BMC Microbiology 2014-01-01

The effects of the natural enzyme, Pectinex Ultra SP-L, on human cell cultures and bacterial biofilms in vitro.

Ian P Olwoch, Oppel B W Greeff, Gisela Jooné, Vanessa Steenkamp

Index: BMC Microbiol. 14 , 251, (2015)

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Abstract

Pectinex Ultra SP-L (Pectinex) is a microbial-derived enzyme that is used in the food industry and that has been shown to inhibit bacterial biofilms. It has been suggested that Pectinex may be useful in the management of biofilm-related bacterial infections and therefore warrants further investigation in this regard. The aim of this study was to investigate the cytotoxicity of Pectinex on cervical adenocarcinoma cells (HeLa), lymphocytes and neutrophils. Cell viability and morphology were assessed using an in vitro spectrophotometric MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay and polarization-optical transmitted light differential interference contrast microscopy. This study also investigated the antibacterial and antibiofilm actions of Pectinex, alone and in combination with antibiotics, on standard and clinical cultures of Staphylococcus aureus and Pseudomonas aeruginosa. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined using p-iodo-nitrotetrazolium violet staining of bacterial cultures and regrowth of subcultures. Biofilm biomass and cell viability were quantified spectrophotometrically after staining with crystal violet and MTT.The IC50 (±SEM) of Pectinex was 193.9 (±22.2) PGU/ml for HeLa cells, 383.4 (±81.5) and 629.6 (±62.8) PGU/ml for fMLP-stimulated and non-stimulated lymphocytes respectively, and 245.9 (±9.4) and 529.7 (±40.7) PGU/ml for fMLP-stimulated and non-stimulated neutrophils, respectively. Induced morphological features characteristic of apoptosis and necrosis included cell membrane blebs and vacuolization in HeLa cells, clumping in lymphocytes, as well as shrunken rounded cells, apoptotic bodies and debris in all cultures. Pectinex (7.42 - 950 PGU/ml-1) was not bactericidal. In clinical cultures of Staphylococcus aureus, co-administration of Pectinex was associated with a 28.0% increase in both the MIC and MBC of amoxicillin-clavulanate. In clinical cultures of P. aeruginosa, there was an 89.0% and 92.8% increase in the MIC and MBC of ciprofloxacin, respectively. Pectinex ≤ 118.75 PGU/ml-1 and incubation periods ≥ 6 h were associated with increased biomass and cell viability in S. aureus or P. aeruginosa biofilms.Pectinex appeared to antagonize the antibacterial effects of amoxicillin-clavulanate and ciprofloxacin and furthermore demonstrated significant cytotoxicity. It was therefore deemed unsuitable for the management of either planktonic or biofilm phenotypes of S. aureus or P. aeruginosa.