The effect of polyoxyethylene stearate (POES) on the growth of mycobacteria in radiometric 7Hl2 middlebrook TB medium

Introduction

Polyoxyethylene stearate (POES; Figure 1) is a non-ionic emulsifying agent. Such agents have been shown to enhance the growth of mycobacteria in vitro. Polyoxyethylene stearate has been recommended as an additive to the radiolabelled 7H12 Middlebrook TB media and as such has been shown to enhance growth of mycobacteria, in the radiometric BACTECe rapid culture system.Polyoxyethylene stearate was reported to enhance the growth of a number of strains of mycobacteria  without causing many of the detrimental effects previously reported with the use of Tween 80, such as clouding the media and alterations in bacterial cell morphology [4] POES can be obtained in a number of different forms. As far as molecular structure is concerned for determining the type of POES, the only variable is condense number of ethylene epoxide. Emulsifying agents are classified by their ability to act either as detergents, (spreading agents) or solubilizers. These abilities are quantified by the Hydrophilic-Lipophilic Balance (HLB) number and each POES has a different HLB value. Cutler et al. examined the growth enhancing effect of six previously untested POE’s (POE(8)S to POE(100)S) whose spreading properties (Hydrophilic-Lipophilic Balances-HLB’s) were known. The aim of this study was to determine which polyoxyethylene stearate has the greatest ability to enhance the growth of different mycobacterial species and how the HLB of each polyoxyethylene stearate related to its effect on the growth of each species. [1-2]

Result overview

They found that POE(50)S produced the greatest enhancement in growth and reduction in the time taken to detect growth for M. tuberculosis and POE(30)S and POE(JL)S for species of mycobacteria other than M. tuberculosis (MOTT). Comparing the effects of polyoxyethylene stearate’s to their HLB’s (in BACTEC 7H12 media) we suggest four factors which may affect the growth enhancing ability of each polyoxyethylene stearate: 1 its dispersal effect on the bacteria (acting as an emulsifier); 2 it’s solubilizer effect on the bacterial cell; 3 the effect the POES on the dispersal of the radiolabelled fatty acid used as a substrate in the media to produce radiolabelled CO2; 4 mycobacteria may metabolise polyoxyethylene stearate.

Dicussion

Cutler et al. compared the effect of polyoxyethylene stearate with different HLB values, ranging from detergent (HLB, 13-15) to solubilizers (HLB, 15-18). With oil/water emulsions, the spreading ability of toluene on the surface of an aqueous solution has been shown to reflect the HLB of the emulsion. Complete spreading is achieved at HLB 16, as HLB decreases the spreading spreading ability decreases [3]. This may be important if the dispersal or spreading of the mycobacteria throughout the media affects their growth.

To reiterate on our original aims, we wished to determine which polyoxyethylene stearate had the greatest ability to enhance the growth of different mycobacterial species and how the HLB of each polyoxyethylene stearate related to its effect on the growth of each mycobacterial species. They found that the enhancing abilities of polyoxyethylene stearate was different for M. tuberculosis complex strains and MOlT. With MOTT the maximum enhancing ability was at HLB 16 (POE(30)S). At this point POES should have reached its maximum spreading ability but is still a detergent and not a solubilizer. This peak enhancement at HLB 16 was greatest with the MAIS group of strains. With M. tuberculosis maximum enhancement was at HLB 17.9 (POE(50)S). There was no enhancement of growth with POE(30)S. The mean enhancement of growth by polyoxyethylene stearate resulted in a 17 % decrease in the time taken to detect growth compared to the control. The MOTT maximum growth enhancement is related to the HLB that gives maximum dispersal (HLB 16). The decrease in enhancing ability with POES of HLB greater than 16 may reflect the increasing solubilizing effect of the POES on the mycobacterial cell.

The variations shown between the effect of polyoxyethylene stearate on different strains may reflect the different factors which could play a role in affecting growth. It has for example, been suggested that Tween 80 enhances mycobacterial growth by increasing dispersal and by providing a metabolite which acts as an additional nutrient. When emulsifying agents which only increase dispersal without being metabolised are added to the media, they do not enhance growth. As well as dispersing the bacteria, emulsification agents may also 3]. Radiolabelled 14C palmitic acid is used in BACTEC 7H12 Middlebrook TB media as a radiolabelled substrate. Increasing the dispersal of this substrate in the media may also enhance the production of 14CO2 by making the substrate more easily available to the mycobacteria and lead to an increase in bacterial growth (as indicated by G.I.).

Growth in the presence of fatty acids produces changes in the morphology of mycobacteria, shown most effectively in the electron micrographs of Schaefer and Lewis, and increases the lipid content of the cells. Further investigations into the effect of polyoxyethylene stearate on cell morphology and an analysis of the cell fractions may help to clarify the polyoxyethylene stearate phenomenon. Polyoxyethylene stearate could also increase the supply of metabolites to the mycobacteria either (as suggested previously) by increasing the available 14C palmitic acid as in the case of Tween 80, or by providing a metabolite on being hydrolysed by the mycobacteria, or possibly both. Although POE(30)S was not the best agent to use with strains of M. tuberculosis, with the increasing isolation of other strains of mycobacteria from immunosuppressed patients (including AIDS patients] the improved growth of the MAIS group of organisms by POE(30)S could prove to be useful in these cases. This raises the question whether they should use two media for culturing M. tuberculosis and MOTT similar to the way Lowenstein-Jensen (LJ) media are used in many laboratories at present. Two LJ slopes (one with added pyruvate and one without] are used to improve the isolation rate: pyruvate being added for M, bovis and isoniazid resistant M. tuberculosis. Two liquid media could be used, one with POE(100)S and one with POE(30)S, for the isolation of M. tuberculosis and MOTT respectively.[1]

References

[1] Cutler RR, Wilson P, Clarke FV. The effect of polyoxyethylene stearate (POES) on the growth of mycobacteria in radiometric 7H12 Middlebrook TB medium. Tubercle. 1987;68(3):209-220. doi:10.1016/0041-3879(87)90057-2

[2] Zhu ZF, Meng N , Xu DS.The Usability of Polyoxyethylene Stearate as Lubricant for Sizing Cotton Warp Yarns[J].Journal of Donghua University (English Edition). 2012; 29(2):4.

[3] Becher P. Emulsions: theory and practice. American Chemical Society.1965;182, 209.