Factors influencing the probiotic potential of an inhibitor-producing Micrococcus luteus

Abstract

A strain of Micrococcus luteus, isolated from the epidermis of an adult human male, produces anti-bacterial activity in vitro. The purpose of this study was to investigate the potential value of this bacterium, designated M. luteus Q24, as a topical probiotic. This objective was pursued by (i) determining the spectrum of inhibitory activity of M. luteus Q24 against a heterogeneous selection of bacteria; (ii) establishing the optimal conditions for production and extraction of the bioactive compound(s); (iii) attempting to characterise the bioactive compound(s); and (iv) monitoring bacterial interactions and population shifts on ex vivo epidermis following application of M. luteus Q24 and various other skin bacteria.

The deferred antagonism test was used to assess the inhibitory activity of M. luteus Q24 against representatives of 32 bacterial species. All tested strains of Staphylococcus aureus were sensitive. Other sensitive bacteria included Staphylococcus saprophyticus, Staphylococcus simulans, Corynebacterium ulcerans, Corynebacterium diphtheriae biotype gravis, Streptococcus spp., Micrococcus spp., Lactococcus lactis, Propionibacterium propionicum and Propionibacterium acnes. Five staphylococci (Staphylococcus aureus CAMP, Staphylococcus aureus Oxford, Staphylococcus cohnii, Staphylococcus saprophyticus and Staphylococcus simulans) were selected as indicator organisms to further characterise the anti-staphylococcal inhibitory activity. M. luteus Q24 appeared to produce more than one bioactive compound in vitro, and the expression of these differed as a function of incubation time and medium composition. While inhibitory activity against various streptococci was optimal on blood agar after 48 hr incubation (at 37 °C in an atmosphere of 5 % CO2 in air), the best non blood-containing medium for recovery against the staphylococcal indicators was Columbia agar base supplemented with CaCl2 (50 mM) (CABCaCl2). Extraction of the anti-staphylococcal compound(s) from cells grown for 24 hr on CABCaCl2 was achieved with acidified 95 % methanol, followed by a further n- propanol extraction. No indication of a peptide component could be detected in the purified product by C18 reversed phase HPLC and subsequent analysis by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). MS data indicated that the inhibitory compound was a long chain, branched molecule, containing sulphur, with an estimated molecular weight of 1061-1079 Da. Late elution from the r-HPLC column and the binding of the compound to the walls of polypropylene microcentrifuge tubes, indicated that the compound was hydrophobic (or held in a hydrophobic matrix). NMR analysis indicated the presence of a lipid component. An ex vivo model was developed using porcine skin to examine bacterial population interactions on an epithelial membrane. Test cultures were inoculated onto the epidermis of porcine skin to mimic infection. After incubation, samples of the bacterial population were recovered from the epidermis and analysed by culture and by PCR-DGGE. The population levels achieved by S. aureus CAMP were reduced by two orders of magnitude when co-cultured with M. luteus Q24 by comparison to the levels obtained from mono-culture inocula. By contrast, the population levels of S. aureus CAMP were not diminished by co-culture with M. luteus T-18 (a non inhibitor- producing strain). These findings provide encouraging evidence for the potential probiotic application of M. luteus Q24 to the prevention of cutaneous membrane infection. Of particular interest is the relatively high susceptibility of S. aureus strains to the M. luteus Q24 inhibitory compound(s). A beneficial public health application would be to assess the efficacy of M. luteus Q24 against methicillin resistant variants of S. aureus (MRSA), with the objective of colonising hospital personnel with M. luteus Q24 to decrease S. aureus carriage. Other important potential targets for M. luteus Q24 control are Propionibacterium spp., key aetiological agents of acne.