Adaptation of Gram-Negative and Gram-Positive Bacteria to a Newly Formulated Hand Sanitizer

• Main Author: Al-Najjar, Eman Ahmad Jaber
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2021
• Subjects: Gram-negative bacteria; Gram-positive bacteria; Hygiene; Medicine; Pharmaceutical sciences; Pharmacology; Sanitizers
• ISBN: 9798460460038

Background Hand sanitizers are an important group of antiseptics that are mostly used by health care workers and are also used by others. Hand contamination is a source of microbes that cause serious infections. Misuse of these antimicrobials holds the potential for developing microbial resistance to them. This study aims at investigating the possible adaptation of selected microbes to a new formula of a hand sanitizer. Methodology Wild type strains of Pseudomonas aeruginosa ATCC27853, E. coli ATCC8739 and staphylococcus aureus ATCC6538 are subjected to sub-MIC levels of a newly formulated hand sanitizer (HS) using the square plate technique, tolerant strains that grew at higher concentrations of the hand sanitizer were considered resistant and were investigated for the adopted mechanism of resistance. Tolerant and wild type strains were compared for the changes in Leakage of a 260 nm substance, pyocyanin pigment synthesis in Pseudomonas aeruginosa, cell surface hydrophobicity , and LPS composition using SDS-PAGE electrophoresis. Effect of microbial HS adaptation on susceptibility to antibiotics is observed using disc diffusion assay. Results The effect of the new formula of HS was predominantly on Gram-negative bacteria (P. aeruginosa and E. coli) and showed a lesser effect on Gram-positive bacteria (S. aureus). P. aeruginosa, tolerated the presence of the hand sanitizer in growth media , as indicated by growth at all concentrations in the gradient square plate and the increased production of the pyocyanin pigment. Tolerant colonies picked at the highest concentration of the HS showed no altered antibiotic sensitivity compared to HS sensitive cells. Growth of E. coli in the gradient square plate was inhibited at 0.28%; however, tolerant colonies were observed and detected at higher concentrations of the hand sanitizer. These colonies were tested for antibiotic susceptibility and compared to susceptibility of HS sensitive cells. E. coli sensitive strain have demonstrated a significant decrease in the zone of inhibition of tetracycline, nitrofurantoin and a significant increase in zone of inhibition of ciprofloxacin in comparison to the sensitive strain. Exposure of Gram negative test strains to the HS resulted in a significant increase in the 260 nm substance in the growth media indicating effect of the hand sanitizer on cell membrane this effect was not observed on Gram positive bacteria, however this effect was significantly reduced in the HS tolerant strains suggesting changes in the outer membrane. Hydrophobicity experiment revealed significant differences in the affinities of HS tolerant P. aeruginosa to hydrocarbons. P. aeruginosa tolerant strains exhibited higher hydrophobicity on their cell surfaces than HS sensitive strains. While no change in cell surface hydrophobicity for both tolerant and sensitive E. coli strains was observed. SDS-PAGE analysis of tolerant and sensitive E. coli strains revealed the emergence of bands with a molecular weight of less than 25 kD in tolerant strains, compared to the sensitive strains. Increase in band intensity with molecular weights ranging from (25-75 kD) in tolerant strains compared to sensitive ones was observed in P. aeruginosa. Conclusion The newly developed hand sanitizer exhibits an inhibitory effect on Gram-negative bacteria; however, frequent exposure to sub-minimal inhibitory concentrations can lead to microbial tolerance and resistance. This induced resistance does not confer resistance to antibiotics used in this study