In situ monitoring of Lentilactobacillus parabuchneri biofilm formation via real-time infrared spectroscopy

Abstract

Foodborne pathogenic microorganisms form biofilms at abiotic surfaces, which is a particular challenge in food processing industries. The complexity of biofilm formation requires a fundamental understanding on the involved molecular mechanisms, which may then lead to efficient prevention strategies. In the present study, biogenic amine producing bacteria, i.e., Lentilactobacillus parabuchneri DSM 5987 strain isolated from cheese were studied in respect with biofilm formation, which is of substantial relevance given their contribution to the presence of histamine in dairy products. While scanning electron microscopy was used to investigate biofilm adhesion at stainless steel surfaces, in situ infrared attenuated total reflection spectroscopy (IR-ATR) using a custom flow-through assembly was used for real-time and non-destructive observations of biofilm formation during a period of several days. The spectral window of 1700-600 cm^-1 provides access to vibrational signatures characteristic for identifying and tracking L. parabuchneri biofilm formation and maturation. Especially, the amide I and II bands, lactic acid produced as the biofilm matures, and a pronounced increase of bands characteristic for extracellular polymeric substances (EPS) provide molecular insight into biofilm formation, maturation, and changes in biofilm architecture. Finally, multivariate data evaluation strategies were applied facilitating the unambiguous classification of the observed biofilm changes via IR spectroscopic data.

Fig. 1. Infrared signatures of L. parabuchneri planktonic cells.

Full-range IR-ATR spectra of dry planktonic L. parabuchneri (red spectrum) and dry MRS medium (dashed black spectrum).

Fig. 2. IR-ATR spectra of L. parabuchneri planktonic cells recorded during the drying process.

IR-ATR spectra were recorded every 5 min until 60 min.

Fig. 3. Molecular changes in the planktonic form of L. parabuchneri vs 24 old biofilms.

Comparison of IR spectra of 24 h old L. parabuchneri biofilm performed in flow-through mode (black spectrum) vs. planktonic cells at static conditions (dashed blue spectrum).

Fig. 4. Oxygen monitoring in growth media.

The obtained oxygen levels are shown as response of the fiberoptic oxygen probe (%) vs. mL of MRS medium.

Fig. 5. Biofilm evolution.

a Scheme of the deposition of L. parabuchneri biofilms at the ATR waveguide surface after the conditioning film was formed. b Optical image of L. parabuchneri bacterial biofilm attached to the surface of the ATR waveguide during 7 days of IR measurements at flow-through conditions. c 3D plot of IR-ATR spectra vs. time revealing the molecular changes occurring during 72 h of biofilm development. d Concentration profiles of oxygen prior to and after inoculation of the L. parabuchneri suspension.

Fig. 6. IR-ATR spectra of L. parabuchneri biofilms monitored for 24, 48, and 72 h during real-time incubation within the flow system.

The observed spectral changes correspond to proteins (amide I: 1700–1618 cm⁻¹, amide II: 1585–1486 cm⁻¹), LA: 1465–1293 cm⁻¹, amide III + NA + PL: 1350–1180 cm⁻¹ and PS + NA + PL: 1189–960 cm⁻¹, respectively (LA = lactic acid or lactate, NA = nucleic acids, PL = phospholipids, PS = polysaccharides).

Fig. 7. Deconvolution of IR spectra for unraveling molecular details during biofilm formation.

a IR-ATR spectrum of MRS medium supplemented with 5 mM L ( + ) L-histidine (red) and experimentally obtained spectra of the individual constituents in the spectral region from 1800 to 900 cm⁻¹ (see color code, top, middle). b Resulting cumulative impulse fit (black) and fitted IR signatures of the individual constituents (see color code, top, right). The integration values (peak center and width) of the individual components (see Table 2) served as restrictions for the cumulative impulse fit (n ≥ 10; Gaussian fit). The experimentally obtained sum spectrum is given in red in (a) and (b).

Fig. 8. Temporal progression of characteristic integrated peak areas during 48 h of L. parabuchneri inoculation.

(Reference spectrum: MRS medium (0.5 g L⁻¹). Integrated peak areas for amide 1 band (1700–1617 cm^‒1, black); amide II band (1577–1464 cm^‒1, red); amid III band (1350–1200 cm^‒1, blue); nucleic acids (1280–1180 cm^‒1, green), and the extracellular polymeric substances (1121–997 cm^‒1, purple) are shown.

Fig. 9. Multivariate classifications of IR spectral datasets of L. parabuchneri biofilms via principal component analysis.

a Scores plot of the classified spectral data recorded after 24, 48, 72, and 96 h via PCA revealing that the obtained spectra clearly cluster along the time axis and that the recorded IR spectra unambiguously identify the maturation stage of the biofilm; b loadings of the two PCs explaining >98% of the total variance.

Fig. 10. Scanning electron microscopy images of L. parabuchneri DSM 5987 biofilms grown at stainless steel surfaces.

Biofilm-forming L. parabuchneri cells: a after 24 h of incubation; b after 48 h of incubation; c after 72 h of incubation, and d by exemplary agglomerates embedded into EPS and adherent to the surface. 1–10 μm scale bars are shown on micrographs.

Fig. 11. Custom IR-ATR flow cell concept.

The continuous flow assembly was equipped with a ZnSe ATR waveguide providing 6 internal reflections for monitoring biofilm formation and evolution via evanescent field absorption spectroscopy.