24 hours of incubation at 37°C, inhibition zones were measured.Only antibiotics to which E. coli exhibited resistance were considered, and the study used antibiotic-alone inhibition diameters as a control.The percentage increase fold was calculated using the specified equation (Fold increase% = (ba)/a×100) by Ghosh et al. (2012).Where (a) represents the inhibition zones (mm) for an antibiotic alone, and (b) represents the inhibition zones (mm) for the antibiotic in combination with mannan.

3.1.Cultural and microscopic examination
Colony morphology was examined on MacConkey agar.Characteristics such as colour, size, and colony edges were noted following a 24-hour incubation at 37°C.Subsequently, positive isolates were validated by culturing them in Eosin-Methylene Blue (EMB) medium at 37°C for 24 hours.Identification was confirmed by the distinctive bright metallic green colour of the colonies on this medium.A single colony of each isolate was fixed on a clean slide to study gram stain under a light microscope (Erjavec, 2019).Out of the total number of samples (n=150) comprising 73 urine,10 catheter,13 wound and 54 stool samples, a total of 112 isolates were carried out in the following order: 54 isolates from urine samples, 9 isolates from catheter samples, 6 isolates from wound samples, and 43 isolates from stool samples.Table (1) shows the proportions of isolates according to the type of total samples and the sources of their isolation.E. coli is the main cause of urinary tract infections (UTIs).It possesses specific virulence genes, including the (fem) gene for type one pili production and Fim H adhesin for invading bladder cells (Pokharel et al., 2023).P-fimbria adhesins attach to uro-epithelial cells, and UPEC carries the (Sfa/Dr family) fimbria of antigens that enhance colonization, increasing the risk of recurring UTIs.This feature is more common in UPEC than in E. coli causing diarrhea (Radera et al., 2023).E. coli causes catheter-associated UTIs by colonizing the intestinal and urinary tracts.Catheter insertion introduces opportunistic organisms, increasing susceptibility.Flagella help E. coli ascend from the catheter to the bladder.Catheter material affects colonization and biofilm formation (Sanchez et al., 2022).E. coli isolates from various wound types exhibit distinct virulent factors compared to other strains.These factors encompass a heightened prevalence of aerobactin  et al., 2020).While this bacterium can remain asymptomatic, under favorable conditions, they utilize these virulence factors to infiltrate extra-intestinal areas in susceptible hosts (Longhi et al., 2022).Finally, E. coli isolates from stool samples differ based on the genes responsible for encoding them (Abdulqader et al., 2022).Cytotoxic Necrotizing Factor 1,2 genes are found in enterotoxigenic E. coli (Jarquin et al., 2022).While Bundle-forming Pilus (bfpA) and Enteropathogenic Attachment and Effacement (eaeA) genes are responsible for encoding Enteropathogenic E. coli (EPEC) (Maniha and Noor, 2020).Additionally, Heat-stable and Heat-labile toxin genes are responsible for encoding Enterotoxigenic E. coli and Shiga-like toxin (Stx1) and ( Stx2) genes are responsible for encoding Shiga-like toxin-producing E. coli (STEC) (Huang et al., 2021).

Antibiotic susceptibility test results
The results of the antibiotic susceptibility test were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Jacobs et al., 2022).Antibiotic susceptibility tests are crucial for evaluating effectiveness against E. coli infections.E. coli strains classified as Multidrug Resistant (MDR) are resistant to three or more antimicrobial agents, posing a significant public health risk (Rafailidis and Kofteridis, 2022).Imipenem, introduced to the market in 1987, is a potent inhibitor of a wide range of Gram-positive and Gram-negative bacteria (Zhanel et al., 1998).However, resistance to imipenem has been recorded in various parts of the world (Karikari et al., 2022).E. coli resistance to aminoglycosides is mainly attributed to the production of aminoglycoside-modifying enzymes (AMEs) (Pradier and Bedhomme, 2023).
The AST results in this study showed that, imipenem (0%) and amikacin (1%), with sensitivity observed for cefotaxime (25%), gentamicin (29%), ceftriaxone (28%), ciprofloxacin (41%), norfloxacin (34%), trimethoprimsulfamethoxazole (48%), and tetracycline (35%).The results of AST are summarized in   and Soloi, 2022).NaOH is commonly employed in extraction procedures due to its affordability, ease of use, and effectiveness in producing optimal alkaline solutions for treating natural fibres (Zhang et al., 2018).It was observed that mannan and surface proteins within the yeast wall exhibit a spontaneous binding tendency at specific pH levels.Additionally, molecular study's findings revealed that the surface proteins had varying binding sites for mannan, dependent on pH, mainly influenced by hydrogen bonds and hydrophobic interactions.These findings affirm that the interaction between surface proteins and mannan at pH 5.0 demonstrates enhanced binding affinity and greater structural stability (Fu et al., 2023).

Antibacterial activity of mannan against E. coli isolates from different source
The results of MIC and MBC of E. coli isolates are defined in Table (2).Mannan, a complex carbohydrate composed of mannose units, affects the growth of E. coli depending on its concentration and type.At low concentrations, mannan promotes growth by acting as a carbon source for the bacteria.E. coli breaks down mannan into smaller sugars and metabolizes them through the glycolytic pathway (Scribano et al., 2020).However, at high concentrations, mannan inhibits growth by forming a viscous gel that limits bacterial motility and cell division.The viscosity of the mannan solution can also restrict nutrient uptake, leading to growth inhibition (Wagenlehner et al., 2022).The effect of mannan on the growth of E. coli is that it increases the production of oxygen free radicals inside the bacterial cell, which kills the bacteria (Smith et al., 2022).This physiological change is characterized by greater peptide abundance of key TCA proteins in response to ROS overproduction in the presence of mannan in the culture medium.
Additionally, mannan may contribute to hyperactivity of the electron transport chain.The MIC of mannan on E. coli may vary depending on the source of bacterial isolates and mannan source (Smith et al., 2020).

Evaluation of combined effect between antibiotics and mannan on E. coli isolates
The results of studying the synergistic effects of antibiotics combined with mannan from S. cerevisiae against MDR-E.coli isolates were recorded and analysed, as illustrated in Table 3.
Table (3 The results revealed variations in the diameters of inhibition zones around antibiotic discs when used alone compared to when impregnated with mannan.The observed differences were recorded the highest antibacterial effect by mannan and gentamycin (G) showing equal 250% in the inhibition zone of E. coli (C3), while lowest effect was 20% in the inhibition zone of E. coli (S12).For mannan and cefotaxime (CTX) combination, the highest fold increase of 160% in the inhibition zone of E. coli (C3), while lowest effect was 22.2% in the inhibition zone of E. coli (U9).The highest antibacterial effect 340% in the inhibition zone of E. coli (U7) by mannan and ceftriaxone (CRO) while lowest effect was 20% in the inhibition zone of E. coli (S11).
For mannan and ciprofloxacin (Cip) combination, the highest fold increase of 344.4% in the inhibition zone of E. coli (C3), while lowest effect was 22.2% in the inhibition zone of E. coli (S11).The highest antibacterial effect 337.5% in the inhibition zone of E. coli (C3) by mannan and norfloxacin (Nor) while lowest effect was 30% in the inhibition zone of E. coli (S12).Finaly For mannan and trimethoprim-sulfamethoxazole (SXT) combination, the highest fold increase of 237.5% in the inhibition zone of E. coli (C1), while lowest effect was 20% in the inhibition zone of E. coli (U9).Usage of a single treatment is frequently ineffective because of factors such as drug resistance, constrained drug absorption, or challenges in reaching the intended site.To challenge these issues, combination therapy, involving the use of two or more drugs or a drug in combination with polymeric compounds, can be utilized (Pena et al., 2021).This approach can help delay the development of resistance or control existing resistance, as it targets multiple pathways or mechanisms of action.By using a combination therapy approach, a lower dose of each drug can be employed, reducing toxicity and minimize side effects.The use of combination therapy has shown promise in improving treatment outcomes for a range of diseases and conditions (Masri et al., 2019).An explanation for the impact of oligosaccharides on E. coli is linked to osmolarity.E. coli can react to changes in osmotic pressure induced by mannan, thereby enhancing antibiotic activity against E. coli( Asadpoor et al., 2021).Another reason for the increased sensitivity of E. coli to the mannan-related antagonist is the multiple ionic interactions between the charged sugars and the surface of E. coli.This stimulates its negative charge on the surface of the E. coli, weakening its movement and agglomeration, and making it more sensitive to the antibiotic (Powell et al., 2014).Although mannan may not inherently have antimicrobial properties, when administered alongside conventional drugs, it can enhance their effectiveness.The and uptake, a unique resistance pattern observed in bacterial isolates from burn wounds in an Iraqi hospital as stated byTayh et al. (2023)   , α-hemolysin, cytotoxic necrotizing factor, and S-fimbriae.The genes encoding these factors are situated on pathogenicity islands (PAI) (Ssekatawa

Figure
resistance to gentamicin in this study closely aligned with the rate reported byWu et al. (2021).Plasmid-mediated quinolone resistance (PMQR) determinants may significantly contribute to bacterial survival in the presence of quinolones.Another resistance mechanism involves the physical blocking of target sites by (Qnr) proteins or the increased expression of efflux pumps to expel antibiotic molecules(van Driel et al., 2019).E. coli develops resistant to cephalosporins upon acquiring Expanded Spectrum Beta-Lactamases (ESBLs) such as TEM-1, TEM-2, and SHV-1(Yasir et al., 2020), or it may be linked to the production of plasmidic class C βlactamases, such as the CMY enzyme(Judge et al., 2023).Often, trimethoprim and sulfamethoxazole are used together in various quantities depending on the target bacteria, providing synergistic bactericidal activity(Kawahara et al., 2023).Changes to the permeability of the cell wall and excessive production of dihydrofolate reductase, either through chromosomal mutations or plasmid-mediated acquisition of trimethoprim-resistant DHFR enzymes (dfr) genes(Saito et al., 2022).Despite the decline in the use of tetracycline in humans and animals, resistance to tetracycline continues to increase.The main reason for this increase is the spread of (tet) genes among bacteria due to the continued use of tetracyclines in animal production worldwide(Çiçek et al., 2022).