Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.     

Rapid quantitative analysis of binary mixtures of Escherichia coli strains using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Pyrolysis mass spectrometry (PyMS) and multivariate calibration were used to show the high degree of relatedness between Escherichia coli HB101 and E. coli UB5201. Next, binary mixtures of these two phenotypically closely related E. coli strains were prepared and subjected to PyMS. Fully interconnected feedforward artificial neural networks (ANNs) were used to analyse the pyrolysis mass spectra to obtain quantitative information representative of the level of E. coli UB5201 in E. coli HB101. The ANNs exploited were trained using the standard back propagation algorithm, and the nodes used sigmoidal squashing functions. Accurate quantitative information was obtained for mixtures with >3% E. coli UB5201 in E. coli HB101. To remove noise from the pyrolysis mass spectra and so lower the limit of detection, the spectra were reduced using principal components analysis (PCA) and the first 13 principal components used to train ANNs. These PCA-ANNs allowed accurate estimates at levels as low as 1% E. coli UB5201 in E. coli HB101 to be predicted. In terms of bacterial numbers, it was shown that the limit of detection for PyMS in conjunction with ANNs was 3 × 10⁴ E. coli UB5201 cells in 1·6 × 10⁷ E. coli HB101 cells. It may be concluded that PyMS with ANNs provides a powerful and rapid method for the quantification of mixtures of closely related bacterial strains.     

Dereplication for biotechnology screening: PyMS analysis and PCR–RFLP–SSCP (PRS) profiling of 16S rRNA genes of marine and terrestrial actinomycetes

The search for exploitable biology is a major task for biotechnology-based industries. In this context, discrimination between previously tested or recovered micro-organisms (dereplication) is imperative, in order to reduce screening costs by sorting large collections of isolates, which are then subjected to further detailed evaluation. Pyrolysis mass spectrometry (PyMS) is a whole-cell fingerprinting technique that enables the rapid and reproducible sorting of micro-organisms, uses small samples and has the advantage of being fully automated. In this study, we compare chemometric fingerprinting with a ribotyping fingerprinting method, in order to investigate the extent to which pyrogroups formed by PyMS analysis relate to genetic diversity, using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS). A mixture of environmental strains of mycolic acid containing actinomycetes was used to mimic the selection of colonies from primary isolation plates. The congruence found between the clusters defined by the chemometric and molecular fingerprinting techniques was very high and demonstrated the effectiveness of PyMS as a rapid sorting and dereplicating procedure for putatively novel strains, criteria that are critical for biotechnological screens. Moreover, PyMS analysis revealed significant variation within pyrogroups that contained strains with the same genotypic (PRS) characteristics, thus emphasising its discriminatory capacity at the infraspecies level.     

Rapid characterisation of deep-sea actinomycetes for biotechnology screening programmes

A continual need in natural product discovery is dereplication, that is the ability to exclude previously tested microorganisms from screening programmes. Whole-cell fingerprinting techniques offer an ideal solution to this problem because of their rapidity and reproducibility, dependence on small samples, and automation. One such technique, Curie-point pyrolysis mass spectrometry (PyMS), has been deployed for the characterisation of a unique collection of actinomycetes recovered from Pacific Ocean sediments approximately 2000 to 6500 m below sea level. This paper addresses the question: to what extent are pyrogroups, defined on the basis of PyMS fingerprinting, related to classifications derived from more conventional microbial systematics? A collection of 44 randomly chosen deep-sea rhodococci were coded and subjected to a double-blind PyMS and numerical taxonomic (NT) analysis; the latter sorted the strains into clusters (taxospecies) using large sets of equally weighted phenotypic data. At the end of the experiment the codes were disclosed and the NT classification shown to generate 6 homogeneous clusters corresponding to different deep-sea sites. The matching of these clusters with the resulting pyrogroups was very high with an overall congruence of nearly 98%. Thus, PyMS characterisation is directly ascribable to the phenotypic variation being sought for biotechnology screens. Moreover, the exquisite discriminatory power of PyMS readily revealed infraspecific diversity in these industrially important bacteria.     

Discrimination of higher plant calluses based on embryogenic capacity and taxonomic classification by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum, and has been widely applied to the discrimination of closely related microbial strains. Minimally prepared samples of embryogenic and non-embryogenic calluses derived from various higher plants (sweet potato, morning glory, Korean ginseng, Siberian ginseng, and balloon flower) were subjected to PyMS for spectral fingerprinting. A dendrogram based on the unweighted pair group method, with arithmetic mean of pyrolysis mass spectra, divided the calluses into Siberian ginseng embryogenic callus and the others, which were subsequently divided into embryogenic and non-embryogenic callus groups, regardless of plant species from which the calluses were derived. In the non-embryogenic callus group, the dendrogram was in agreement with the known taxonomy of the plants. These results indicate that PyMS analysis could be applied for discriminating plant calluses based on embryogenic capacity and taxonomic classification.     

Identification and Discrimination of Oral Asaccharolytic Eubacterium spp. by Pyrolysis Mass Spectrometry and Artificial Neural Networks

Curie-point pyrolysis mass spectra were obtained from 29 oral asaccharolytic Eubacterium strains and 6 abscess isolates previously identified as Peptostreptococcus heliotrinreducens. Pyrolysis mass spectrometry (PyMS) with cluster analysis was able to clarify the taxonomic position of this group of organisms. Artificial neural networks (ANNs) were then trained by supervised learning (with the back-propagation algorithm) to recognize the strains from their pyrolysis mass spectra; all Eubacterium strains were correctly identified, and the abscess isolates were identified as un-named Eubacterium taxon C₂ and were distinct from the type strain of P. heliotrinreducens. These results demonstrate that the combination of PyMS and ANNs provides a rapid and accurate identification technique.     

Taxonomic discrimination of higher plants by pyrolysis mass spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum and has been widely applied to the discrimination of closely related microbial strains. Leaf samples of six species and one variety of higher plants (Rosa multiflora, R. multiflora var. platyphylla, Sedum kamtschaticum, S. takesimense, S. sarmentosum, Hepatica insularis, and H. asiatica) were subjected to PyMS for spectral fingerprinting. Principal component analysis of PyMS data was not able to discriminate these plants in discrete clusters. However, canonical variate analysis of PyMS data separated these plants from one another. A hierarchical dendrogram based on canonical variate analysis was in agreement with the known taxonomy of the plants at the variety level. These results indicate that PyMS is able to discriminate higher plants based on taxonomic classification at the family, genus, species, and variety level.Abbreviations ANNs Artificial neural networks - CVA Canonical variate analysis - GC/MS Gas chromatography/mass spectrometry - PCA Principal component analysis - PyMS Pyrolysis mass spectrometry - UPGMA Unweighted pair group method with arithmetic mean Communicated by I.S. Chung     

Plasma corticosterone concentrations in males of the skink Egernia whitii during acute and chronic confinement, and over a diel period

This study examined adrenal and gonadal responses to capture and to longer term captivity in males of the scincid lizard, Egernia whitii. Animals subjected to acute capture stress in the field exhibited a rapid rise in plasma corticosterone concentrations that had not attenuated by 4 h after capture; plasma testosterone concentrations decreased significantly over this period. Animals returned to captive conditions in the laboratory showed little change in plasma corticosterone concentrations by 1 week, but concentrations were significantly lower at 4 weeks. Plasma testosterone did not vary significantly during this period. Sampling over a diel period indicated that plasma corticosterone concentrations vary little over the daylight hours; however, there was a significant decrease between the samples taken at 17:00 h and 21:00 h. These results suggest that, as in other species, the acute adrenal response to capture stress may confound assessment of other physiological parameters, although if experiments are carried out during daylight hours, time of sampling should have little influence on plasma corticosterone concentrations. The results also suggest that males of E. whitii require at least a week to adapt to captivity, although further studies investigating different captive conditions are warranted for this social species.     

A Rapid,Simple Method for the Determination of 6-Benzyladenine by Pyrolysis Mass Spectrometry

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum. PyMS was used for the quantitative determination of 6-benzyladenine (BA) supplemented to agar-solidified culture media (ASM) in this study. When subjected to PyMS, pure BA generated prominent fingerprint peaks. The peaks at m/z 68 and 123 were chosen for the quantitative measurement of BA because of the highest signal among those generated from pure BA and because of one of the highest masses among those with a prominent signal, respectively. To establish a standard curve for BA concentration in ASM, the combined peak intensity at m/z 68 and 123 was plotted against BA concentration ranged from as low as 0.44 μM after logarithmic transformation of both parameters. A linear regression line was yielded, which indicates BA concentration in ASM is directly proportional to the peak intensity, with R ² = 0.9052, significant at the 99% level. These results suggest that PyMS enables the quantitative determination of growth regulators and other related compounds in plant materials in a rapid, simple, sensitive, accurate manner.     

Inter-strain comparison by pyrolysis mass spectrometry of Staphylococcus aureus isolates associated with nosocomial infection

Pyrolysis mass spectrometry was used to characterise Staphylococcus aureus isolates from an outbreak of post-operative wound infections on a mixed surgical ward. The PyMS results were compared with those of phage typing. Both suggested a single strain of S. aureus, of phage type 3C, 55,71, was responsible for six of the 13 wound infections. PyMS differentiated an isolate from a member of staff of similar phage type to the epidemic strain, which had previously been considered to be the point source for the outbreak. PyMS is a rapid and inexpensive technique for investigating nosocomial outbreaks, including those caused by S. aureus and, in this instance, was more discriminatory than phage typing.     

The differentiation of Carnobacterium divergens using the random amplification of polymorphic DNA polymerase chain reaction technique

The potential of the randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique to differentiate Carnobacterium divergens from other members of the genus Carnobacterium was examined. A numerical analysis of the genomic profiles obtained demonstrated that it was possible to differentiate the C. divergens strains from other Carnobacterium strains using this technique. The heterogeneity observed in the representatives of the species C. piscicola adds further weight to the suggestion in other taxonomic studies that subspecies of this species exist.     

Prediction of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus populations in yoghurt by Curie point pyrolysis-mass spectrometry.

We evaluated the potential of pyrolysis-mass spectrometry (PyMS) for quantifying the binary mixed population of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yoghurt. For this purpose, a new analytical approach was developed. The yoghurt was transparised and its total bacterial population was recovered by centrifugation and estimated by turbidimetric measurement. The quantity of each population (L. bulgaricus, S. thermophilus) was then estimated in the pellet by PyMS, and the data were analysed by artificial neural networks (ANNs). In parallel, streptococci and lactobacilli were numerated on SYL agar and these data were used as reference values to predict the bacterial counts of each population by PyMS. A close correlation was established between the streptococci and the lactobacilli counts on SYL agar and PyMS measurements (r(2)=0.98 for S. thermophilus and r(2)=0.96 for L. bulgaricus). Combined turbidimetric measurement and PyMS/ANNs seemed to be a powerful method for obtaining rapid counts of binary mixtures of bacteria in yoghurt.     

Improved microfluorometric DNA determination in biological material using 33258 Hoechst.

A simple and rapid microfluorometric method is described for the determination of DNA in submicrogram quantities using 33258 Hoechst fluorochrome. A high degree of reproducibility was obtained using calf thymus and phage DNA, mouse liver chromatin, and HeLa cells homogenate preparations. None or very little interference by the routinely used preparation reagents or by the cellular components was found. Compared to other commonly used procedures this innovative and versatile technique can be conveniently applied to DNA microdetermination for the high sensibility/reproducibility ratio and can also be used without the need of previous purification steps.     

Rapid analysis of the expression of heterologous proteins in Escherichia coli using pyrolysis mass spectrometry and Fourier transform infrared spectroscopy with chemometrics: application to alpha 2-interferon production.

Cell pastes and supernatant Escherichia coli samples, taken from an industrial bioprocess overproducing recombinant alpha 2 IFN were analysed using pyrolysis mass spectrometry (PyMS) and diffuse reflectance-absorbance Fourier transform infrared spectroscopy (FT-IR). PyMS and FT-IR are physico-chemical methods which measure predominantly the bond strengths of molecules and the vibrations of bonds within functional groups, respectively. They therefore give quantitative information about the total biochemical composition of the bioprocess sample. The interpretation of these hyperspectral data, in terms of the quantity of alpha 2 IFN in the cell pastes and supernatant samples was possible only after the application of the 'supervised learning' methods of artificial neural networks (ANNs) and partial least squares (PLS) regression. Both PyMS and FT-IR are novel, rapid and economical methods for the screening and the quantitative analysis of complex biological bioprocess over producing recombinant proteins. Models established using either spectral data set had a similarly satisfactory predictive ability. This shows that whole-reaction mixture spectral methods, which measure all molecules simultaneously, do contain enough information to allow their quantification when the entire spectra are used as the inputs to methods based on supervised learning. Moreover, this is the first study where FT-IR in the mid-IR range has been used to quantify the expression of a heterologous protein directly from fermentation broths and the first study to compare the abilities of PyMS and FT-IR for the quantitative analyses of an industrial bioprocess.     

Identification of Carnobacterium species by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region and species-specific PCR

The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C. divergens, C. gallinarum, C. mobile, C. funditum, C. alterfunditum, C. inhibens, and C. viridans. An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species. Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species. The L-ISR sequence revealed the presence of two tRNA genes, tRNA(Ala) and tRNA(Ile), which were separated by a spacer region that varied from 24 to 38 bp long. This region was variable among the species, allowing the design of species-specific primers. These primers were tested and proved to be species specific. The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria.     

Rapid screening for metabolite overproduction in fermentor broths, using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Binary mixtures of model systems consisting of the antibiotic ampicillin with either Escherichia coli or Staphylococcus auresu were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra, so as to obtain quantitative information on the concentration of ampicilin in the mixtures, partial least squares regression (PLS), principal components regression (PCR), and fully interconnected feedforward artificial neural networks (ANNs) were studied. In the latter case, the weights were modified using the standard backpropagation algorithm, and the nodes used a sigmoidal squsahing funciton. It was found that each of the methods could be used to provide calibration models which gave excellent predictions for the concentrations of ampicillin in samples on which they had not been trained. Furthermore, ANNs trained to predict the amount of ampicilin in E. coli were able to generalise so as to predict the concentration of ampicillin in a S. aureus background, illustrating the robustness of ANNs to rather substantial variations in the biological background. The PyMS of the complex mixture of ampicilin in bacteria could not be expressed simply in terms of additive combinations of the spectra describing the pure components of the mixtures and their relative concentrations. Intermolecular reactions took place in the pyrolysate, leading to a lack of superposition of the spectral components and to a dependence of the normalized mass spectrum on sample size. Samples from fermentations of a single organism in a complex production medium were also analyzed quantitatively for a drug of commercial interest. The drug could also be quantified in a variety of mutant-producing strains cultivated in the same medium. The combination of PyMS and ANNs constitutes a novel, rapid, and convenient method for exploitation in strain improvement screening programs. (c) 1994 John Wiley & Sons, Inc.     

Reproducibility of the circadian rhythms of serum cortisol and melatonin in healthy subjects: a study of three different 24-h cycles over six weeks

Plasma melatonin and cortisol are characterized by a marked circadian rhythm, but little information is available about the reproducibility and stability of these rhythms over several weeks in the same subjects. This study examined the characteristics of these rhythms in 31 healthy human subjects 20 to 30 years of age. They were synchronized with a diurnal activity from 0800 to 2300 and nocturnal rest. They participated in three 24-hour sessions (S1, S2, and S3): S2 took place two weeks after S1 and S3 4 weeks after S2. Blood samples were taken during each session at 3-hour intervals from 1100 to 2000 and hourly from 2200 to 0800. Comparison of the circadian rhythms between groups used repeated measures 2-way ANOVA, the cosinor method, and Bingham's test. Intraindividual variations were compared by the cosinor method and Bingham's test. The groups did not differ, but a slight difference in the amplitude or acrophase of individual circadian rhythms was observed in 5 of 31 subjects for melatonin and 1 of 31 for cortisol. The circadian means did not differ over the three sessions. These results show that the circadian profile of cortisol and melatonin are highly reproducible over a six-week period, in both individuals and groups. Our study clearly shows that these hormones can be considered to be stable markers of the circadian time structure and therefore useful tools to validate rhythms' synchronisation of human subjects.     

Specific molecular detection of Carnobacterium piscicola SF668 in cold smoked salmon

AIMS: To establish a rapid and reliable multiplex PCR (mPCR)-based method allowing specific identification of Carnobacterium piscicola SF668 during storage of cold smoked salmon (CSS). METHODS AND RESULTS: CSS was inoculated with C. piscicola SF668 and stored at 4 degrees C. Samples were withdrawn at regular time intervals and analysed by counting the number of viable cells. About 25-100% of colonies grown on Elliker plates were subjected to mPCR amplification. The results show that strains presumably identified as C. piscicola SF668 were predominant over the test period. CONCLUSIONS: mPCR is a powerful tool to study competitiveness of C. piscicola SF668, which inhibits the growth of Listeria monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study demonstrates the importance of molecular methods in studying competitiveness of strains with potential food applications.