Properties of the Extracellular Polymeric Substance Layer from Minimally Grown Planktonic Cells of Listeria monocytogenes

Abstract

The bacterium Listeria monocytogenes is a serious concern to food processing facilities because of its persistence. When liquid cultures of L. monocytogenes were prepared in defined media, it was noted that planktonic cells rapidly dropped out of suspension. Zeta potential and hydrophobicity assays found that the cells were more negatively charged (-22, -18, -10 mV in defined media D10, MCDB 202 and brain heart infusion [BHI] media, respectively) and were also more hydrophobic. A SEM analysis detected a capsular-like structure on the surface of cells grown in D10 media. A crude extract of the extracellular polymeric substance (EPS) was found to contain cell-associated proteins. The proteins were removed with pronase treatment. The remaining non-proteinaceous component was not stained by Coomassie blue dye and a further chemical analysis of the EPS did not detect significant amounts of sugars, DNA, polyglutamic acid or any other specific amino acid. When the purified EPS was subjected to attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, the spectra obtained did not match the profile of any of the 12 reference compounds used. An x-ray diffraction (XRD) analysis showed that the EPS was amorphous and a nuclear magnetic resonance (NMR) analysis detected the presence of glycerol. An elemental energy dispersive x-ray (EDX) analysis showed traces of phosphorous as a major component. In conclusion, it is proposed that the non-proteinaceous component may be phospholipid in nature, possibly derived from the cell wall lipoteichoic acid.

Keywords: L. monocytogenes; bond stretching; capsule; extracellular polymeric substance; minimal media; proteinaceous and non-proteinaceous moiety; spectroscopy.

Figure 1

Cells of L. monocytogenes (Lm1040S) grown in MCDB 202 (showing flocculated cells) and brain heart infusion (BHI) media at 37 °C for 18 h (a). SEM micrographs were captured for cells grown in BHI (b) and D10 (c) and MCDB 202 (d) media. Cells were grown for 18 h with shaking at 37 °C and then harvested from the broth by centrifugation. Cells were re-suspended in PBS, fixed on electron stubs and coated with platinum before viewing. The white arrow (c) indicates an area where a thick layer of an extracellular polymeric substance (EPS) can be seen joining cells.

Figure 2

Representative cell hydrophobicity (a) assay showing affinity to chloroform solvent (500 µL) and zeta potential measurements (b) of strain Lm 10403S cells. Cells (a) were grown in MCDB 202 (□), D10 (■) and BHI (■) media before an affinity analysis was carried out with the microbial attachment to hydrocarbon (MATH) method. The zeta potential (b) was measured and the results represent values from triplicate independent samples. Error bars represent two standard errors in both panels. Statistical modelling showed that the solvent type and volume, media and strain interaction were significant terms (p < 0.05).

Figure 3

Staining of the EPS of the L. monocytogenes crude extract with Coomassie blue (a) and methylene blue (b). Lane L in both panels is the ladder. In panels (a,b) samples were treated with pronase, (lanes 1, 2, 3) whereas samples in lanes 4, 5 and 6 were not.

Figure 4

A SEM analysis (500×) of the purified EPS (a). The cell culture was pelleted by centrifugation after which the EPS was precipitated, dialysed and treated with pronase to remove cell-associated proteins. The material was freeze dried and stored at −85 °C. Panel (b) shows the trace of energy dispersive x-ray elemental analyses of the purified EPS of L. monocytogenes.

Figure 5

X-ray diffraction traces of PGA (a) and the EPS from L. monocytogenes (b) Diffractograms were recorded on powdered samples with a scanning time of 6 s per angular interval at 20 °C.

Figure 6

¹³C NMR spectrum of the EPS of L. monocytogenes. The EPS of L. monocytogenes (30 mg) was dissolved in 4% deuterated water. Spectra were acquired with ¹³C NMR and peaks due to the carbons in glycerol were visible.

Figure 7

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of purified L. monocytogenes and other compounds. In panel (a), the EPS (the EPS of L. monocytogenes) was compared with bovine serum albumin (BSA), DNA and glycerol. Panel (b) shows a comparison with the defined media components, which included aluminium chloride (AlCl₃), sodium hydroxide (NaOH) sodium dihydrogen phosphate (NaH₂PO₄), ferric chloride (FeCL₃) and potassium phosphate (K₂HPO₄). These ionic materials were used to test for the presence of impurities. Further comparisons were made with sugars (c); namely, galactose, sucrose, mannose and glucose.

Figure 7

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of purified L. monocytogenes and other compounds. In panel (a), the EPS (the EPS of L. monocytogenes) was compared with bovine serum albumin (BSA), DNA and glycerol. Panel (b) shows a comparison with the defined media components, which included aluminium chloride (AlCl₃), sodium hydroxide (NaOH) sodium dihydrogen phosphate (NaH₂PO₄), ferric chloride (FeCL₃) and potassium phosphate (K₂HPO₄). These ionic materials were used to test for the presence of impurities. Further comparisons were made with sugars (c); namely, galactose, sucrose, mannose and glucose.