Dominik Ziegler

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry.

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.

Prevalence and characteristics of methicillin-resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons

BackgroundMethicillin-resistant coagulase-negative staphylococci (MR-CNS) are of increasing importance to animal and public health. In veterinary medicine and along the meat and milk production line, only limited data were so far available on MR-CNS characteristics. The aim of the present study was to evaluate the prevalence of MR-CNS, to identify the detected staphylococci to species level, and to assess the antibiotic resistance profiles of isolated MR-CNS strains.ResultsAfter two-step enrichment and growth on chromogenic agar, MR-CNS were detected in 48.2% of samples from livestock and chicken carcasses, 46.4% of samples from bulk tank milk and minced meat, and 49.3% of human samples. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 414 selected MR-CNS strains belonged to seven different species (S. sciuri, 32.6%; S. fleurettii, 25.1%; S. haemolyticus, 17.4%; S. epidermidis, 14.5%, S. lentus, 9.2%; S. warneri, 0.7%; S. cohnii, 0.5%). S. sciuri and S. fleurettii thereby predominated in livestock, BTM and minced meat samples, whereas S. epidermidis and S. haemolyticus predominated in human samples. In addition to beta-lactam resistance, 33-49% of all 414 strains were resistant to certain non-beta-lactam antibiotics (ciproflaxacin, clindamycin, erythromycin, tetracycline).ConclusionsA high prevalence of MR-CNS was found in livestock production. This is of concern in view of potential spread of mecA to S. aureus (MRSA). Multiresistant CNS strains might become an emerging problem for veterinary medicine. For species identification of MR-CNS isolated from different origins, MALDI-TOF MS proved to be a fast and reliable tool and is suitable for screening of large sample amounts.

Evaluation of matrix-assisted laser desorption/ionization time of flight mass spectrometry for characterization of Culicoides nubeculosus biting midges.

Matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS) has shown promise in species identification of insect species. We evaluated its potential to consistently characterize laboratory‐reared biting midges of the species Culicoides nubeculosus (Meigen) (Diptera: Ceratopogonidae). Twenty‐one reproducible potential biomarker masses for C. nubeculosus were identified under different experimental treatments. These treatments included the homogenization of insects in either water or known concentrations of formic acid. The biomarker masses were present independent of age, gender and different periods of storage of individuals in 70% ethanol (a standard preservation method). It was found that the presence of blood in females reduced the intensity of the MALDI‐TOF pattern, necessitating the removal of the abdomen before analysis. The protein profiles of a related non‐biting midge, Forcipomyia sp. (Diptera: Ceratopogonidae), and of Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) mosquitoes were also examined and were distinctly different. These findings provide preliminary data to optimize future studies in differentiation of species within the Culicoides genus using MALDI‐TOF MS which is a rapid, simple, reliable and cost‐effective technique.

Rapid Genus- and Species-Specific Identification of Cronobacter spp. by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Cronobacter spp. are Gram-negative opportunistic food-borne pathogens and are known as rare but important causes of life-threatening neonatal infections. Rapid and reliable identification of Cronobacter species and their differentiation from phenotypically similar, nonpathogenic Enterobacter turicensis, Enterobacter helveticus, and Enterobacter pulveris have become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid genus and species identification of the six Cronobacter species recognized so far. To this end, we developed a reference MS database library that includes 54 Cronobacter target strains as well as 17 nontarget strains. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2,000 to 30,000 Da). Genus- and species-specific biomarker protein mass patterns were determined. The defined biomarker mass patterns (Spectral Archive and Microbial Identification System [SARAMIS] SuperSpectrum) were validated using 36 strains from various Cronobacter species as well as eight nontarget strains. For all strains the mass spectrometry-based identification scheme yielded identical results as with a PCR-based identification system. All strains were correctly identified, and no nontarget strain was misidentified as Cronobacter. Our study demonstrates that MALDI-TOF MS is a reliable and powerful tool for the rapid identification of Cronobacter strains to the genus and species level.

Identification of field-caught Culicoides biting midges using matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Culicoides biting midges are of great importance as vectors of pathogens and elicitors of allergy. As an alternative for the identification of these tiny insects, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was evaluated. Protein mass fingerprints were determined for 4-5 field-caught reference (genetically confirmed) individuals of 12 Culicoides species from Switzerland, C. imicola from France, laboratory-reared C. nubeculosus and a non-biting midge. Reproducibility and accuracy of the database was tested in a validation study by analysing 108 mostly field-caught target Culicoides midges and 3 specimens from a non-target species. A reference database of biomarker mass sets containing between 24 and 38 masses for the different species could be established. Automated database-based identification was achieved for 101 of the 108 specimens. The remaining 7 midges required manual full comparison with the reference spectra yielding correct identification for 6 specimens and an ambiguous result for the seventh individual. Specimens of the non-target species did not yield identification. Protein profiling by MALDI-TOF, which is compatible with morphological and genetic identification of specimens, can be used as an alternative, quick and inexpensive tool to accurately identify Culicoides biting midges collected in the field.

Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling

Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

A rapid MALDI-TOF MS identification database at genospecies level for clinical and environmental Aeromonas strains.

The genus Aeromonas has undergone a number of taxonomic and nomenclature revisions over the past 20 years, and new (sub)species and biogroups are continuously described. Standard identification methods such as biochemical characterization have deficiencies and do not allow clarification of the taxonomic position. This report describes the development of a matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI-TOF MS) identification database for a rapid identification of clinical and environmental Aeromonas isolates.

In Situ Identification of Plant-Invasive Bacteria with MALDI-TOF Mass Spectrometry

Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues.

Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS

Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.

Tracing of Two Pseudomonas Strains in the Root and Rhizoplane of Maize, as Related to Their Plant Growth-Promoting Effect in Contrasting Soils

TaqMan-based quantitative PCR (qPCR) assays were developed to study the persistence of two well-characterized strains of plant growth-promoting rhizobacteria (PGPR), Pseudomonas fluorescens Pf153 and Pseudomonas sp. DSMZ 13134, in the root and rhizoplane of inoculated maize plants. This was performed in pot experiments with three contrasting field soils (Buus, Le Caron and DOK-M). Potential cross-reactivity of the qPCR assays was assessed with indigenous Pseudomonas and related bacterial species, which had been isolated from the rhizoplane of maize roots grown in the three soils and then characterized by Matrix-Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) mass spectrometry (MS). Sensitivity of the qPCR expressed as detection limit of bacterial cells spiked into a rhizoplane matrix was 1.4 × 102 CFU and 1.3 × 104 CFU per gram root fresh weight for strain Pf153 and DSMZ 13134, respectively. Four weeks after planting and inoculation, both strains could readily be detected in root and rhizoplane, whereas only Pf153 could be detected after 8 weeks. The colonization rate of maize roots by strain Pf153 was significantly influenced by the soil type, with a higher colonization rate in the well fertile and organic soil of Buus. Inoculation with strain DSMZ 13134, which colonized roots and rhizoplane to the same degree, independently of the soil type, increased yield of maize, in terms of biomass accumulation, only in the acidic soil of Le Caron, whereas inoculation with strain Pf153 reduced yield in the soil Buus, despite of its high colonization rate and persistence. These results indicate that the colonization rate and persistence of inoculated Pseudomonas strains can be quantitatively assessed by the TaqMan-based qPCR technique, but that it cannot be taken for granted that inoculation with a well-colonizing and persistent Pseudomonas strain has a positive effect on yield of maize.