Evaluation of Phage-Antibiotic synergy against Klebsiella pneumonaie and encapsulation of phages to improve their stability under gastronomic conditions.

Abstract

The rise of multidrug-resistant Klebsiella pneumoniae in recent years has heightened global public health considerations. The capacity of this disease to produce hypervirulent strains with enhanced virulence features complicates control. Klebsiella pneumoniae, a member of the Klebsiella genus, causes a variety of infections, including respiratory, urinary tract, and septicemia, primarily affecting susceptible people in both community and hospital environments. Klebsiella pneumoniae, an ESKAPE pathogen known for producing nosocomial infections, uses virulence features such as capsules, fimbriae, lipopolysaccharides, adhesins, and iron absorption pathways for pathogenesis. However, the bacterium's increasing antibiotic resistance jeopardizes treatment efficacy, allowing it to bypass the immune system and cause serious infections. The investigation of Klebsiella specific bacteriophages was prompted by the urgent need to combat the rise of virulent and antibiotic-resistant K. pneumoniae strains. These natural viruses are possible bacterial growth inhibitors and antibacterial sources against Klebsiella infections. Antibiotics, previously the gold standard for treating bacterial illnesses, are becoming less effective due to the prevalence of antibiotic-resistant bacteria. Antibiotic misuse and overuse have promoted resistance. Innovative treatments for illnesses caused by multi-drug resistance bacteria are desperately needed to combat this. Phage therapy has emerged as an attractive option for therapy for such illnesses. Studies on phage-antibiotic synergy against multidrug-resistant bacterial pathogens are gaining increasing attention worldwide. In this study, two previously isolated and characterized bacteriophages KPA and KPM, and their cocktail were used to evaluate their synergistic effect with subinhibitory concentrations of cefepime, gentamicin, and meropenem against multidrug-resistant, biofilm forming, uropathogenic Klebsiella pneumoniae isolated from urine samples from catheterized patients. In this study, a significant reduction in host bacterial counts was observed when treated with KPA, KPM, and their cocktail with the subinhibitory concentrations of cefepime and meropenem, and no synergistic effect of phages was observed in host bacterial eradication when used with gentamicin, Evaluation of Phage-Antibiotic synergy against Klebsiella pneumonaie and encapsulation of phages to improve their stability under gastronomic conditions. 13 but a cocktail of both phages worked synergistically with gentamicin. Cefepime at a concentration of 8 µg and 4 µg with KPA at MOI 0.1 and a cocktail of KPA and KPM caused a 100% reduction in the host bacterium and meropenem at a concentration of 1µg, and 0.5µg with KPA, KPM and their cocktail caused a 100% reduction in the host bacterium and the cocktail of KPA and KPM at 0.25 µg Meropenem killed 100% of the host bacterial cells. All antibiotics tested with KPA, KPM, and their cocktail inhibited biofilm formation by host bacteria, but the biofilm inhibition potential of both phage and their cocktail was higher with meropenem, followed by cefepime and gentamicin. A cocktail of KPA and KPM completely destroyed the biofilm formed on glass slides, followed by KPA and KPM. Throughout the study, the antibacterial and antibiofilm activity of the phage cocktail was higher, followed by KPA and KPM. Controlling gastrointestinal flora is an effective technique for treating illnesses caused by microbiome abnormalities. While traditional probiotics have been used in the past, recent attention has shifted to phage delivery for changing gut bacteria and gene regulation. However, phage proteins might be damaged during storage and delivery, reducing their effectiveness. This research focuses on improvements in phage encapsulation and distribution investigation, emphasizing carrier systems' protective capacities and focused colon delivery. Encapsulation techniques enhance phage stability and targeted delivery within the body. These strategies could reshape phage therapy and contribute to fighting antibiotic-resistant infections. This research delves into the resilience of phage KPA and KPM within simulated gastrointestinal conditions. It uncovers that the encapsulation of these phages utilizing polymers such as alginate + agarose is a successful approach to safeguard them from the harsh acidic surroundings of the stomach. This encapsulation strategy ensures phage survival by mitigating the impact of low pH settings, a critical factor for their successful oral administration. The study demonstrates remarkable loading efficiencies of 99.3% and 99.6% of the phages KPA and KPM respectively, within matrices like alginate + agarose indicating the effectiveness of encapsulation. Evaluation of Phage-Antibiotic synergy against Klebsiella pneumonaie and encapsulation of phages to improve their stability under gastronomic conditions. 14 Furthermore, the research underlines the significance of gastric emptying times during the phages' transit through the stomach. It suggests that the encapsulated phages' fate is intricately tied to factors such as formulation and particle size when introduced alongside a solid meal. The findings emphasize that the encapsulation process not only preserves phage viability in extreme gastric conditions but also allows for controlled release in the intestine's more favorable pH environment. In our study, the maximum phage entrapment was reported in the case of alginate + agarose at pH 2, followed by the remaining pH values, whereas maximum KPA and KPM phage releases have been observed in case of alginate + gelatin because at the intestinal pH of 7.4 alginate + gelatin capsules swell and release the encapsulated phages in a pH-dependent manner. However, the stability and release of phages in the simulated gastric fluid (pepsin) and simulated intestinal fluid (pancreatin) has also been determined. Maximum phage entrapment in the case of pepsin has been observed in alginate + agarose while no release has been reported in the case of pepsin that indicates the successful encapsulation of phages because the free phages are highly sensitive to extreme gastric conditions of the stomach, however, the highest phage entrapment in case of pancreatin has been observed in case of alginate + agarose and maximum release has been reported in case of alginate + gelatin. This work contributes to the advancement of microencapsulation techniques for phage based therapies and highlights the potential of using encapsulated phages to navigate the challenges of oral delivery and targeted release within the gastrointestinal tract