ANTAGONISTIC EFFECT OF LACTOBACILLUS ACIDOPHILUS AGAINST SOME PATHOGENIC BACTERIA

Aim : This study intended to ascertain whether probiotics added to various food products exhibited antimicrobial and antibiofilm properties against common microbial pathogens. Materials and methods: Two hundred samples were collected (130 from Assiut and 70 from New Valley) from different sources including yogurt (90), rayeb (80), and milk powder (30). Lactobacilli strains were isolated and biochemically identified. Then genotypic characterization of the samples was done. The lactobacilli CFS's (CFS) antimicrobial activity was measured by agar well diffusion. The ability of these pathogens to form biofilms was tested. Furthermore, the microplate titer method was employed to analyze the antibiofilm characteristics of CFS. Result: Lactobacillus strains were isolated from different milk products and genotypically investigated and nineteen L. acidophilus have been isolated. Most concentration CFS show antibacterial activity against tested microorganisms with an inhibition zone of diameter greater than 25 mm. Moreover, The CFS of lactobacilli inhibited the biofilm formation by these pathogens.


INTRODUCTION
Probiotics are viable microorganisms that, when supplied in enough doses, profit the host's health.Probiotic microorganism that has been used safely in both fermented and non-fermented foods for a long time is named Lactic Acid Bacteria (LAB) (Quigley, 2019).It is commonly known that probiotics have antibacterial properties against harmful microorganisms.The synthesis of organic acids (which antagonize bacteria, bind to bacteria, and decrease bacterial adherents), bacteriocin, and hydrogen peroxide is what gives probiotics their antibacterial properties (Mahdavi and Isazadeh, 2019).Furthermore, 147 LAB and their ingredients have been recommended as a possible biofilm biocontrol agent (Khiralla, et al., 2015).
Staphylococcus is the reason for many disorders in people and affects animals.Because Staphylococcus aureus produces a variety of endotoxins when it grows on different food commodities, it is well-known as a common food poisoning pathogen (Lee, et al., 2015).Furthermore, the infection of the urinary system impacts 150 million people globally each year.UPEC is the most popular microorganism linked to urinary tract infections (UTTIs), accounting for 90% and 50% of community-acquired and nosocomial infections of the urinary tract (oval et al., 2014).E. coli creates microcolonies called biofilms on the urethral catheters, and also on the urinary bladder mucosa.By encasing them in an extracellular matrix, biofilm shields bacteria from the immune system and drugs.Furthermore, the easy transmission of resistance factors amongst the bacteria in the biofilm is made possible by close bacterial association.Drug resistance will increase as a result, making UTI treatment more challenging (Eberly et al., 2017).
The rise of new strains that are not affected by antibiotics, new therapy protocols away from antibiotics have become increasingly popular in recent years.Probiotic use is one of these treatment modalities that shows promise for infection control.

Preparation of samples (APHA, 1992):
Preparation of pasteurized yogurt, rayeb milk and milk powder for isolation of Lactobacillus by mixing 1gm of the sample with 9 ml peptone physiological saline solution.

Isolation of Lactobacilli (Lamiaa, 2014):
The diluted test sample was streaked on the surface of De Man Rogosa Sharpe (MRS) agar and incubated for 40 -42 hours at 37⁰C in a 10% Co2 incubator.Colonies appear as white smooth and convex with regular edges varying in size 1-5 mm.After being purified by recurred subculturing, Lactobacillus spp.were confirmed depending on Gram staining, sporulation, morphology and biochemical tests.A copy of the strains was cultured on MRS agar slants at 4 °C and subcultured every four weeks (Shruthy et al., 2011).

Identification of Lactobacillus Sp. by PCR (Kim et al., 2020):
To identify the bacteria's species, Bacterial genomic DNA was extracted by boiling method.The supernatant was then taken as a template and preserved at 20° C. Amplified of the 16S ribosomal RNA gene was done by using L. acidophilus 16S-23S rRNA (F:5ꞌ-CCT TTC TAA GGA AGC GAA GGA T-3ꞌ\R:3ꞌ-ACG CTT GGT ATT CCA AAT CGC-5ꞌ) primer.The PCR was done as follows: Primary denaturation for 5 min at 94˚C, then 35 cycles of at 94˚C for 30 sec, at 60˚C for 30 sec and at 72˚C for 30 sec and a final extension for 7 min at 72˚C.

Preparation of pathological bacterial strains:
The used bacterial strains were Escherichia coli and Staph.aureus.All strains were gained from the High-Quality Media unit (HQM) in the Animal Health Research Institute in Dokki, Egypt.Three to five colonies were emulsified from each pathogenic strain in 3-4ml tryptone soya broth.The turbidity was adjusted to approximately 10 8 cfu/ml by comparing it to a McFarland barium sulfate standard 0.5 incubated for 24 hours at 37⁰C.

Antibiotic susceptibility testing of pathological agents (CLSI, 2017):
Susceptibility of E. coli and S. aureus bacteria to antibiotics involving, Aztreonam, Ampicillin, Amikacin, Gentamicin, Nitrofurantoin, Ceftriaxone, Ceftazidin, Imipenem, Meropenem, Tetracycline, Cefoxitin, Sulpha\Trimethoprim, Cefoperazone and Cefpodoxime were examined using the disc diffusion method, and the inhibition zone diameters were evaluated and contrasted with the zones that had been previously reported.

Method
for Making Cell-Free Supernatants: Probiotics were cultured anaerobically in MRS broth for 24 h at 37°C.For 20 minutes at 4 °C and 5000 rpm, cultures were centrifuged to produce a cell-free supernatant.subsequently used after being passed through a filter with a pore size of about 0.2 um.

The antibacterial activity of L. acidophilus (Njoki et al., 2015):
Two plates were done, one for E.coli and the Other for S. aureus.Using 1 N NaOH, the supernatant was neutralized (pH adjusted to 4) in order to eliminate the organic acid's antibacterial properties.A sterile cork borer was used to create 10 mm wells in the Muller-Hinton agar after each overnight cultures of the indicator strains of S. aureus and E. coli were swabbed.The sterile neutral supernatant was added to the agar wells in approximately 100 ul aliquots.For every test isolate, this was done twice.The plates were kept at 48 hours at 34 °C, that is the ideal condition for indicator microorganisms, after being kept at 4 °C for two hours to permit for prediffusion.Inhibition zones were noted, along with their diameters and presence or absence.

Inhibition of biofilm formation (Plyuta et al., 2013):
After ascertaining that the tested microorganisms form biofilm according to Stepanović et al. (2007).The assessment of biofilm inhibition involved employing the spectrophotometric method.In 96-well microtiter plates, 180 µL of various concentrations of the probiotic extract was prepared and distributed.Subsequently, 20 µL of overnight growth cultures of the tested strains were added to each well and kept at 37 °C for 24 hours.Following the removal of the suspensions, 200 µL of phosphate buffer saline (PBS) was added to the wells in order to wash away any free-floating bacteria.Adherent cells forming biofilms on the plate were fixed with 200 µL of methanol for 20 minutes, followed by staining with 200 µL of 0.1% crystal violet at room temperature for 15 minutes.A thorough PBS wash was used to remove any remaining stains.After airdrying, we added 200 µL of ethanol (96%) were added to each well to solubilize the biofilm dye and the mixture was incubated for 15 minutes.The resulting reaction was assessed spectrophotometrically at 570 nm.Each treatment was triplicated, and the experiment was run three times.Control samples, consisting of wells inoculated with bacteria without any treatment, were included in each case.The percentage of biofilm inhibition = [(OD control -OD treatment)/OD control] × 100.

Isolation and Identification of Lactobacilli:
Various yogurt, Rayeb, and milk powder samples were gathered.The colonies that Gram-positive rod-shaped morphology, catalase and oxidase activity, cell motility, and sporulation are considered Lactobacillus spp.Lactobacillus spp.were the predominant organisms among all bacterial isolates.Further identification of Lactobacillus spp. was achieved using 16S ribosomal RNA, revealing 19 isolates of L. acidophilus in this study.In every experiment, the CFS was made from a single pure culture of L. acidophilus to maintain consistency and reproducibility.

Assessment of the Antibiofilm Activity
The CFS of ten L. acidophilus strains resulted in preventing the formation of biofilms by E. coli isolates (Table3).twelve out of 18 strains showed an antibiofilm effect against S. aureus and the greatest inhibition was observed by LAB 6 (93.75%).

DISCUSSION
Harmless bacteria known as lactobacilli are frequently used as probiotics and are intimately linked to the human microbiota.
Their probiotic effect depends on their capacity to produce antimicrobial molecules, improve the function of the epithelium or modify the human immune system to prevent pathogen colonization through rivalry (Raheem et al., 2021).The utilization of antimicrobial agent probiotics and even its products for battling infectious diseases is reinforced by the reality that these healthpromoting bacteria produce inhibitory effects against resistant bacteria; moreover, it is predicted that the inability to fight against the intended pathogens is an uncommon instance, contrary to the often creation of resistance by pathogenic organisms in humans towards antibiotics often employed in the clinic (Simons et al., 2020).Therefore, the potential use of probiotics as antimicrobial substances in the age of antibiotic resistance with a rapidly usage become of great importance (Silva et al., 2020).A variety of pathogens, such as L. monocytogens, E. faecalis, Salmonella typhimurium, S. typhimurium, E. cloacae, P. aeruginosa, Cl. difficile, H. pylori, E. coli O157:H7, S. aureus, S. epidermis, B. subtilis, and Campylobacter jejuni, were demonstrated to be susceptible to antimicrobial effects by lactobacillus CFS.(Muhammad et al., 2019).Indeed, the noteworthy aspect is that the antimicrobial activity of probiotics extends beyond bacteria.Probiotics were shown by Yasui et al., (1993) to offer immunity to diarrhea caused by rotavirus.Furthermore, it was shown that neither environmental variables nor storage circumstances influence the inhibitory properties of lactobacilli supernatants (Koohestani et al., 2018), which is advantageous if CFS is marketed as a medicinal agent.The majority of research on probiotics' antimicrobial effects now has concentrated on CFS (Jeyanathan et al., 2021).
The lactobacilli used in this investigation were taken out of industrial items to test strains that had already received human use permission.

Generally,
Greater antibacterial and antibiofilm effects against E. coli and S. aureus were demonstrated by the CFS of the L. acidophilus and the result indicated that 94.74% and 84.21% of L.acidophilus strains have antibacterial effects on S.aureus and E.coli.This was in agreement with Ebrahim (2017) who showed that 94.4% and 87.0% of lactobacillus strains have antibacterial effects on S.aureus and E.coli and a similar result was obtained by Karthikeyan and Santhosh (2009) However, Ghane et al. (2020) found that the CFS from all kefir isolates led to a decrease in biofilm formation by uropathogenic strains.However, the effectiveness of antibiofilm activity varied notably among different lactobacilli strains.Likewise, the CFS from all LAB led to over 30% inhibition of biofilm formation by E. coli isolates.Notably, from twelve strains only seven showed more than 50% inhibition against all four urinary strains.
In light of our findings, we propose utilizing the CFS of L. acidophilus to manage or stop infection and colonization brought on by pathological microbes, such as S. aureus and E. coli.

Table 1 :
Susceptibility of tested pathogens to antimicrobial agents S. aureus and E. coli respectively but with variable degrees.The average diameter of the inhibition zone was more if S. aureus in comparison to the E. coli isolates.

Table 2 :
Antibacterial effect of the isolated L. acidophilus on the growth of pathogenic bacteria (diameter of the inhibition zone).

Table 3 :
The antibiofilm effect of L. acidophilus strains against some pathogenic organisms.
Soltani et al. (2022)18)approved that L. acidophilus removed 70.60 of S. aureus biofilm.Consistent with our results,Rao et al. (2015), stated that the CFS from Lactobacillus exhibited significant antibiofilm activity.Additionally,Khiralla et al. (2015), recommended three Lactobacillus strains isolated from conventional products as effective biocontrol agents, emphasizing their potential for inhibiting the formation of biofilms by pathogens.Soltani et al. (2022)indicated that L. acidophilus can impede the formation of biofilms by E. coli isolates.