Bacterial genome-wide association study substantiates papGII of Escherichia coli as a major risk factor for urosepsis.

BACKGROUND: Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, often caused by uropathogenic Escherichia coli. Multiple bacterial virulence factors or patient characteristics have been linked separately to progressive, more invasive infections. In this study, we aim to identify pathogen- and patient-specific factors that drive the progression to urosepsis by jointly analysing bacterial and host characteristics. METHODS: We analysed 1076 E. coli strains isolated from 825 clinical cases with UTI and/or bacteraemia by whole-genome sequencing (Illumina). Sequence types (STs) were determined via srst2 and capsule loci via fastKaptive. We compared the isolates from urine and blood to confirm clonality. Furthermore, we performed a bacterial genome-wide association study (bGWAS) (pyseer) using bacteraemia as the primary clinical outcome. Clinical data were collected by an electronic patient chart review. We concurrently analysed the association of the most significant bGWAS hit and important patient characteristics with the clinical endpoint bacteraemia using a generalised linear model (GLM). Finally, we designed qPCR primers and probes to detect papGII-positive E. coli strains and prospectively screened E. coli from urine samples (n = 1657) at two healthcare centres. RESULTS: Our patient cohort had a median age of 75.3 years (range: 18.00-103.1) and was predominantly female (574/825, 69.6%). The bacterial phylogroups B2 (60.6%; 500/825) and D (16.6%; 137/825), which are associated with extraintestinal infections, represent the majority of the strains in our collection, many of which encode a polysaccharide capsule (63.4%; 525/825). The most frequently observed STs were ST131 (12.7%; 105/825), ST69 (11.0%; 91/825), and ST73 (10.2%; 84/825). Of interest, in 12.3% (13/106) of cases, the E. coli pairs in urine and blood were only distantly related. In line with previous bGWAS studies, we identified the gene papGII (p-value < 0.001), which encodes the adhesin subunit of the E. coli P-pilus, to be associated with 'bacteraemia' in our bGWAS. In our GLM, correcting for patient characteristics, papGII remained highly significant (odds ratio = 5.27, 95% confidence interval = [3.48, 7.97], p-value < 0.001). An independent cohort of cases which we screened for papGII-carrying E. coli at two healthcare centres further confirmed the increased relative frequency of papGII-positive strains causing invasive infection, compared to papGII-negative strains (p-value = 0.033, chi-squared test). CONCLUSIONS: This study builds on previous work linking papGII with invasive infection by showing that it is a major risk factor for progression from UTI to bacteraemia that has diagnostic potential.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth.

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods.

Whole-genome sequence-informed MALDI-TOF MS diagnostics reveal importance of Klebsiella oxytoca group in invasive infections: a retrospective clinical study.

BACKGROUND: Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence. METHODS: We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models. RESULTS: Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), "K. quasivariicola" (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and "K. quasivariicola" (odds ratio = 0.17, p < 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53]). CONCLUSIONS: Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options.

Factors Associated With MALDI-TOF Mass Spectral Quality of Species Identification in Clinical Routine Diagnostics.

Background: An accurate and timely identification of bacterial species is critical in clinical diagnostics. Species identification allows a potential first adaptation of empiric antibiotic treatments before the resistance profile is available. Matrix assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS) is a widely used method for bacterial species identification. However, important challenges in species identification remain. These arise from (i) incomplete databases, (ii) close relatedness of species of interest, and (iii) spectral quality, which is currently vaguely defined. Methods: We selected 47 clinically relevant bacterial isolates from 39 species, which can be challenging to identify by MALDI-TOF MS. We measured these isolates under various analytical conditions on two MALDI-TOF MS systems. First, we identified spectral features, which were associated with correct species identification in three different databases. Considering these features, we then systematically compared spectra produced with three different sample preparation protocols. In addition, we varied quantities of bacterial colony material applied and bacterial colony age. Results: We identified (i) the number of ribosomal marker peaks detected, (ii) the median relative intensity of ribosomal marker peaks, (iii) the sum of the intensity of all detected peaks, (iv) a high measurement precision, and (v) reproducibility of peaks to act as good proxies of spectral quality. We found that using formic acid, measuring bacterial colonies at a young age, and frequently calibrating the MALDI-TOF MS device increase mass spectral quality. We further observed significant differences in spectral quality between different bacterial taxa and optimal measurement conditions vary per taxon. Conclusion: We identified and applied quality measures for MALDI-TOF MS and optimized spectral quality in routine settings. Phylogenetic marker peaks can be reproducibly detected and provide an increased resolution and the ability to distinguish between challenging species such as those within the Enterobacter cloacae complex, Burkholderia cepacia complex, or viridans streptococci.

A simple, rapid typing method for Streptococcus agalactiae based on ribosomal subunit proteins by MALDI-TOF MS.

Streptococcus agalactiae (Group B Streptococcus, GBS), is a frequent human colonizer and a leading cause of neonatal meningitis as well as an emerging pathogen in non-pregnant adults. GBS possesses a broad animal host spectrum, and recent studies proved atypical GBS genotypes can cause human invasive diseases through animal sources as food-borne zoonotic infections. We applied a MALDI-TOF MS typing method, based on molecular weight variations of predefined 28 ribosomal subunit proteins (rsp) to classify GBS strains of varying serotypes into major phylogenetic lineages. A total of 249 GBS isolates of representative and varying capsular serotypes from patients and animal food sources (fish and pig) collected during 2016-2018 in Hong Kong were analysed. Over 84% (143/171) noninvasive carriage GBS strains from patients were readily typed into 5 globally dominant rsp-profiles. Among GBS strains from food animals, over 90% (57/63) of fish and 13% (2/15) of pig GBS matched with existing rsp-profiles, while the remainder were classified into two novel rsp-profiles and we failed to assign a fish strain into any cluster. MALDI-TOF MS allowed for high-throughput screening and simultaneous detection of novel, so far not well described GBS genotypes. The method shown here is rapid, simple, readily transferable and adapted for use in a diagnostic microbiology laboratory with potential for the surveillance of emerging GBS genotypes with zoonotic potential.

Agrobacterium spp. nosocomial outbreak assessment using rapid MALDI-TOF MS based typing, confirmed by whole genome sequencing.

Background: A number of episodes of nosocomial Agrobacterium spp. bacteremia (two cases per year) were observed at Bern University Hospital, Switzerland, from 2015 to 2017. This triggered an outbreak investigation. Methods: Cases of Agrobacterium spp. bacteremias that occurred between August 2011 and February 2017 were investigated employing line lists, environmental sampling, rapid protein- (MALDI-TOF MS), and genome-based typing (pulsed field gel electrophoresis and whole genome sequencing) of the clinical isolates. Results: We describe a total of eight bacteremia episodes due to A. radiobacter (n = 2), Agrobacterium genomovar G3 (n = 5) and A. pusense (n = 1). Two tight clusters were observed by WGS typing, representing the two A. radiobacter isolates (cluster I, isolated in 2015) and four of the Agrobacterium genomovar G3 isolates (cluster II, isolated in 2016 and 2017), suggesting two different point sources. The epidemiological investigations revealed two computer tomography (CT) rooms as common patient locations, which correlated with the two outbreak clusters. MALDI-TOF MS permitted faster evaluation of strain relatedness than DNA-based methods. High resolution WGS-based typing confirmed the MALDI-TOF MS clustering. Conclusions: We report clinical and epidemiological characteristics of two outbreak clusters with Agrobacterium. spp. bacteremia likely acquired during CT contrast medium injection and highlight the use of MALDI-TOF MS as a rapid tool to assess relatedness of rare gram-negative pathogens in an outbreak investigation.

Bacterial identification using a SCIEX 5800 TOF/TOF MALDI research instrument and an external database.

In our current study we were identifying 26 bacterial isolates using a SCIEX 5800 TOF/TOF MALDI instrument and an external database. The results were compared with the results of a Vitek® MS system and in case of discrepancies at the species level 16s rRNA sequencing was performed for further verification.

Subspecies Typing of Streptococcus agalactiae Based on Ribosomal Subunit Protein Mass Variation by MALDI-TOF MS.

Background: A ribosomal subunit protein (rsp)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was developed for fast subspecies-level typing of Streptococcus agalactiae (Group B Streptococcus, GBS), a major cause of neonatal sepsis and meningitis. Methods: A total of 796 GBS whole genome sequences, covering the genetic diversity of the global GBS population, were used to in silico predict molecular mass variability of 28 rsp and to identify unique rsp mass combinations, termed "rsp-profiles". The in silico established GBS typing scheme was validated by MALDI-TOF MS analysis of GBS isolates at two independent research sites in Europe and South East Asia. Results: We identified in silico 62 rsp-profiles, with the majority (>80%) of the 796 GBS isolates displaying one of the six rsp-profiles 1-6. These dominant rsp-profiles classify GBS strains in high concordance with the core-genome based phylogenetic clustering. Validation of our approach by in-house MALDI-TOF MS analysis of 248 GBS isolates and external analysis of 8 GBS isolates showed that across different laboratories and MALDI-TOF MS platforms, the 28 rsp were detected reliably in the mass spectra, allowing assignment of clinical isolates to rsp-profiles at high sensitivity (99%) and specificity (97%). Our approach distinguishes the major phylogenetic GBS genotypes, identifies hyper-virulent strains, predicts the probable capsular serotype and surface protein variants and distinguishes between GBS genotypes of human and animal origin. Conclusion: We combine the information depth of whole genome sequences with the highly cost efficient, rapid and robust MALDI-TOF MS approach facilitating high-throughput, inter-laboratory, large-scale GBS epidemiological and clinical studies based on pre-defined rsp-profiles.

Species diversity, host preference and arbovirus detection of Culicoides (Diptera: Ceratopogonidae) in south-eastern Serbia.

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) is a genus of small biting midges (also known as "no-see ums") that currently includes 1368 described species. They are proven or suspected vectors for important pathogens affecting animals such as bluetongue virus (BTV) and Schmallenberg virus (SBV). Currently little information is available on the species of Culicoides present in Serbia. Thus, the aim of this study was to examine species diversity, host preference and the presence of BTV and SBV RNA in Culicoides from the Stara Planina Nature Park in south-eastern Serbia. RESULTS: In total 19,887 individual Culicoides were collected during three nights of trapping at two farm sites and pooled into six groups (Obsoletus group, Pulicaris group, "Others" group and further each group according to the blood-feeding status to freshly engorged and non-engorged). Species identification was done on subsamples of 592 individual Culicoides specimens by morphological and molecular methods (MALDI-TOF mass spectrometry and PCR/sequencing). At least 22 Culicoides species were detected. Four animal species (cow, sheep, goat and common blackbird) as well as humans were identified as hosts of Culicoides biting midges. The screening of 8291 Culicoides specimens in 99 pools for the presence of BTV and SBV RNA by reverse-transcription quantitative PCR were negative. CONCLUSIONS: The biodiversity of Culicoides species in the natural reserve Stara Planina was high with at least 22 species present. The presence of C. imicola Kieffer was not recorded in this area. Culicoides showed opportunistic feeding behaviour as determined by host preference. The absence of SBV and BTV viral RNA correlates with the absence of clinical disease in the field during the time of sampling. These data are the direct outcome of a training programme within the Institutional Partnership Project "AMSAR: Arbovirus monitoring, research and surveillance-capacity building on mosquitoes and biting midges" funded by the programme SCOPES of the Swiss National Science Foundation.

Frankia canadensis sp. nov., isolated from root nodules of Alnus incana subspecies rugosa.

Strain ARgP5T, an actinobacterium isolated from a root nodule present on an Alnus incana subspecies rugosa shrub growing in Quebec City, Canada, was the subject of polyphasic taxonomic studies to clarify its status within the genus Frankia. 16S rRNA gene sequence similarities and ANI values between ARgP5T and type strains of species of the genus Frankiawith validly published names were 98.8 and 82 % or less, respectively. The in silico DNA G+C content was 72.4 mol%. ARgP5T is characterised by the presence of meso-A2pm, galactose, glucose, mannose, rhamnose (trace), ribose and xylose as whole-organism hydrolysates; MK-9(H8) as predominant menaquinone; diphosphatidylglycerol, phosphatidylinositol and phosphatidylglycerol as polar lipids and iso-C16 : 0 and C17 : 1ω8c as major fatty acids. The proteomic results confirmed the distinct position of ARgP5T from its closest neighbours in Frankiacluster 1. ARgP5T was found to be infective on two alder (Alnus glutinosa and Alnusalnobetula subsp. crispa) and on one bayberry (Morella pensylvanica) species and to fix nitrogen in symbiosis and in pure culture. On the basis of phylogenetic (16S rRNA gene sequence), genomic, proteomic and phenotypic results, strain ARgP5T (=DSM 45898=CECT 9033) is considered to represent a novel species within the genus Frankia for which the name Frankia canadensis sp. nov., is proposed.

Functional characterization and phenotypic monitoring of human hematopoietic stem cell expansion and differentiation of monocytes and macrophages by whole-cell mass spectrometry.

The different facets of macrophages allow them to play distinct roles in tissue homeostasis, tissue repair and in response to infections. Individuals displaying dysregulated macrophage functions are proposed to be prone to inflammatory disorders or infections. However, this being a cause or a consequence of the pathology remains often unclear. In this context, we isolated and expanded CD34+ HSCs from healthy blood donors and derived them into CD14+ myeloid progenitors which were further enriched and differentiated into macrophages. Aiming for a comprehensive phenotypic profiling, we generated whole-cell mass spectrometry (WCMS) fingerprints of cell samples collected along the different stages of the differentiation process to build a predictive model using a linear discriminant analysis based on principal components. Through the capacity of the model to accurately predict sample's identity of a validation set, we demonstrate that WCMS profiles obtained from bona fide blood monocytes and respectively derived macrophages mirror profiles obtained from equivalent HSC derivatives. Finally, HSC-derived macrophage functionalities were assessed by quantifying cytokine and chemokine responses to a TLR agonist in a 34-plex luminex assay and by measuring their capacity to phagocytise mycobacteria. These functional read-outs could not discriminate blood monocytes-derived from HSC-derived macrophages. To conclude, we propose that this method opens new avenues to distinguish the impact of human genetics on the dysregulated biological properties of macrophages in pathological conditions.

Draft Genome Sequences of Seven Streptococcus agalactiae Strains Isolated from Camelus dromedarius at the Horn of Africa.

We present draft whole-genome sequences of seven Streptococcus agalactiae strains isolated from Camelus dromedarius in Kenya and Somalia. These data are an extension to the group B Streptococcus (GBS) pangenome and might provide more insight into the underlying mechanisms of pathogenicity and antibiotic resistance of camel GBS.

Comparison of biomarker based Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and conventional methods in the identification of clinically relevant bacteria and yeast.

BACKGROUND: MALDI-TOF MS is an analytical method that has recently become integral in the identification of microorganisms in clinical laboratories. It relies on databases that majorly employ pattern recognition or fingerprinting. Biomarker based databases have also been developed and there is optimism that these may be superior to pattern recognition based databases. This study compared the performance of ribosomal biomarker based MALDI-TOF MS and conventional methods in the identification of selected bacteria and yeast. METHODS: The study was a cross sectional study identifying clinically relevant bacteria and yeast isolated from varied clinical specimens submitted to a clinical laboratory. The identification of bacteria using conventional Vitek 2™ automated system, serotyping and MALDI-TOF MS was performed as per standard operating procedures. Comparison of sensitivities were then carried out using Pearson Chi-Square test and p-value of <0.05 was considered statistically significant. Secondary outcomes analyzed included the major and minor error rates. RESULTS: Of the 383 isolates MALDI-TOF MS and conventional methods identified 97.6 and 95.7% (p = 0.231) to the genus level and 97.4 and 88.0% (p = 0.000) to the species level respectively. Biomarker based MALDI-TOF MS was significantly superior to Vitek 2™ in the identification of Gram negative bacteria and Gram positive bacteria to the species level. For the Gram positive bacteria, significant difference was observed in the identification of Coagulase negative Staphylococci (p = 0.000) and Enterococcus (p = 0.008). Significant difference was also observed between serotyping and MALDI-TOF MS (p = 0.005) and this was attributed to the lack of identification of Shigella species by MALDI-TOF MS. There was no significant difference observed in the identification of yeast however some species of Candida were unidentified by MALDI-TOF MS. CONCLUSION: Biomarker based MALDI-TOF MS had good performance in a clinical laboratory setting with high sensitivities in the identification of clinically relevant microorganisms.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry for comprehensive indexing of East African ixodid tick species.

BACKGROUND: The tick population of Africa includes several important genera belonging to the family Ixodidae. Many of these ticks are vectors of protozoan and rickettsial pathogens including Theileria parva that causes East Coast fever, a debilitating cattle disease endemic to eastern, central and southern Africa. Effective surveillance of tick-borne pathogens depends on accurate identification and mapping of their tick vectors. A simple and reproducible technique for rapid and reliable differentiation of large numbers of closely related field-collected ticks, which are often difficult and tedious to discriminate purely by morphology, will be an essential component of this strategy. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is increasingly becoming a useful tool in arthropod identification and has the potential to overcome the limitations of classical morphology-based species identification. In this study, we applied MALDI-TOF MS to a collection of laboratory and field ticks found in Eastern Africa. The objective was to determine the utility of this proteomic tool for reliable species identification of closely related afrotropical ticks. METHODS: A total of 398 ixodid ticks from laboratory maintained colonies, extracted from the hides of animals or systematically collected from vegetation in Kenya, Sudan and Zimbabwe were analyzed in the present investigation. The cytochrome c oxidase I (COI) genes from 33 specimens were sequenced to confirm the tentatively assigned specimen taxa identity on the basis of morphological analyses. Subsequently, the legs of ticks were homogenized and analyzed by MALDI-TOF MS. A collection of reference mass spectra, based on the mass profiles of four individual ticks per species, was developed and deposited in the spectral database SARAMIS™. The ability of these superspectra (SSp.) to identify and reliably validate a set of ticks was demonstrated using the remaining individual 333 ticks. RESULTS: Ultimately, ten different tick species within the genera Amblyomma, Hyalomma, Rhipicephalus and Rhipicephalus (Boophilus) based on molecular COI typing and morphology were included into the study analysis. The robustness of the 12 distinct SSp. developed here proved to be very high, with 319 out of 333 ticks used for validation identified correctly at species level. Moreover, these novel SSp. allowed for diagnostic specificity of 99.7 %. The failure of species identification for 14 ticks was directly linked to low quality mass spectra, most likely due to poor specimen quality that was received in the laboratory before sample preparation. CONCLUSIONS: Our results are consistent with earlier studies demonstrating the potential of MALDI-TOF MS as a reliable tool for differentiating ticks originating from the field, especially females that are difficult to identify after blood feeding. This work provides further evidence of the utility of MALDI-TOF MS to identify morphologically and genetically highly similar tick species and indicates the potential of this tool for large-scale monitoring of tick populations, species distributions and host preferences.

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial.

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification.

Identification of phlebotomine sand flies using one MALDI-TOF MS reference database and two mass spectrometer systems.

BACKGROUND: Rapid, accurate and high-throughput identification of vector arthropods is of paramount importance in surveillance programmes that are becoming more common due to the changing geographic occurrence and extent of many arthropod-borne diseases. Protein profiling by MALDI-TOF mass spectrometry fulfils these requirements for identification, and reference databases have recently been established for several vector taxa, mostly with specimens from laboratory colonies. METHODS: We established and validated a reference database containing 20 phlebotomine sand fly (Diptera: Psychodidae, Phlebotominae) species by using specimens from colonies or field-collections that had been stored for various periods of time. RESULTS: Identical biomarker mass patterns ('superspectra') were obtained with colony- or field-derived specimens of the same species. In the validation study, high quality spectra (i.e. more than 30 evaluable masses) were obtained with all fresh insects from colonies, and with 55/59 insects deep-frozen (liquid nitrogen/-80 °C) for up to 25 years. In contrast, only 36/52 specimens stored in ethanol could be identified. This resulted in an overall sensitivity of 87 % (140/161); specificity was 100 %. Duration of storage impaired data counts in the high mass range, and thus cluster analyses of closely related specimens might reflect their storage conditions rather than phenotypic distinctness. A major drawback of MALDI-TOF MS is the restricted availability of in-house databases and the fact that mass spectrometers from 2 companies (Bruker, Shimadzu) are widely being used. We have analysed fingerprints of phlebotomine sand flies obtained by automatic routine procedure on a Bruker instrument by using our database and the software established on a Shimadzu system. The sensitivity with 312 specimens from 8 sand fly species from laboratory colonies when evaluating only high quality spectra was 98.3 %; the specificity was 100 %. The corresponding diagnostic values with 55 field-collected specimens from 4 species were 94.7 % and 97.4 %, respectively. CONCLUSIONS: A centralized high-quality database (created by expert taxonomists and experienced users of mass spectrometers) that is easily amenable to customer-oriented identification services is a highly desirable resource. As shown in the present work, spectra obtained from different specimens with different instruments can be analysed using a centralized database, which should be available in the near future via an online platform in a cost-efficient manner.

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

BACKGROUND: Conventionally, 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. RESULTS: We 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. CONCLUSIONS: Whole-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.