Whole-cell MALDI-TOF MS Research Articles

Rapid identification of Salmonella serovars Enteritidis and Typhimurium using whole cell matrix assisted laser desorption ionization – Time of flight mass spectrometry (MALDI-TOF MS) coupled with multivariate analysis and artificial intelligence

Salmonella is a common food-borne pathogen with Enteritidis and Typhimurium being among the most important serovars causing numerous outbreaks. A rapid method was investigated to identify these serovars using whole-cell MALDI-TOF MS coupled with multivariate analysis and artificial intelligence and 113 Salmonella strains, including 38 Enteritidis (SE), 38 Typhimurium (ST) and 37 strains from 32 other Salmonella serovars (SG). Datasets of ions (presence/absence) with high discriminative power were created using newly developed criteria and subject to multivariate analyses and eight artificial intelligence (AI) tools. Principal Component Analysis based on 55 or 88 selected ions separated SE, ST and SG without overlap on the first three principal components. Datasets were partitioned using five partitioning methods with 70% of samples for AI model training and 30% for validation. Of the eight AI models evaluated, high performance (HP) SVM and HP Neural were the top performers, identified three serovar groups 97% correctly on average (range 82%–100%) according to the validation results. Selection of serovar specific ions facilitated differentiation of serotypes using unsupervised model PCA and improved the accuracy of classification using AI significantly (p < 0.01). MALDI-TOF MS incorporated with advanced data processing and classification tools is a promising method to allow rapid identification of Salmonella serovars of concern in routine diagnostic laboratories.

Phenotypic and molecular differentiation of Lactococcus garvieae and Lactococcus petauri isolated from trout

Lactococcosis caused by Lactococcus garvieae leads to infective endocarditis, urinary tract infections, and peritonitis for humans and massive deaths for rainbow trout, an important zoonotic disease affecting fish, other animals and humans. However, the description of Lactococcus petauri revealed that most L. garvieae isolates belonged to L. petauri by genome analysis. In the present study, we revealed a comprehensive genomic comparison between L. garvieae and L. petauri species covering the determination of genome characteristics, overall genome-relatedness indices, genomic islands including virulence genes and antimicrobial resistance models, protein families, habitat preference and ecological distribution features. Besides, L. garvieae and L. petauri strains were also evaluated by whole-cell MALDI-TOF MS analysis. Notably, we assessed a species-specific primer design, named LG_IBS and LP_IBS, based on the genome analysis to distinguish L. garvieae and L. petauri for the first time by basic PCR. In addition to genotypic analyses, 177 L. garvieae isolates and four reference strains were tested to ferment sucrose which was determined as the main phenotypic test to differentiate these species for the first time. Our study revealed that L. garvieae and L. petauri have unique genes, and the designed primers, LG_IBS and LP_IBS, clearly differentiate both isolates. In phenotypical comparison, L. garvieae was observed as negative for sucrose fermentation, whereas L. petauri gave a positive reaction. Therefore, we confirmed that the sucrose fermentation test and designed PCR primers have 100% differentiation power for the isolates from two different laboratories for the first time. Moreover, a comparative analysis of protein families for L. garvieae and L. petauri strains revealed that the genomes of L. garvieae strains do not encode the genes required for sucrose metabolism, whereas L. petauri genomes encode genes for fructokinase, sucrose operon repressor and sucrose-6-phosphate hydrolase. Although both species exhibit similar habitat preferences, they also differ in the distribution of various genomic islands, including virulence factors and antimicrobial resistance genes. In conclusion, we propose that sucrose fermentation and newly designed primers yielding different amplicons for L. garvieae and L. petauri could be employed to differentiate the members of both species.

Pseudomonas lactucae sp. nov., a pathogen causing bacterial rot of lettuce in Japan.

Two phytopathogenic bacteria, MAFF 301380T and MAFF 301381, isolated from rot lesions of lettuce (Lactuca sativa L. var. capitata L.) in Japan, were characterized using a polyphasic approach. The cells were Gram-reaction-negative, aerobic, non-spore-forming, rod-shaped and motile with one to three polar flagella. Analysis of the 16S rRNA gene sequences showed that the strains belong to the genus Pseudomonas and are closely related to Pseudomonas cedrina subsp. cedrina CFML 96-198T (99.72 %), Pseudomonas cedrina subsp. fulgida P515/12T (99.65 %), Pseudomonas gessardii DSM 17152T (99.51 %), Pseudomonas synxantha DSM 18928T (99.44 %), Pseudomonas libanensis CIP 105460T (99.44 %) and Pseudomonas lactis DSM 29167T (99.44 %). The genomic DNA G+C content was 60.4 mol% and the major fatty acids consisted of summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). Phylogenetic analysis using the rpoD gene sequences and phylogenomic analyses based on the whole genome sequences demonstrated that the strains are members of the Pseudomonas fluorescens subgroup but formed a monophyletic and robust clade separated from their closest relatives. The average nucleotide identity and digital DNA-DNA hybridization values between the strains and their closely related species were 88.65 % or less and 36.3 % or less, respectively. The strains could be distinguished from their closest relatives by phenotypic characteristics, pathogenicity towards lettuce and whole-cell MALDI-TOF MS profiles. The evidence presented in this study supports the classification of the strains as representing a novel Pseudomonas species, for which we propose the name Pseudomonas lactucae sp. nov., with the type strain MAFF 301380T (=ICMP 23838T).

Pseudomonas arcuscaelestis sp. nov., isolated from rainbow trout and water.

Cells of strains P66T, V1 and W15Feb18 are Gram-stain-negative short rods and motile by one polar flagellum. Strain P66T was isolated from rainbow trout (Oncorhynchus mykiss) cultivated at a fish farm in Turkey. Strain V1 was isolated from sand of an intertidal shore on the Galicia coast in Spain and strain W15Feb18 was isolated from water collected at the Woluwe River in Belgium. Based on 16S rRNA sequence similarity values, the strains were grouped under the genus Pseudomonas and the Pseudomonas putida phylogenetic group of species. The DNA G+C content ranged from 58.5 to 58.9 mol%. The strains were characterized phenotypically by the API 20NE and Biolog GEN III tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and fatty acid contents. The absence of the hydrolysis of gelatin and the assimilation of arabinose, mannose and mannitol differentiated these strains from the closest species, Pseudomonas alkylphenolica. The major fatty acid components were C16:0 (29.91-31.68 %) and summed feature 3 (36.44-37.55 %). Multilocus sequence analysis with four and 83 housekeeping gene sequences and a core proteome analysis showed that these strains formed a phylogenetic cluster in the P. putida group of species. Genome comparisons by the average nucleotide identity based on blast and the Genome-to-Genome Distance Calculator demonstrated that the three strains belonged to the same genomic species and were distant from any known species, with similarity values lower than the thresholds established for species in the genus Pseudomonas. These data permitted us to conclude that strains P66T, V1 and W15Feb18 belong to a novel species in the genus Pseudomonas, for which the name Pseudomonas arcuscaelestis sp. nov. is proposed. The type strain is P66T (=CECT 30176T=CCUG 74872T). The other strains have been deposited in the CECT with the corresponding collection numbers: V1 (=CECT 30356) and W15Feb18 (=CECT 30355).

Pseudomonas anatoliensis sp. nov and Pseudomonas iridis sp. nov. isolated from fish

In a study carried out between 2013 and 2018 in fish farms in Turkey, several putative novel species were isolated. The 16S rRNA nucleotide sequences of fourteen strains of Gram-negative rods, which were isolated from asymptomatic and symptomatic rainbow trouts (Onchorhynchus mykiss), placed them under the genus Pseudomonas. The similarity values of the concatenated nucleotide sequences of the rpoD, rpoB, gyrB and 16S rRNA genes clustered these isolates into the P. fluorescens phylogenetic group of species and into the Pseudomonas koreensis subgroup, close to Pseudomonas helmanticensis and Pseudomonas baetica. An isolate of a totally different origin, strain CCUG 67011, clustered with these isolates. Phenotypic characterization, together with the chemotaxonomic data, whole-cell MALDI-TOF MS and fatty acids methyl esters analyses were performed. The DNA G + C content was 58.7 mol% for isolate P9T and 58.8 mol% for isolate P42T. The phylogenomic analysis and whole genome nucleotide sequences of four of these isolates confirmed that the isolates P9T, P25 and P141, represent a novel species for which the name Pseudomonas anatoliensis sp. nov. is proposed, with P9T as the type strain (=CCUG 74755T = CECT 3172T). The isolates P1, P2, P10, P27, P30, P24a, P42T, P117, P139, P152 and CCUG 67011 represent another novel sècies, for which the name Pseudomonas iridis sp. nov. is proposed, with P42T as the type strain (=CCUG 74870T = CECT 3174T).

Pseudomonas nosocomialis sp. nov., isolated from clinical specimens.

Strains A31/70T, CCUG 58779 and SD129 were Gram-stain-negative, short rods, motile by one polar flagellum and isolated from clinical specimens in Botswana, Sweden and Spain, respectively. The 16S rRNA sequence similarity values grouped them in the Pseudomonas stutzeri phylogenetic group of species. The DNA G+C content ranged from 65.5 to 65.7 mol%. The strains were characterized phenotypically by the API 20NE and Biolog GEN III tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and by their fatty acid contents. The absence of the arginine dihydrolase and the hydrolysis of gelatin differentiated these strains from the closest species, Pseudomonas azotifigens. The major fatty acid contents were summed feature 8 (38.6 %), C16 : 0 (22.6 %), summed feature 3 (20.5 %) and C12 : 0 (8.4 %). Multilocus sequence analysis with three housekeeping gene sequences (rpoD, gyrB and 16S rRNA) together with whole-genome comparisons indicated that these strains cluster together in the phylogenetic analysis and their similarity values were lower than the thresholds established for species in the genus Pseudomonas. These results permit us to conclude that strains A31/70T, CCUG 58779 and SD129 belong to a novel species in the genus Pseudomonas for which the name Pseudomonas nosocomialis sp. nov. is proposed. The type strain is A31/70T (=CECT 9981T=CCUG 73638T).

Pseudomonas piscium sp. nov., Pseudomonas pisciculturae sp. nov., Pseudomonas mucoides sp. nov. and Pseudomonas neuropathica sp. nov. isolated from rainbow trout

Six Gram negative, motile bacteria were isolated from rainbow trout (Oncorhynchus mykiss). The 16S rRNA sequence similarity values grouped them in the Pseudomonas mandelii (strains P49, P50T, 154aT and P154b), Pseudomonas fluorescens (strain P115T) and Pseudomonas koreensis (strain P155T) phylogenetic subgroups in the genus Pseudomonas. The DNA G+C content ranged from 58.5 to 60 mol%. The strains were characterized phenotypically using API 20NE and Biolog GENIII tests, and chemotaxonomically by their whole-cell MALDI-TOF MS protein profiles and fatty acid contents. Multi-locus sequence analysis with four housekeeping gene sequences (rpoD, rpoB, gyrB and 16S rRNA) together with genome comparisons by average nucleotide identity and genome-to-genome distance calculations were performed. Results showed that the similarity values of these strains to known species type strains were lower than the thresholds established for species in the genus Pseudomonas. Based on these data, we concluded that strains P49, P50T, P115T, P154aT, P154b and P155T belonged to four novel species. The names proposed are: Pseudomonas piscium sp. nov. for strains P49 and P50T with P50T (=CECT 30175T=CCUG 74871T) as the type strain; Pseudomonas pisciculturae sp. nov. for strain P115T (CECT 30173T=CCUG 74873T); Pseudomonas mucoides sp. nov. for strains P154aT and P154b with P154aT (=CECT 30177T=CCUG 74874T) as the type strain; and Pseudomonas neuropathica sp. nov. for strain P155T (=CECT 30178T=CCUG 74875T).

Analysis and Characterization of Immune Cells and Their Activation Status by Whole-Cell MALDI-TOF Mass Spectrometry.

For 40years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in proteomics and biochemistry. It has been demonstrated in the last decade that MALDI-TOF MS can be used routinely to identify and classify numerous bacterial species or subspecies. We applied MALDI-TOF MS directly to intact mammalian cells, and we found that this method is valuable to identify human circulating cells and cells involved in the immune response including macrophages. We then stimulated human macrophages with cytokines, bacterial products, and a variety of bacteria. We found that MALDI-TOF MS discriminated unstimulated and stimulated macrophages and also detected multifaceted activation of macrophages. We conclude that whole-cell MALDI-TOF MS is an accurate method to identify various cell types and to detect subtle modifications in cell activity and therefore it can be beneficial in clinical practices for a rapid patient classification based on their immune profile.

Whole-Cell MALDI-TOF MS Versus 16S rRNA Gene Analysis for Identification and Dereplication of Recurrent Bacterial Isolates.

Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well-established bacterial identification and classification methods based on nearly complete 16S rRNA gene sequence analyses: a phylotype-based approach, using a closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4–10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.

Polyphasic characterization of a novel species in the Lactobacillus casei group from cow manure of Taiwan: Description of L. chiayiensis sp. nov.

Two Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strains, NCYUAST and BCRC 18859 (=NRIC 1947), were isolated from cow manure of Taiwan and coconut juice of Philippines, respectively. Comparative sequence analysis of 16S rRNA gene revealed that the novel strains were members of the genus Lactobacillus. These two strains had 100% of 16S rRNA gene sequence similarity and 98.6% of average nucleotide identity (ANI) value based on whole genome sequences. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (99.6% similarity), Lactobacillus paracasei subsp. paracasei (99.1%), L. paracasei subsp. tolerans (99.1%), Lactobacillus rhmnosus (99.0%) and ‘Lactobacillus zeae’ (99.7%) were the closest neighbors to these novel strains. The results of phenotypic and chemotaxonomic characterization, multilocus sequence analysis (MLSA) based on the sequences of three housekeeping genes (dnaK, pheS and yycH), whole-genome sequence (WGS)-based comparison by ANI and in silico DNA–DNA hybridization (isDDH), species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses demonstrated that the novel two strains represented a single, novel species within the L. casei group, for which the name Lactobacillus chiayiensis sp. nov., is proposed. The type strain is NCYUAST (=BCRC 81062T=NBRC 112906T).

Establishment and application of an analytical in-house database (IHDB) for rapid discrimination of Bacillus subtilis group (BSG) using whole-cell MALDI-TOF MS technology

Members of the Bacillus subtilis group (BSG) possess industrial applicability; unfortunately, B. subtilis and its phylogenetically closest species are indistinguishable from one another using 16S rDNA sequencing, physiological and biochemical tests. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique for the fast and reliable identification of microorganisms. The aim of this study was to construct a unique analytical in-house database (IHDB) for BSG discrimination based on whole-cell protein fingerprinting using MALDI-TOF MS, as well as to discover biomarkers from the MS peaks to generate a classification model for further differentiation using the ClinProTools software. Type strains of 12 species (included five subspecies) of the BSG were used to build a main spectrum profile (MSP) to create an IHDB under the optimized parameters. The BSG isolates obtained from partial recA gene sequencing were used for IHDB validation. A total of 84 (100%) isolates were correctly identified to the species level and had high score values (mean score: 2.52). However, the IHDB had ambiguous identification at the subspecies level of Bacillus amyloliquefaciens. After implementation of the classification models, the strains could be clearly differentiated. We have successfully developed a rapid, accurate and cost-effective platform for the species- and subspecies-level discrimination of BSG based on the implementation of the IHDB and coupled with ClinProTools, which can be employed as an alternative technology to DNA sequencing and applied for efficient quality control of the microbial agent.

Mass spectrometry methods for predicting antibiotic resistance.

Developing elaborate techniques for clinical applications can be a complicated process. Whole-cell MALDI-TOF MS revolutionized reliable microorganism identification in clinical microbiology laboratories and is now replacing phenotypic microbial identification. This technique is a generic, accurate, rapid, and cost-effective growth-based method. Antibiotic resistance keeps emerging in environmental and clinical microorganisms, leading to clinical therapeutic challenges, especially for Gram-negative bacteria. Antimicrobial susceptibility testing is used to reliably predict antimicrobial success in treating infection, but it is inherently limited by the need to isolate and grow cultures, delaying the application of appropriate therapies. Antibiotic resistance prediction by growth-independent methods is expected to reduce the turnaround time. Recently, the potential of next-generation sequencing and microarrays in predicting microbial resistance has been demonstrated, and this review evaluates the potential of MS in this field. First, technological advances are described, and the possibility of predicting antibiotic resistance by MS is then illustrated for three prototypical human pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Clearly, MS methods can identify antimicrobial resistance mediated by horizontal gene transfers or by mutations that affect the quantity of a gene product, whereas antimicrobial resistance mediated by target mutations remains difficult to detect.

Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants.

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria.

Quantitative whole-cell MALDI-TOF MS fingerprints distinguishes human monocyte sub-populations activated by distinct microbial ligands.

BackgroundConventionally, human monocyte sub-populations are classified according to surface marker expression into classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) lineages. The involvement of non-classical monocytes, also referred to as proinflammatory monocytes, in the pathophysiology of diseases including diabetes mellitus, atherosclerosis or Alzheimer’s disease is well recognized. The development of novel high-throughput methods to capture functional states within the different monocyte lineages at the whole cell proteomic level will enable real time monitoring of disease states.ResultsWe isolated and characterized (pan-) monocytes, mostly composed of classical CD16− monocytes, versus autologous CD16+ subpopulations from the blood of healthy human donors (n = 8) and compared their inflammatory properties in response to lipopolysaccharides and M.tuberculosis antigens by multiplex cytokine profiling. Following resting and in vitro antigenic stimulation, cells were recovered and subjected to whole-cell mass spectrometry analysis. This approach identified the specific presence/absence of m/z peaks and therefore potential biomarkers that can discriminate pan-monocytes from their CD16 counterparts. Furthermore, we found that semi-quantitative data analysis could capture the subtle proteome changes occurring upon microbial stimulation that differentiate resting, from lipopolysaccharides or M. tuberculosis stimulated monocytic samples.ConclusionsWhole-cell mass spectrometry fingerprinting could efficiently distinguish monocytic sub-populations that arose from a same hematopoietic lineage. We also demonstrate for the first time that mass spectrometry signatures can monitor semi-quantitatively specific activation status in response to exogenous stimulation. As such, this approach stands as a fast and efficient method for the applied immunology field to assess the reactivity of potentially any immune cell types that may sustain health or promote related inflammatory diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0140-1) contains supplementary material, which is available to authorized users.

Acinetobacter gandensis sp. nov. isolated from horse and cattle.

We previously reported the presence of an OXA-23 carbapenemase in an undescribed species of the genus Acinetobacter isolated from horse dung at the Faculty of Veterinary Medicine, Ghent University, Belgium. Here we include six strains to corroborate the delineation of this taxon by phenotypic characterization, DNA-DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C determination, MALDI-TOF MS and fatty acid analysis. The nearly complete 16S rRNA gene sequence of strain UG 60467(T) showed the highest similarities with those of the type strains of Acinetobacter bouvetii (98.4 %), Acinetobacter haemolyticus (97.7 %), and Acinetobacter schindleri (97.2 %). The partial rpoB sequence of strain UG 60467(T) showed the highest similarities with 'Acinetobacter bohemicus' ANC 3994 (88.6 %), A. bouvetii NIPH 2281 (88.6 %) and A. schindleri CIP 107287T (87.3 %). Whole-cell MALDI-TOF MS analyses supported the distinctness of the group at the protein level. The predominant fatty acids of strain UG 60467(T) were C12 : 0 3-OH, C12 : 0, C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). Strains UG 60467(T) and UG 60716 showed a DNA-DNA relatedness of 84 % with each other and a DNA-DNA relatedness with A. schindleri LMG 19576(T) of 17 % and 20 %, respectively. The DNA G+C content of strain UG 60467(T) was 39.6 mol%. The name Acinetobacter gandensis sp. nov. is proposed for the novel taxon. The type strain is UG 60467(T) ( = ANC 4275(T) = LMG 27960(T) = DSM 28097(T)).

Source-identifying biomarker ions between environmental and clinical Burkholderia pseudomallei using whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates) with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs), including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs) consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei.

Whole-cell MALDI-TOF MS: A new tool to assess the multifaceted activation of macrophages

Whole-cell MALDI-TOF MS is routinely used to identify bacterial species in clinical samples. This technique has also proven to allow identification of intact mammalian cells, including macrophages. Here, we wondered whether this approach enabled the assessment human macrophages plasticity. The whole-cell MALDI-TOF spectra of macrophages stimulated with IFN-γ and IL-4, two inducers of M1 and M2 macrophage polarisation, consisted of peaks ranging from 2 to 12 kDa. The spectra of unstimulated and stimulated macrophages were clearly different. The fingerprints induced by the M1 agonists, IFN-γ, TNF, LPS and LPS+IFN-γ, and the M2 agonists, IL-4, TGF-β1 and IL-10, were specific and readily identifiable. Thus, whole-cell MALDI-TOF MS was able to characterise M1 and M2 macrophage subtypes. In addition, the fingerprints induced by extracellular (group B Streptococcus, Staphylococcus aureus) or intracellular (BCG, Orientia tsutsugamushi, Coxiella burnetii) bacteria were bacterium-specific. The whole-cell MALDI-TOF MS fingerprints therefore revealed the multifaceted activation of human macrophages. This approach opened a new avenue of studies to assess the immune response in the clinical setting, by monitoring the various activation patterns of immune cells in pathological conditions.

Characterization of a Yersinia enterocolitica biotype 1A strain harbouring an ail gene

The chromosomal ail gene (attachment and invasion locus) is commonly used as target gene for the detection of pathogenic Y. enterocolitica strains in food testing. The ail PCR does not detect strains of biotype 1A (BT1A), which are regarded as non-pathogenic because BT1A strains lack the virulence plasmid and chromosomally encoded virulence genes. In some recent reports, however, BT1A strains were discovered that harboured the ail gene. We isolated an ail-positive strain and characterized this strain with phenotypic and genotypic methods to study its possible relation to pathogenic Y. enterocolitica strains. The ail region of the BT1A strain was sequenced and compared with the corresponding region of nonpathogenic BT1A strains and pathogenic strains. Pulsed field gel electrophoresis (PFGE) analysis was applied revealing no similarity of the PFGE pattern of this strain to the patterns of pathogenic strains. Virulence-gene-based PCR analyses showed the strain to be positive for ystB, but negative for virulence genes ystA, virF and yadA. Whole-cell MALDI-TOF MS combined with a shrinkage discriminant analysis approach was applied and clearly classified the ail-positive biotype 1A strain within the cluster of BT1A strains. PCR detection of ail sequences in food matrices should be followed by the isolation of the responsible strain and its characterization using phenotypic or genotypic methods. The ail gene may be present in Y. enterocolitica BT1A strains, which are commonly considered as nonpathogenic. Efficient methods such as PCR typing of other virulence genes or rapid MALDI-TOF MS-based bacterial profiling allow a more comprehensive assessment of the pathogenicity potential of Yersinia strains.

Evaluation of matrix-assisted laser desorption ionization-time of flight whole cell profiles for assessing the cultivable diversity of aerobic and moderately halophilic prokaryotes thriving in solar saltern sediments

The moderately halophilic, cultivable fraction of prokaryotes thriving in hypersaline sediments of a solar saltern in Mallorca, Spain, has been studied by means of different cultivation media. A set of 374 isolates retrieved with six different culture conditions was screened, using whole-cell MALDI-TOF MS analysis to classify them into 25 phenotypic clusters at 52% similarity. The phylogenetic inference, made from comparative sequence analyses of the 16S rRNA genes of selected strains, indicated that each phenotypic cluster was comprised of a genealogically homogeneous set of strains. DNA–DNA hybridization (DDH) results among selected strains confirmed that each MALDI-TOF cluster encompassed members of the same species. On the other hand, the intra-cluster diversity, measured by several RAPD (Random Amplified Polymorphic DNA) amplifications, indicated that the clusters corresponded to several populations of the same phylogenetic unit coexisting in the same environment. The results encourage the use of MALDI-TOF MS for further exhaustive studies of the cultivable diversity of hypersaline environments.

Whole-cell spectroscopy is a convenient tool to assist molecular identification of cultivatable marine bacteria and to investigate their adaptive metabolism.

Recent developments in whole-cell spectroscopic methods allow rapid characterization of microorganisms of interest to human health, but have yet to be widely applied to marine microbiological studies. In this study of bacteria associated with the kelp Laminaria digitata, we have isolated 18 epiphytic bacterial strains from several thalli, sequenced their 16S rDNA, built corresponding phylogenetic trees, and characterized them using spectroscopic methods. Molecular taxonomy revealed Gram(+)Actinobacteria and Gram(-)Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. Twelve marine reference strains (Gram(+)Firmicutes, and Gram(-)Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes) were treated accordingly. Whole-cell MALDI-TOF MS spectral profiles of 29 of the 30 strains were built into a database against which 16 replicate spectra of each strain were compared and categorized into groups. The proton HR-MAS NMR stack plots allowed visual delineation into taxonomic groups according to their most common peaks, in agreement with identifiable compounds from corresponding D(2)O solution spectra. With both methods, these groups corresponded to taxa identified by 16S rDNA sequences, MALDI-TOF MS being more discriminative than HR-MAS NMR. Culture age did not influence the spectral signatures in both approaches. Most cells grown under minimal conditions (VNSS medium) afforded HR-MAS NMR profiles markedly different to those grown in enriched conditions (ZoBell medium), indicating different adaptive metabolic responses between the two media. Spectral signatures obtained under strictly controlled conditions can be used as rapid and reliable tools for taxonomic purposes and as markers of physiological status.