Ya-Di Wang ·Xue-Ling Li ·Jun Hu ·Jun-Hong Lü

Abstract Fourier-transform infrared(FTIR)spectroscopy has emerged as a viable alternative to biochemical and molecular biology techniques for bacterial typing with advantages such as short analysis time,low cost and laboratorial simplicity.In this study,synchrotron radiationbased FTIR(SR-FTIR)spectroscopy with higher spectral quality was successfully applied to type 16 foodborne pathogenic bacterial strains.Combined with principal component analysis(PCA)and hierarchical cluster analysis(HCA),we found that the speci fic spectral region 1300-1000 cm-1,which re flects the information of phosphate compounds and polysaccharides,can be used as the signature region to cluster the strains into groups similar with genetic taxonomic method.These findings demonstrated that FTIR spectra combined with HCA have a great potential in quickly typing bacteria depending on their biochemical signatures.

Keywords Synchrotron radiation·FTIR ·Spectral signature·Bacterial typing·PCA ·HCA

1 Introduction

Rapid identi fication and typing microorganisms are an increasingly important task in food safety and epidemiological diagnosis[1].Traditional bacteria classi fication methods,such as serological test,PCR,DNA-DNA hybridization,and DNA sequencing[2],are always timeconsuming and need laboriousculturing procedures.Moreover,these phenotype or gene-based methods have dif ficulty in discriminating closely related strains which have similar phenotypic or genotypic properties[1].

Fourier-transform infrared(FTIR)spectroscopy has emerged as a real alternative of molecular biology and biochemical methods to describe microorganisms dependent on chemical components[3-5].Due to the advantages of short analysis time and low cost[6],since it was introduced to characterize microorganisms by Naumann and co-workers[7,8],FTIR spectroscopy was successfully applied to discriminate bacteria at genus,species,and even subspecies level[9].The commonly used infrared spectra(Figure S1,Supporting Information)include a lipid region,protein region,mixed region,and polysaccharide region(3000-2800,1800-1500,1500-1200,and 1200-900 cm-1,respectively)[10].Since infrared spectra contain complex chemicalinformation,multivariate statisticalanalysis methods,such as principal component analysis(PCA)and hierarchical cluster analysis(HCA),are usually applied to explain their diversities and to further type FTIR spectra of bacteria[11-13].

The development of synchrotron source for infrared spectroscopy has greatly improved the accuracy and signalto-noise ration of the measurements[14].In the previous work,we have successfully applied synchrotron radiationbased FTIR(SR-FTIR)spectroscopy to discriminate 10 bacterial strains.Herein,we tried to further classify and type 16 foodborne bacteria from seven genera by the combination of SR-FTIR and chemometric methods.Whole spectra(3000-2800,1800-900 cm-1)and spectra of four divided regions(lipid region,protein region,mixed region,and polysaccharide region)were used,respectively.We found that the spectra region between 1300 and 1000 cm-1can be recognized as the signature region to cluster the bacteria from the same taxonomic levels into one group.With high reproducibility and low sample amount,SR-FTIR spectroscopy shows great potential in fast identifying and typing microorganisms based on the speci fic infrared signature.

2 Experimental section

2.1 Bacterial strains

A total of 16 foodborne bacteria were used,and their culture conditionsare listed in Table 1 (Supporting Information).

2.2 Sample preparation

Bacterial strains were incubated overnight and collected by centrifugation,respectively.After re-suspended in 50 μl ethyl alcohol,5 μl sample was dropped on the BaF2window and air-dried at room temperature[9,14,15].

2.3 Synchrotron FTIR spectroscopy

Transmission mode was chosen,and aperture was 20 μm × 20 μm.Datacollecting wasmonitored by OMNIC 9.2(Thermo Fisher Scienti fic),and the collected spectra were further processed by baseline correcting,15-point smoothing,and normalization[11,16].The Savitzky-Golay method was used to calculate the first derivative spectra.

2.4 Data analysis

PCA and HCA were carried out on first derivative spectra using SPSS Statistics 22.0(IBM).For PCA,the first two PCs(PC1 and PC2)were used to draw scatter plots.For HCA,Ward's linkage algorithm and Euclidian distance measurements(or Pearson's correlation coef ficient)were chosen[11],the top horizontal axis of a dendrogram depicted the distance coef ficient values,and the actual distance was assigned 0-25 in proportion.

3 Results and Discussion

Spectra were recorded from 16 different bacterial isolates(Table S1,Supporting Information).A typical SRFTIR spectrum of Vibrio parahaemolyticus is shown in Fig.S1(Supporting Information).The recorded spectra of these 16 bacterial strains showed similar bands in the whole spectral region(3000-2800 and 1800-900 cm-1),and little differences were observed(Fig.S2a,Supporting Information).To further resolve the spectral differences among these strains,the first derivative transformations were performed.As shown in Fig.S2b(Supporting Information),the first derivatives of reduced FTIR spectra displayed minor differences in the mixed region(1500-1200 cm-1) and in the polysaccharide region(1200-900 cm-1).

To find out the better discriminating results,the first derivative spectra of the 16 bacterial strains were used and multivariate analysis methods PCA and HCA were carried out on the whole spectral region and speci fic regions(such aslipid region,protein region,mixed region,and polysaccharide region),respectively.

We firstly tried to carry out PCA on whole spectra of 16 bacteria while performing HCA to explain their af finitydisaf finity.As shown in Fig.1,it can be seen that all Listeria strains are well separated by principle component one and two.The dendrograms from HCA of the average spectra showed close clustering of these species,suggesting that Listeria strains have a similar biochemical pro file and all of them are from the same order.Two Staphylococcus strains were also grouped together.However,both Vibrio strains and Salmonella strains were decentralized and could not cluster into their corresponding group.These results indicated that the whole spectra could be dif ficult to distinguish all the bacterial strains at distinct taxonomic levels.

Since the infrared spectra of the microorganism re flect the information of several classes of biomolecules,PCA and HCA were carried out on these speci fic spectral regions, respectively. For the lipid region(3000-2800 cm-1)and protein region(1800-1500 cm-1),most strains with the same genus did not cluster correctly and only Listeria strains were partly grouped together(Fig.S3 and Fig.S4,Supporting Information).These results indicated that either the lipid region or protein region contained low speci fic information among these strains and could not be used to classify bacteria.

Fig.1 Score plots from a PCA( first and second components,left)and HCA(right)of the whole infrared spectra of the 16 bacterial strains

We further typed the bacteria based on the spectra of the mixed region 1500-1200 cm-1(Fig.2).PCA displayed that four Listeria strains and three Vibrio strains were signi ficantly separated from other bacteria.HCA illustrated that Listeria,Vibrio,Staphylococcus,and most Salmonella(except S.dysenteriae)were formed as separate genusspeci fic clusters.Although not all strains were classi fied into their corresponding sub-clusters,this result was very close to the phylogenetic relatedness.Interestingly,similar results were obtained after PCA and HCA of the spectral region 1200-900 cm-1,which mainly represent the information about nucleic acids and polysaccharides components(Fig.3).For example,four Listeria strains were clearly differentiated from the others and three of four Salmonella species were classi fied into a same cluster.However,some strains,such as Staphylococcus epidermidis,S.typhimurium(CICC10420),and Vibrio vulni ficus,were misclassi fied.

Fig.2 PCA(left)and HCA(right)of the 16 bacterial strains based on the infrared spectra of the mixed region(1500-1200 cm-1)

Fig.3 PCA(left)and HCA(right)of the 16 bacterial strains based on the infrared spectra of the polysaccharide region(1200-900 cm-1)

The above results suggested that the spectra information in the mixed region(1500-1200 cm-1)and polysaccharide region(1200-900 cm-1)had relatively good discriminating capability,which have been previously reported as the speci fic regions for characterizing these kinds of bacterial strains[17,18].To acquire the most appropriate spectra for typing bacteria within their corresponding taxonomy,the wavenumber,ranging from 1500 to 900 cm-1,was carefully optimized and selected. Finally, the region 1300-1000 cm-1was found of particular interest because of its proximity to the best clustering results(Fig.4).This region mainly re flected the chemical information of P=O symmetric and asymmetric stretching vibrations of＞PO2-in nucleic acids or phospholipids and C-O-C,C-O vibrations in glycogen and carbohydrates[19].The results showed that these bacteria were typed into three larger clusters,including four Listeria strains,three Vibrio strains,and two Staphylococcus strains which were clearly classifi ed.Although the Salmonella species,Yersinia enterocolitica and Shigella dysenteriae were grouped together,the fact that all three of them belong to members of Enterobacteriales could explain the dif ficulty of discrimination,which was consistent with previous reports[12,20].

4 Conclusion

We reported on the use of SR-FTIR spectroscopy to type 16 foodborne bacteria from seven genera.A whole spectral region and four subdivided spectral regions were analyzed,respectively.We found that the wavenumber range of 1300-1000 cm-1,which mainly re flects the information of nucleic acids and polysaccharides,contained more speci fic information and could be referred to as the signature region.The spectral signature proceeded with PCA and HCA obtained a delineation among the bacterial classes,and cluster results matched with their phylogenetic relationship.These results proved that SR-FTIR spectroscopy with high spectral resolution has the greatest potential in fast identifying and typing microorganisms based on the speci fic infrared signature.

Fig.4 PCA(left)and HCA(right)of 16 bacterial strains based on the infrared spectral region 1300-1000 cm-1

AcknowledgementsWe thank the staff from BL01B beamline of National Center for Protein Science Shanghai(NCPSS)at Shanghai Synchrotron Radiation Facility,for assistance during data collection.