Differentiation of Listeria monocytogenes Serovars by Using Artificial Neural Network Analysis of Fourier-Transformed Infrared Spectra

A classification system based on Fourier transform infrared(FTIR) spectroscopy combined with artificial neural networkanalysis was designed to differentiate 12 serovars of Listeriamonocytogenes using a reference database of 106 well-definedstrains. External validation was performed using a test setof another 166 L. monocytogenes strains. The O antigens (serogroup)of 164 strains (98.8%) could be identified correctly, and Hantigens were correctly determined in 152 (91.6%) of the teststrains. Importantly, 40 out of 41 potentially epidemic serovar4b strains were unambiguously identified. FTIR analysis is superiorto PCR-based systems for serovar differentiation and has potentialfor the rapid, simultaneous identification of both species andserovar of an unknown Listeria isolate by simply measuring awhole-cell infrared spectrum.

INTRODUCTION

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Introduction

Human listeriosis, caused by the pathogen Listeria monocytogenes,mainly is a consequence of the ingestion of contaminated foodproducts and remains a significant public health problem (8,14, 30). Since many different contamination routes for thisubiquitous bacterium have been reported (19, 27, 34), the developmentof rapid and accurate typing methods is of particular importance.A substantial number of sensitive, discriminatory, and reproducibletyping technologies have therefore been developed (20).

L. monocytogenes expresses O-somatic and H-flagellar antigenswhich, by their unique combination, determine the serovar (serotype)of individual strains. Thirteen serovars have been found inthis species by using specific and standardized sera (32). However,traditional serotyping presents a number of limitations suchas the commercial availability and high cost of sera, as wellas limited reproducibility. Palumbo et al. (28) therefore proposeda low-cost enzyme-linked immunosorbent assay combined with commercialantisera for the differentiation of L. monocytogenes serovars.This method, nevertheless, is of limited potential for applicationin routine laboratories due to the complex and laborious protocol.Consequently, very few diagnostic laboratories offer conventionalserotyping of L. monocytogenes.

Most clinical isolates belong to serovars 1/2a, 1/2b, and 4b;among these, the majority of strains which have caused largeoutbreaks are serovar 4b (15). In contrast, most food strainsbelong to serovar 1/2c (12, 36). There is now considerable evidencethat epidemic serovar 4b clones express specific virulence biomarkers(11) and carry distinct genetic markers (21, 36). So far, onlya few well-known strains of serovars 1/2a and 4b have been usedto study the virulence of L. monocytogenes. Virulence attributesspecific to other clinical or food-related serovars cannot yeteffectively be addressed. Although there are no specific legalspecifications for serotyping in terms of food safety or riskassessment at this time, serovar differentiation may be helpfulin the future when additional knowledge concerning the relationbetween the serovar and pathogenic potential of Listeria monocytogenesbecomes available (14).

Infrared spectra of microorganisms reflect the overall structureof the molecular constituents of the cell (24, 25), and Fouriertransform infrared spectroscopical (FTIR) analysis of intactcells has been used to distinguish bacteria at different taxonomiclevels (10). Extensive reference libraries containing thousandsof spectra of well-characterized microorganisms can be usedfor the rapid identification of unknown isolates at the specieslevel (9, 17, 23, 26, 35). A powerful method for data processingto interpret these complex spectral patterns is an importantkey for a successful identification. Advanced multivariate methodssuch as artificial neural networks (ANN) have been shown tobe especially advantageous for the analysis of subtle differencesat, and partially below, the species level (7, 31, 33). Recently,Rebuffo et al. (29) described a superior ANN-based FTIR methodfor the identification of Listeria monocytogenes and relatedListeria species.

FTIR studies on Escherichia coli and Salmonella enterica fordifferentiation of serotypes based on variations in their lipopolysaccharidehave been undertaken (10, 16). However, neither study used asufficient number of strains to be statistically relevant orresolved the differentiation of all serovars. Therefore, itremains unclear whether FTIR spectroscopy potentially may differentiatethe serovars of pathogens, in particular if the technique isapplied to a large number of strains covering a significantpart of the intraspecific biodiversity. In this study, we thereforeapplied FTIR combined with ANN to a large and diverse collectionof strains representing 12 serovars of L. monocytogenes in orderto evaluate whether this method is suitable to discriminatespecific spectral patterns which differentiate between L. monocytogenesserovars.

FTIR spectra of Listeria monocytogenes reflect serogroup- and serovar-specific markers.

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Ftir spectra of listeria...

The Listeria monocytogenes strains used in this work were grownunder standardized conditions on tryptone soy agar plates, andspectra were measured as described elsewhere (29). Figure 1shows typical first derivatives of infrared spectra of 12 Listeriamonocytogenes serovars. At a first glance, the polysaccharideregion between 900 and 1,200 cm^–1 displays the most prominentspectral differences among the four serogroups (1/2, 3, 7, and4). This indicates that carbohydrate-containing structures areinvolved in serogroup discrimination. Moreover, subtle spectraldifferences within each serogroup due to serovar-specific markerswere observed. FTIR spectra of 106 L. monocytogenes strains,including the 69 reference strains of L. monocytogenes studiedby Rebuffo et al. (29) and comprising all known serovars (seeTable S1 in the supplemental material) were used for hierarchicalcluster analysis (HCA) (Fig. 2A). This HCA was performed asdescribed by Rebuffo et al. (29) using the first derivativeof the original spectra covering the regions from 900 to 1,200and 1,400 to 1,800 cm^–1. One major cluster correspondedto serogroups 1/2, 3, and 7, while another cluster correspondedto serogroup 4. This observation is in accordance with two distinctstructural types of teichoic acids found previously for serogroups1/2, 3, and 7 versus serogroup 4 (6). A serogroup 4-specificgene cassette which is absent in serovar 1/2b strains has beenfound (18), and DNA array studies of 13 genes involved in cellwall biosynthesis revealed the same grouping of L. monocytogenesserovars (4). These two major serovar groups were used to establishlevel 1 of a four-layer neural net (Fig. 2B). The constructionand optimization of the ANN are described in more detail inthe supplemental material.

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FIG. 1. Typical first derivatives of infrared spectra of 12 Listeria monocytogenes serovars. Black dotted line, WSLC 1427, 1/2a; black solid line, WSLC 1030, 1/2b; gray solid line, WSLC 1377, 1/2c; black dotted line, WSLC 1485, 3a; black solid line, WSLC 1444, 3b; gray solid line, WSLC 11082, 3c; black dotted line, WSLC 1939, 4a; black solid line, WSLC 1634, 4b; gray solid line, WSLC 11094, 4c; gray dotted line, WSLC 1045, 4d; black dashed line, WSLC 1761, 4e; black solid line, WSLC 1932, 7. Each strain is represented by two independent spectra to show the reproducibility of the measurements. Spectra are stacked to clearly show the spectral differences among the four serogroups (1/2, 3, 7, and 4). The polysaccharide region (1,200 to 900 cm^–1) has been expanded to show the subtle differences between and within the serogroups. WSLC, Weihenstephan Listeria Collection.

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FIG. 2. (A) Hierarchical cluster analysis of the first derivatives of 106 L. monocytogenes strains belonging to 12 serovars and included in the reference data set. Spectral regions used were 900 to 1,200 cm^–1 and 1,400 to 1,800 cm^–1 (correlation with scaling to first range according to the OPUS FT-IR program [Bruker, Ettlingen, Germany]). Cluster analysis was performed using Ward's algorithm. (B) Artificial neural network classification scheme for the discrimination of serogroups and serovars.

Validation of FTIR-based serovar differentiation.

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Validation of ftir-based serovar...

The identification potential of this ANN was evaluated by aninternal validation (compare reference 29). This internal validation(Table 1) resulted in a correct identification of 100% of thesomatic antigens (serogroup level). However, only 94.3% of theflagellar antigens (serovar level) could be identified. Then,an external validation was performed using a test set of 166Listeria monocytogenes strains isolated from food, the environment,animals, and humans. These represented 12 serovars and includedthe 130 L. monocytogenes strains used for external validationby Rebuffo et al. (29) (see Table S2 in the supplemental material).These strains were either identified at the species level asdescribed by Rebuffo et al. (29) and serotyped based on agglutinationreactions with antisera for L. monocytogenes (Denka Seiken Co.,Japan) according to the instructions of the manufacturer orhad been previously serotyped by a reference laboratory. Theexternal validation (Table 2) resulted in the correct typingrate of 98.8% at the serogroup level and 91.6% at the serovarlevel. Importantly, 40 out of 41 potentially outbreak-causingserovar 4b strains were identified correctly. From 87 serovar1/2a plus 1/2b strains, 84 were assigned to either one of thesetwo serovars. Five serovar 1/2b strains were misidentified as1/2a. In total, only 3.1% of the potentially pathogenic serovars1/2a, 1/2b, and 4b were grouped with a potentially nonpathogenicserovar. However, both internal and external validations weresomewhat biased since only a few strains of serovars 3c, 4a,4c, 4e, and 7 were available, which is due to the fact thatthese are rarely isolated from food and never from patients.

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TABLE 1. Internal validation of the infrared spectral reference database

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TABLE 2. External validation of the infrared spectral reference database

Comparison of FTIR- and PCR-based serovar differentiation.

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Comparison of ftir- and...

Due to the importance of serotyping L. monocytogenes, a fewPCR-based methods have been proposed. However, some are limitedto differentiation of strains into only two or three serovargroups (2, 13, 22) or to differentiation of only two serovarsfrom the others (37). Others are more complicated since twoor even three independent PCRs are needed (1, 13). In contrast,the PCR system developed by Doumith et al. (3), in a one-stepmultiplex PCR, allows the differentiation of L. monocytogenesstrains into four "serovar groups". Group 1 comprises serovars1/2a and 3a; group 2 comprises serovars 1/2c and 3c; group 3contains serovars 1/2b, 3b, and 7; and group 4 comprises serovars4b, 4d, and 4e. However, individual serovars cannot be separated.We applied this PCR system to the 166 strains of our external-validationstrain set. The PCR-based method correctly differentiated 159(4 not typeable and 3 incorrectly typed [see Table S2 in thesupplemental material]) out of the 166 strains (95.8%) to theserovar group level, which is comparable to its previous validation(5). FTIR-based serotyping was able to correctly discriminate164 out of the 166 strains (98.8%) at the serogroup level. Inaddition, FTIR is much more discriminatory since 91.6% of theindividual serovars can be determined (Table 2).

Conclusion.

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Conclusion.

Rebuffo et al. (29) previously described an ANN-based FTIR methodfor the reliable identification of all Listeria species in only25 h. Here, we report on the development of an L. monocytogenesANN subnet in order to additionally identify serogroups andserovars. The integration of both classification systems nowoffers the possibility to simultaneously identify Listeria atthe species level, L. monocytogenes at the serogroup level,and most L. monocytogenes strains at the serovar level in asingle step by simply measuring an infrared spectrum of a pureListeria culture. Our data indicate that this method is superiorto molecular approaches for L. monocytogenes serovar determination.We suggest that FTIR identification and serotyping constitutea rapid and inexpensive tool which may be suitable for diagnosticlaboratories. This tool may be used routinely in food controlto gain additional information on the pathogenic potential ofstrains isolated from the food-processing chain.

ACKNOWLEDGMENTS

This work was supported in part by the Forschungskreis der Ernährungsindustriee. V. (Bonn), the Arbeitskreis für industrielle Forschung,and the Ministry of Economics and Technology, project no. 14126N.

We thank H. Hof, Mannheim, Germany, C. Montel, Aurillac, France,and K. Pellicer, La Plata, Argentina, for kindly providing Listeriamonocytogenes strains.

FOOTNOTES

* Corresponding author. Mailing address: Abteilung Mikrobiologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung, Technische Universität München, Weihenstephaner Berg 3, D-85350 Freising, Germany. Phone: 49 8161 713516. Fax: 49 8161 714512. E-mail: siegfried.scherer{at}wzw.tum.de.

Published ahead of print on 1 December 2006.

Supplemental material for this article may be found at http://aem.mcb.org/.

REFERENCES

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