The insightful fusion of chemistry and microbiology to understand Lentilactobacillus parabuchneri biofilms

Unrevealing molecular details of histamine-producing species via in situ IR-ATR spectroscopy - An aimed spotlight of “bad biofilms”
The insightful fusion of chemistry and microbiology to understand Lentilactobacillus parabuchneri biofilms

Anyone may wonder, why exactly Lentilactobacillus parabuchneri?

Food contaminant microorganisms form robust biofilms along with exceptionally high histamine production. Biogenic amine producers (Bas) formed in food via the activity of lactic acid bacteria invoke prominent toxicological effects on the digestive and respiratory systems. Among them, the risky biofilms of Lactobacillus parabuchneri bacteria exhibit food poisoning by high-histamine producing ability, therefore is essential to understand the involvement of molecular mechanisms of biofilm formation and metabolic pathways. Lentilactobacillus parabuchneri is a Gram-positive, facultatively anaerobic bacterium isolated from cheese and other food products, subsequently leading to severe food spoilage resulting in foodborne illness. Biofilm formation within amino-acyl decarboxylase activity of bacteria are introduced by contamination during manufacture, equipment’s surfaces and post-ripening processing such as grating slicing, cutting, etc. To date only a few studies, report on the molecular mechanisms and properties of Lentilactobacillus parabuchneri biofilms owing to the complexity of these systems. In the present study, we use as a model the microbial microorganism L. parabuchneri DSM 5987 strain isolated from cheese to investigate its ability to form biofilms which are of substantial relevance given their contribution to the presence of histamine in dairy products.


What did we do?

Infrared attenuated total reflection spectroscopy (IR-ATR) using a custom flow-through assembly was applied for real-time and non-destructive observations of biofilm formation at unprecedented molecular detail during in situ monitoring of changes associated with molecular fingerprints of L. parabuchneri. To get a better understanding of what’s going on, we have elaborated the molecular changes of biofilms grown in ATR waveguide surface compared to biofilm adhesion studies by scanning electron microscopy (SEM) at common industry-relevant surfaces of real applications i.e., stainless steel. Herein, the biofilms' chemistry and architecture were evaluated by a combination of IR-ATR spectroscopy and microscopy. Multivariate data analysis and classification strategies (PCAs) were applied to facilitate the multiparametric mining of the molecular processes involved in biofilm formation. We also studied the viability by the crystal violet staining method (CV) as a biofilm-biomass quantification procedure to make the classification of the biofilm formation ability for the L. parabuchneri DSM 5987 strain.


What did we find?

We found out that indeed we can use real-time infrared spectroscopy as an analytical tool for in situ and non-destructively monitoring of L. parabuchneri biofilms during the extended unperturbed observation periods. . Detailed IR vibrational peak attributions of L. parabuchneri constituents explains the dominating contributions to biofilm formation. The spectral range between 1700 and 600 cm‒1 is unique of interest providing information on molecular processes via significantly increased changes of the amide I and II bands representing the protein signature of the biomass, lactate, nucleic acids and lipids along with the characteristic features of extracellular polymeric substances indicating formation, growth, and maturation of the biofilm. Overall, the representative IR spectral window was found to be most suitable for the identification of Lactobacillus biofilms enabling to study bioadhesion mechanisms, as well as physico-chemical property changes of L. parabuchneri during not only the early stages of biofilm formation but for an extended period. The comprehensive characterization of the biochemical properties concerning the overlap of IR features owing to the complexity of the bacterial matrix was completely resolved via cumulative fit functions and integrated peak value strategy (IPVs). Multivariate data analysis applied for efficient data mining of such extensive datasets by the classification models showed distinct data clusters enabling the unambiguous assignment of the biofilm age.  Finally, the studies we derived from these findings were transferred to L. parabuchneri biofilm formation at biotic and abiotic surfaces with a focus on stainless-steel surfaces relevant to food processing and dairy industries. L. parabuchneri DSM 5987 strain aggregates into micro clusters, chains of undivided cells, and eventually mature biofilms that are of concern for surfaces used in dairy industry.


Take away

Here it is observed that physico-chemical changes and molecular mechanisms of L. parabuchneri biofilms evaluated during a long-term period of versatile IR-ATR monitoring are innovative approaches providing molecular insight into early-stage detection of food spoilage by histamine-producing species. By understanding the biofilm formation involved in the initial adhesion processes, the L. parabuchneri biofilm has been dynamically monitored, molecularly analyzed, and structurally understood facilitating the subsequent development of suitable prevention strategies for microbial contaminations. We can collectively anticipate that from this knowledge concrete steps for prevention and disintegration via suitable antimicrobials may be driven for future studies.


The complete story is recently published in npj Biofilms and Microbiomes. Check out here to read more:


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