Anti-infective efficacy of Psidium guajava L. leaves against certain pathogenic bacteria

Abstract

Water extracts of Psidium guajava leaves prepared by three different extraction methods were compared with respect to their anti-infective activity against Pseudomonas aeruginosa and Staphylococcus aureus in the nematode host Caenorhabditis elegans. The water extract prepared by Microwave Assisted Extraction method was found to have better anti-infective activity, and its activity was further compared with hydroalcoholic extract prepared using the same extraction method against five different pathogenic bacteria. Both these extracts could attenuate virulence of P. aeruginosa, S. aureus, Serratia marcescens, and Chromobacterium violaceum, towards C. elegans. Anti-infective efficacy of P. guajava leaf extract seems partly to stem from its quorum-modulatory property, as it could modulate production of quorum sensing-regulated pigments in all the susceptible bacteria.

Keywords: Anti-virulence; Antimicrobial Resistance (AMR); Caenorhabditis elegans; Guava leaf; Microwave Assisted Extraction (MAE); Quorum Sensing (QS).

Figure 1.

Comparison of in vivo anti-infective efficacy of P. guajava leaf extracts prepared by three different extraction methods, against P. aeruginosa ( A– C), and S. aureus ( D– F). Catechin (50 μg/mL) and gentamicin (0.1 μg/mL) employed as positive controls conferred 100% and 80% protection on the worm population, respectively. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans. DMSO (0.5%v/v) and GLE at tested concentrations showed no toxicity towards C. elegans. MAE: Microwave Assisted Extraction; VAE: Vacuum Assisted Extraction; GLE: Guava Leaf Extract.

Figure 2.. Comparison of the in vivo anti-infective potential of water extract and hydroalcoholic extract of P. guajava leaf extracts prepared by Microwave Assisted Extraction method, against five different pathogenic bacteria.

Figures A– E shows data against P. aeruginosa, S. aureus, S. marcescens, C. violaceum and S. pyogenes respectively. Catechin (50 μg/mL) employed as a positive control conferred 100% protection on worm population against all the pathogenic bacteria except S. pyogenes. Against S. pyogenes, catechin could not offer any protection to host worms. Gentamicin (0.1 μg/mL) allowed survival of worm population to the extent of 80% in face of P. aeruginosa, S. aureus, or S. pyogenes challenge; and 100% against the remaining two pathogens. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacteria towards C. elegans. DMSO (0.5%v/v) and GLE at tested concentrations showed no toxicity towards C. elegans.

Figure 3.. Effect of hydroalcoholic extract of P. guajava leaves prepared by Microwave Assisted Extraction method on bacterial growth and QS-regulated pigment production.

( A) P. aeruginosa ( B) S. aureus ( C) S. marcescens ( D) C. violaceum ( E) S. pyogenes. Bacterial cell density and pigment production were quantified as earlier described by us ( Joshi et al., 2016). Bacterial growth was measured as OD ₇₆₄ for the four pigmented bacteria, while for S. pyogenes OD ₆₆₀ was used. OD of pyoverdine was measured at 405 nm, and that of pyocyanin at 520 nm; Pyoverdine Unit was calculated as the ratio OD ₄₀₅/OD ₇₆₄ (an indication of pyoverdine production per unit of growth); Pyocyanin Unit was calculated as the ratio OD ₅₂₀/OD ₇₆₄ (an indication of pyocyanin production per unit of growth. OD of staphyloxanthin was measured at 450 nm, and Staphyloxanthin Unit was calculated as the ratio OD ₄₅₀/OD ₇₆₄ (an indication of staphyloxanthin production per unit of growth). OD of prodigiosin was measured at 535 nm, and Prodigiosin Unit was calculated as the ratio OD ₅₃₅/OD ₇₆₄ (an indication of prodigiosin production per unit of growth). OD of violacein was measured at 585 nm, and Violacein Unit was calculated as the ratio OD ₅₈₅/OD ₇₆₄ (an indication of violacein production per unit of growth). QS: Quorum sensing