Fourier Transform Infrared and Raman Spectroscopy for Characterization of Listeria monocytogenes Strains

The purpose of this study was to characterize the variation in biochemical composition of 89 strains of Listeria monocytogenes with different susceptibilities towards sakacin P, using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. The strains were also analyzed using amplified fragment length polymorphism (AFLP) analysis. Based on their susceptibilities to sakacin P, the 89 strains have previously been divided into two groups. Using the FTIR spectra and AFLP data, the strains were basically differentiated into the same two groups. Analyses of the FTIR and Raman spectra revealed that the strains in the two groups contained differences in the compositions of carbohydrates and fatty acids. The relevance of the variation in the composition of carbohydrates with respect to the variation in the susceptibility towards sakacin P for the L. monocytogenes strains is discussed.     

Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei

AIMS: To evaluate the potential of sakacin P and sakacin P-producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food? METHODS AND RESULTS: Listeria monocytogenes, a Lact. sakei strain and/or the bacteriocin sakacin P were added to chicken cold cuts, vacuum packed and incubated at 4 or 10 degrees C for 4 weeks. Each of two isogenic Lact. sakei strains, one producing sakacin P and the other not, had an inhibiting effect on the growth of L. monocytogenes. The effect of these two isogenic strains on the growth of L. monocytogenes was indistinguishable, even though sakacin P was produced in the product by one of the two Lact. sakei strains. The addition of purified sakacin P had an inhibiting effect on the growth of L. monocytogenes. A high dosage of sakacin P (3.5 microg x g(-1)) had a bacteriostatic effect throughout the storage period of 4 weeks, while a low dosage (12 ng x g(-1)) permitted initial growth, but at a slow rate. After 4 weeks of storage, the number of L. monocytogenes in the samples with a low dosage of sakacin P was 2 logs below that in the untreated control. When using a high dosage of sakacin P, the bacteriocin was detected in samples stored for up to 6 weeks. CONCLUSIONS: (i) Sakacin P is produced by a Lact. sakei strain when growing on vacuum-packed chicken cold cuts. (ii) Inhibiting effects of Lact. sakei, other than sakacin P, are active in inhibiting the growth of L. monocytogenes growing on chicken cold cuts. (iii) Sakacin P is stable on chicken cold cuts over a period of 4 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: Both sakacin P and Lact. sakei were found to have potential for use in the control of L. monocytogenes in chicken cold cuts.     

Differences in susceptibility of Listeria monocytogenes strains to sakacin P,sakacin A,pediocin PA-1, and nisin

Two hundred strains of Listeria monocytogenes collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin. The individual 50% inhibitory concentrations (IC(50)) were determined in a microtiter assay and expressed in nanograms per milliliter. The IC(50) of sakacin P ranged from 0.01 to 0.61 ng ml(-1). The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml(-1), respectively. The use of a large number of strains and the accuracy of the IC(50) determination revealed patterns not previously described, and for the first time it was shown that the IC(50) of sakacin P divided the L. monocytogenes strains into two distinct groups. Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism. The results from these studies essentially confirmed the grouping based on the IC(50) of sakacin P. A high correlation was found between the IC(50) of sakacin P and that of pediocin PA-1 for the 200 strains. Surprisingly, the correlation between the IC(50) of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC(50) of sakacin A and the class I bacteriocin nisin.     

Rapid epidemiological analysis of Acinetobacter strains by Raman spectroscopy

From earlier publications, we noticed that Raman spectra could potentially be used for subspecies identification of microorganisms. Here we evaluated the technique for its use as a typing tool of Acinetobacter species, using a collection of well-characterised strains from five hospital outbreaks. The strains were previously analysed using molecular techniques as cell envelope protein profiling and ribotyping. In this study, we have typed the strains by AFLP analysis and Raman spectroscopy. We compared the results using hierarchical cluster analysis, which showed highly similar groupings by both techniques. There seemed to be some misclassification between two sets of outbreak strains in the Raman analysis. We ascribe this to the clonal relationship between the strains of both outbreaks, described earlier. This results from a highly similar biochemical composition of the strains involved, and hence a highly similar Raman spectrum. We conclude that Raman spectroscopy could be an easy-to-use alternative in epidemiological studies of Acinetobacter strains and a promising starting point for the development of epidemiological studies in general.     

Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes

AFLP analysis using four selective primers was performed on a set of 33 Listeria monocytogenes including strains from patients and foods implicated in outbreaks, human sporadic cases or foods. Strains were tested belonging to serovars 1/2a, 1/2b, 1/2c, 3b, and 4b. Using one of the primers, the AFLP technique generated 20 different sized DNA fragments. The 33 cultures segregated into 14 different patterns, each comprising 7-12 different fragments. Although the method was not sufficiently discriminatory for epidemiological typing, AFLP analysis reconfirmed the observation that L. monocytogenes comprises two major genetic groups: group 1 includes strains of serovars 1/2a and 1/2c, while group 2 serovars 1/2b, 3b and 4b.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

Typing of food-borne Listeria monocytogenes by polymerase chain reaction-restriction enzyme analysis and amplified fragment length polymorphism

The applicability of polymerase chain reaction-restriction enzyme analysis (PCR-REA) and amplified fragment length polymorphism (AFLP) for typing of food borne Listeria monocytogenes strains was tested. A panel of 43 L. monocytogenes strains isolated from food, mostly serovars 1/2b or 1/2a, were analysed by the optimized PCR-REA oriented to inlA and inlB genes and by AFLP. By PCR-REA, five types of profiles were obtained. By AFLP, the strains were separated into 11 types and 18 subtypes forming two major clusters. PCR REA was a relatively straightforward method for typing food-borne L. monocytogenes with a moderate discrimination power. AFLP was a more complex but a highly discriminative and reproducible method.     

Reliable and rapid identification of Listeria monocytogenes and Listeria species by artificial neural network-based Fourier transform infrared spectroscopy

Differentiation of the species within the genus Listeria is important for the food industry but only a few reliable methods are available so far. While a number of studies have used Fourier transform infrared (FTIR) spectroscopy to identify bacteria, the extraction of complex pattern information from the infrared spectra remains difficult. Here, we apply artificial neural network technology (ANN), which is an advanced multivariate data-processing method of pattern analysis, to identify Listeria infrared spectra at the species level. A hierarchical classification system based on ANN analysis for Listeria FTIR spectra was created, based on a comprehensive reference spectral database including 243 well-defined reference strains of Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri. In parallel, a univariate FTIR identification model was developed. To evaluate the potentials of these models, a set of 277 isolates of diverse geographical origins, but not included in the reference database, were assembled and used as an independent external validation for species discrimination. Univariate FTIR analysis allowed the correct identification of 85.2% of all strains and of 93% of the L. monocytogenes strains. ANN-based analysis enhanced differentiation success to 96% for all Listeria species, including a success rate of 99.2% for correct L. monocytogenes identification. The identity of the 277-strain test set was also determined with the standard phenotypical API Listeria system. This kit was able to identify 88% of the test isolates and 93% of L. monocytogenes strains. These results demonstrate the high reliability and strong potential of ANN-based FTIR spectrum analysis for identification of the five Listeria species under investigation. Starting from a pure culture, this technique allows the cost-efficient and rapid identification of Listeria species within 25 h and is suitable for use in a routine food microbiological laboratory.     

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 network analysis was designed to differentiate 12 serovars of Listeria monocytogenes using a reference database of 106 well-defined strains. External validation was performed using a test set of another 166 L. monocytogenes strains. The O antigens (serogroup) of 164 strains (98.8%) could be identified correctly, and H antigens were correctly determined in 152 (91.6%) of the test strains. Importantly, 40 out of 41 potentially epidemic serovar 4b strains were unambiguously identified. FTIR analysis is superior to PCR-based systems for serovar differentiation and has potential for the rapid, simultaneous identification of both species and serovar of an unknown Listeria isolate by simply measuring a whole-cell infrared spectrum.     

Characterisation of persistent and sporadic Listeria monocytogenes strains by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP)

This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants. In each plant persistent and sporadic strains were selected on the basis of PFGE typing results. A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study. PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information. The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively. The combination of PFGE and AFLP typing results yielded a total of 48 genotypes. Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only. Our results showed that L. monocytogenes strains causing persistent contamination differ from sporadic strains. In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups. These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.     

糙皮侧耳(Pleurotus ostreatus)的AFLP指纹图谱分析

在对糙皮侧耳(Pleurotus ostreatus)的AFLP分析条件进行优化的基础上,利用该技术建立了14株产自不同地区的糙皮侧耳：DNA指纹图谱,并进行了数据分析。结果表明,在合成的14条引物的不同组合中,引物对E-3／M-3可以产生较多的DNA多态片段,E-AGC／M-CAT引物对的扩增效果最好,共获得184条DNA扩增带,其中多态性条带i101条,占54．89％。利用UPGMA法对所获数据进行聚类分析,计算得到糙皮侧耳菌株之间的遗传距离,发现不同品种间遗传距离差异较大,从0．192到0．754,说明糙皮侧耳的遗传多样性比较丰富。绘制的指纹分析树状图表明,14个糙皮侧耳菌株被分为6个组群,其中P17和杂3的相似性系数最高,达到了81．2％,而侧5与其他菌株的亲缘关系相对最远。     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

Molecular characterization of ochratoxin A producing strains of the genus Penicillium

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.     

The application of micro-FTIR spectroscopy to analyze nutrient stress-related changes in biomass composition of phytoplankton algae.

Micro-Fourier transform infrared (FTIR) spectroscopy was used to study changes in spectral features of three species of Cyanobacteria (Microcystis aeruginosa, Croococcus minutus, and Nostoc sp.) and two Bacillariophyceae (Cyclotella meneghiniana, and Phaeodactylum tricornutum) in response to nutrient stress. The change of physiological state of the cells was followed during a 4-week starvation period on the basis of physiological key parameters and by means of FTIR spectroscopy. Changes in the integrated FTIR bands of cell spectra assigned to proteins, lipids, carbohydrates and silicate were used to calculate relative biomass composition. The results show that short-term acclimatization become visible at first in pigmentation and photosynthetic efficiency, whereas changes in biomass composition reflect long term modulation in the metabolism. Simultaneous monitoring of short term and long term stress acclimatization showed evidence that the metabolic strategies to cope with increasing nutrient limitation are highly species-specific. This species-specificity can only be resolved in natural phytoplankton samples by single cell techniques. The results show that the FTIR technique has the potential to become applicable for the determination of single cell biomass composition from natural phytoplankton communities.     

FTIR-spectral analysis of two photosynthetic H2-producing strains and their extracellular polymeric substances

The Fourier transform infrared (FTIR) spectra of the cells of two photosynthetic H₂-producing strains, Rhodoblastus acidophilus and Rhodobacter capsulatus, as well as their extracellular polymeric substances (EPS), were evaluated. The FTIR spectra of R. capsulatus and its EPS during its cultivation were also recorded. The main peaks in the spectra, including 1,080 cm⁻¹ (carbohydrates), 1,250 cm⁻¹ (nucleic acids), 2,830–2,930 cm⁻¹ (lipids), 1,660–1,535 cm⁻¹ (Amide I and II of proteins), were observed. The relative heights of these peaks in the spectra of the two strains were different, showing the difference in contents of various components in the cells or EPS. The ratios among the main components in the EPS obtained from the FTIR spectra were in good agreement with those from a conventional quantitative chemical analysis. As an easy, rapid, and direct technique, the FTIR spectroscopy could be used to characterize the components and their relative contents of EPS of photosynthetic bacteria.An erratum to this article can be found at     

Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake Lb674

Abstract Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes , was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 ( sakR ) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.     

A high‐throughput FTIR spectroscopy approach to assess adaptive variation in the chemical composition of pollen

The two factors defining male reproductive success in plants are pollen quantity and quality, but our knowledge about the importance of pollen quality is limited due to methodological constraints. Pollen quality in terms of chemical composition may be either genetically fixed for high performance independent of environmental conditions, or it may be plastic to maximize reproductive output under different environmental conditions. In this study, we validated a new approach for studying the role of chemical composition of pollen in adaptation to local climate. The approach is based on high‐throughput Fourier infrared (FTIR) characterization and biochemical interpretation of pollen chemical composition in response to environmental conditions. The study covered three grass species, Poa alpina, Anthoxanthum odoratum, and Festuca ovina. For each species, plants were grown from seeds of three populations with wide geographic and climate variation. Each individual plant was divided into four genetically identical clones which were grown in different controlled environments (high and low levels of temperature and nutrients). In total, 389 samples were measured using a high‐throughput FTIR spectrometer. The biochemical fingerprints of pollen were species and population specific, and plastic in response to different environmental conditions. The response was most pronounced for temperature, influencing the levels of proteins, lipids, and carbohydrates in pollen of all species. Furthermore, there is considerable variation in plasticity of the chemical composition of pollen among species and populations. The use of high‐throughput FTIR spectroscopy provides fast, cheap, and simple assessment of the chemical composition of pollen. In combination with controlled‐condition growth experiments and multivariate analyses, FTIR spectroscopy opens up for studies of the adaptive role of pollen that until now has been difficult with available methodology. The approach can easily be extended to other species and environmental conditions and has the potential to significantly increase our understanding of plant male function.     

A Multiscale Vibrational Spectroscopic Approach for Identification and Biochemical Characterization of Pollen

Background

Analysis of pollen grains reveals valuable information on biology, ecology, forensics, climate change, insect migration, food sources and aeroallergens. Vibrational (infrared and Raman) spectroscopies offer chemical characterization of pollen via identifiable spectral features without any sample pretreatment. We have compared the level of chemical information that can be obtained by different multiscale vibrational spectroscopic techniques.

Methodology

Pollen from 15 different species of Pinales (conifers) were measured by seven infrared and Raman methodologies. In order to obtain infrared spectra, both reflectance and transmission measurements were performed on ground and intact pollen grains (bulk measurements), in addition, infrared spectra were obtained by microspectroscopy of multigrain and single pollen grain measurements. For Raman microspectroscopy measurements, spectra were obtained from the same pollen grains by focusing two different substructures of pollen grain. The spectral data from the seven methodologies were integrated into one data model by the Consensus Principal Component Analysis, in order to obtain the relations between the molecular signatures traced by different techniques.

Results

The vibrational spectroscopy enabled biochemical characterization of pollen and detection of phylogenetic variation. The spectral differences were clearly connected to specific chemical constituents, such as lipids, carbohydrates, carotenoids and sporopollenins. The extensive differences between pollen of Cedrus and the rest of Pinaceae family were unambiguously connected with molecular composition of sporopollenins in pollen grain wall, while pollen of Picea has apparently higher concentration of carotenoids than the rest of the family. It is shown that vibrational methodologies have great potential for systematic collection of data on ecosystems and that the obtained phylogenetic variation can be well explained by the biochemical composition of pollen. Out of the seven tested methodologies, the best taxonomical differentiation of pollen was obtained by infrared measurements on bulk samples, as well as by Raman microspectroscopy measurements of the corpus region of the pollen grain. Raman microspectroscopy measurements indicate that measurement area, as well as the depth of focus, can have crucial influence on the obtained data.     

Production of sakacin P by Lactobacillus sakei in a completely defined medium

In order to investigate factors influencing the production of the bacteriocin, sakacin P, Lactobacillus sakei CCUG 42687 was grown in a completely defined medium (DML-B) with 33 components. Although the maximum sakacin P concentration obtained was higher on a complex medium due to higher cell mass, the production per cell mass was higher in DML-B. Sakacin P was produced at 4-30 degrees C, with the highest specific production at low temperatures. More sakacin P was produced at uncontrolled pH compared with cultivation at pH 6.3. Tween-80 had a positive effect on sakacin P production, while addition of sodium chloride and trace metals had negative effects. The decrease in sakacin P concentration during the late growth and stationary phases was shown to be cell-independent and promoted at high temperature and pH. Some differences in production levels of sakacin P were found among six strains of Lactobacillus sakei tested.