Vibrational Spectrum Characterization of Outer Surface of Helicobacter Pylori Biofilms by Functionally-Enhanced Derivative Spectroscopy (Feds)

Abstract

Mid-infrared spectroscopy in conjunction with Functionally-Enhanced Derivative Spectroscopy (IR+FEDS) is a powerful analytical tool for the improvement of analysis of microorganism IR spectra. The objective of this research is to characterize the outer surface of two Helicobacter pylori strains by IR+FEDS. This work is a key stage for the study of cell-cell and cell-surface interactions between microorganisms, as well as, for polymicrobial biofilm characterizations where H. pylori species are involved. Artificial bacterial biofilms were deposited on ultrafiltration cellulose membranes covalently modified by insertion of one spectral marker and used as sensing surface for analysis of bacterial biolayers. Biolayers were analyzed using an infrared spectrophotometer with ATR. Data were analyzed by classic procedures and by deconvolution based on FEDS transform. It is concluded that, for correct application of technique is required a minimum amount of noise in the working spectra which can be achieved by simple smoothing algorithms; in addition, reproducibility must be warranted by the implementation of standardized protocols and the use of an appropriate number of samples. It is concluded that in addition to typical signals associated with the IR spectrum of microorganisms, by FEDS, a better and more detailed description of the outer membrane of H. pylori biofilms can be performed. In particular, it is concluded that the detecting and monitoring of cysteine-rich proteins can be satisfactorily performed by IR+FEDS.

Multiple surface interaction mechanisms direct the anchoring, co-aggregation and formation of dual-species biofilm between Candida albicans and Helicobacter pylori

“…Selecting of region for spectral analysis: A strong overlap of the signals is expected due to the compositional complexity of the biofilms [30] , [31] , [32] . A first comparison of differentiation capacity of IR spectra was performed by chemometric analysis using Principal Component Analysis (PCA).…”

“…A first comparison of differentiation capacity of IR spectra was performed by chemometric analysis using Principal Component Analysis (PCA). To study the biofilms, the region between 3100 and 2400 cm −1 was selected, in this region, signals associated with cysteine units have been previously described for H. pylori by FEDS [30] . In particular, H. pylori contains cysteine-rich proteins on its external membrane which are associated to disulfide bonds (—S—S—) which can be monitored from IR spectra in conjunction with deconvolution techniques.…”

“…In particular, H. pylori contains cysteine-rich proteins on its external membrane which are associated to disulfide bonds (—S—S—) which can be monitored from IR spectra in conjunction with deconvolution techniques. Disulfide bonds are important in this research because they have been related with the anchoring of H. pylori to surfaces [30] , [33] . In addition, in this spectral range, signals associated with invariant structural units can be easily identified, such as CH, CH 2 and CH 3 , which can be used as internal markers for the analysis of signal displacements since they are weakly affected by neighboring electronegative groups.…”

“…By the use of FEDS is possible to carry out the transformation of the spectrum of the biofilms allowing the construction of “ spectral fingerprints ” with higher resolution of signals in comparison with IR spectra. Details of this procedure have recently been published previously for H. pylori and C. albicans using artificial biofilms [30] , [31] . Here, the analysis is focused in the building spectral fingerprints of biofilms and dual-species biofilms promoted from aqueous environments.…”

“…Samples obtained from C. albicans , H. pylori and (Ca + Hp)BF were analyzed by Scanning Electron Microscopy (SEM) (JEOL 6380LV with resolution of 3.0 nm), Transmission Electron Microscopy (TEM) (JEOL-JEM 1200EXII with resolution of 0.15 nm), and optic microscopy (Nikon Eclipse E600 microscope). Samples were pretreated by the following procedure: For SEM, chemical fixation was performed with paraformaldehyde, followed of dehydration with acetone, dried at 40 °C in temperature-controlled oven and coated with an ultrathin layer of gold; whereas for TEM, chemical fixation was performed using paraformaldehyde and osmium tetroxide, followed of dehydration with acetone and embedding in EMbed-812 [30] , [31] .…”