Characterization of the Lactobacillus Casei Group Based on Profiling of Ribosomal Proteins Coded in S10-spc-alpha Operons as Observed by MALDI-TOF MS

Significance of the topic

The Lactobacillus casei group encompasses closely related strains widely used in food fermentation and probiotics. Their genetic similarity makes accurate subspecies discrimination challenging by conventional methods. Profiling ribosomal proteins via MALDI-TOF MS offers a rapid molecular phylogenetic approach to resolve taxonomy, support quality control in food and clinical laboratories, and improve strain management.

Objectives and study overview

This study aimed to develop and validate a method based on ribosomal proteins encoded in the S10-spc-alpha operon, measured by MALDI-TOF MS, for fast and precise discrimination of L casei group members at subspecies level. The approach was implemented in the Strain Solution software and benchmarked against established genetic techniques.

Methodology and instrumentation

• Strains and culture conditions: Fifteen reference strains of L casei, L paracasei subspecies and L rhamnosus obtained from JCM and NBRC collections were cultured per vendor protocols
• Operon sequencing: S10-spc-alpha operon sequenced by PCR; ribosomal protein masses predicted from amino acid sequences with processing adjustments
• Instrument: AXIMA MALDI-TOF MS in linear positive mode; sinapic acid matrix; cell lysates prepared by bead beating in TMA I buffer
• Data analysis: Theoretical mass list registered in Strain Solution; peak matching produced binary presence profiles; cluster analysis by UPGMA in BioNumerics

Main results and discussion

Four ribosomal proteins (L36, S19, L22, S11) exhibited invariant masses across group members serving as group markers. Additional proteins (L23, L24, S13) showed strain specific mass shifts. MALDI-TOF MS spectra resolved these variations, enabling assignment of unknown strains. Strain Solution matching identified JCM8129 as identical to L casei NBRC15883T with all 14 marker peaks matching. UPGMA clustering reproduced known phylogeny consistent with ribotyping and MLST results.

Benefits and practical applications of the method

• Rapid subspecieslevel resolution within L casei group where fingerprint methods lack consistency
• Reduced sample preparation and analysis time compared to DNAbased techniques
• Applicable to probiotic quality control, food safety monitoring and microbial diagnostics

Future trends and potential applications

Extension of S10GERMS profiling to other bacterial genera could broaden phylogenetic analyses. Integration with high throughput MALDI workflows and automated databases may enable real time strain monitoring in industrial and clinical settings. Advances in software automation will further streamline microbial identification.

Conclusion

The S10GERMS approach combined with MALDI-TOF MS provides a theoretically grounded, fast and accurate method for discriminating L casei group members at subspecies level. Implementation in Strain Solution demonstrates its practical value for diverse applications in analytical and industrial microbiology.

References

1. Sato H. et al Characterization of the Lactobacillus casei group based on the profiling of ribosomal proteins coded in S10-spc-alpha operons as observed by MALDI-TOF MS Syst Appl Microbiol 2012 35 447–454
2. Ryu CS Czajka JW Sakamoto M Benno Y Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping Microbiol Immunol 2001 45 271–275
3. Huys G et al Accuracy of species identity of commercial bacterial cultures intended for probiotic or nutritional use Res Microbiol 2006 157 803–810