Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis.

BACKGROUND: Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. RESULTS: As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. CONCLUSIONS: In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

Molecular Typing of Burkholderia mallei Isolates from Equids with Glanders, India.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013-2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area-related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.

A Whole-Genome-Based Gene-by-Gene Typing System for Standardized High-Resolution Strain Typing of Bacillus anthracis.

Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.

A Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species.

Burkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (>95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay.

Serological and molecular investigation for brucellosis in swine in selected districts of Uganda.

Brucellosis is a notifiable zoonotic disease affecting livestock, humans, and wildlife in Uganda. Pigs can be infected with human pathogenic Brucella suis biovars 1 and 3 and can be a significant source of brucellosis for humans. Uganda has a rapidly growing pig population, and the pork consumption per capita is the highest in East Africa. The objective of this work was to determine the seroprevalence of brucellosis in Ugandan pigs. A cross-sectional serosurvey of pigs was conducted in three of the major pig-keeping districts in Uganda (Masaka (n = 381 samples), Mukono (n = 398), and Kamuli (n = 414)). In addition, pigs originating from these districts were sampled in the major pig abattoir in Kampala (n = 472). In total, 1665 serum samples were investigated by serological and molecular tests. Only three putative brucellosis-positive samples were detected serologically using indirect ELISA. These sera were found negative for Brucella antibodies by CFT; however, two had antibodies against Yersinia enterocolitica as determined by SAT. Presence of antibodies against Yersiniae was confirmed by Y. enterocolitica antibody-specific ELISA. The two Yersiniae ELISA-positive samples were brucellosis negative using real-time PCR. We tested additional 142 sera from the 1665 samples with real-time PCR. All tested negative. Under this type of production system, we expect a maximum B. suis prevalence of less than 1 % at 95 % confidence level, and therefore, the risk of acquiring brucellosis from the pigs or their products is negligible. However, pigs may harbor the zoonotic Y. enterocolitica. This is the first study to investigate the occurrence of brucellosis in pigs in Uganda and the first study to report Y. enterocolitica antibodies in swine in Uganda.

Raman spectroscopic detection and identification of Burkholderia mallei and Burkholderia pseudomallei in feedstuff.

Burkholderia mallei (the etiologic agent of glanders in equines and rarely humans) and Burkholderia pseudomallei, causing melioidosis in humans and animals, are designated category B biothreat agents. The intrinsically high resistance of both agents to many antibiotics, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Current methods to identify these organisms may require up to 1 week, as they rely on phenotypic characteristics and an extensive set of biochemical reactions. In this study, Raman microspectroscopy, a cultivation-independent typing technique for single bacterial cells with the potential for being a rapid point-of-care analysis system, is evaluated to identify and differentiate B. mallei and B. pseudomallei within hours. Here, not only broth-cultured microbes but also bacteria isolated out of pelleted animal feedstuff were taken into account. A database of Raman spectra allowed a calculation of classification functions, which were trained to differentiate Raman spectra of not only both pathogens but also of five further Burkholderia spp. and four species of the closely related genus Pseudomonas. The developed two-stage classification system comprising two support vector machine (SVM) classifiers was then challenged by a test set of 11 samples to simulate the case of a real-world-scenario, when "unknown samples" are to be identified. In the end, all test set samples were identified correctly, even if the contained bacterial strains were not incorporated in the database before or were isolated out of animal feedstuff. Specifically, the five test samples bearing B. mallei and B. pseudomallei were correctly identified on species level with accuracies between 93.9 and 98.7%. The sample analysis itself requires no biomass enrichment step prior to the analysis and can be performed under biosafety level 1 (BSL 1) conditions after inactivating the bacteria with formaldehyde.

Fate of Bacillus anthracis during production of laboratory-scale cream cheese and homemade-style yoghurt.

The viability of Bacillus anthracis during production and storage of cream cheese and yoghurt was evaluated. Experimental cheeses were manufactured from whole milk inoculated with a suspension of B. anthracis vegetative cells and spores at a final concentration of 10(4) cfu/ml. Lactic acid bacteria (LAB) and lab ferment were used to induce milk ripening and milk coagulation. The pH-value of the contaminated milk dropped below 4.5 within the first 6 h and the amount of LAB increased by approximately 2-logs. During cheese production and storage at 5-9 °C for 24 days no growth of B. anthracis was observed. The amount of vegetative cells and spores fluctuated by 1-log. Inoculation of whole milk with heat-treated spores at 10(4) cfu/ml resulted in a slight increase of vegetative cell counts during the first 6 h. This indicated that germination occurred, but replication of vegetative cells was still inhibited in the produced cheese. Incubation of cheeses at room temperature or heating after milk coagulation strongly reduced the amount of LAB but had no effect on the growth behaviour of B. anthracis. The vegetative cell and spore content remained steady at 10(4) cfu/100 mg. During yoghurt production the pH-value decreased within 5 h below 5 and growth of B. anthracis was inhibited throughout storage. A pH-value of 5 or less is likely a critical factor to control the growth of B. anthracis. However, spores remained viable in experimental cream cheeses and yoghurts and are a potential risk of infection.

Isolation of the highly pathogenic and zoonotic agent Burkholderia pseudomallei from a pet green Iguana in Prague, Czech Republic.

BACKGROUND: Melioidosis caused by Burkholderia (B.) pseudomallei is an endemic zoonotic disease mainly reported from northern Australia and Southeast Asia. In Europe, cases of human melioidosis have been reported only from patients travelling to endemic regions. Besides humans, B. pseudomallei has a very broad host range in domestic and wild animals. There are some reports about importation of B. pseudomallei-infected animals from endemic areas into Europe. The present report describes the first case of B. pseudomallei infection of a pet iguana in Europe. CASE PRESENTATION: In a 5-year-old pet Iguana iguana living in a private household in Prague, Czech Republic, B. pseudomallei was isolated from pus of an abscess. The isolate VB976100 was identified by Vitek®2, MALDI-TOF mass spectrometry and polymerase chain reaction as B. pseudomallei. The molecular typing resulted in multi-locus sequence type 436 hitherto, which has been found only once worldwide in a B. pseudomallei strain isolated in the USA and originating from Guatemala. The identification as internal transcribed spacer type G indicates a close relatedness to strains mainly isolated in the Western Hemisphere. These findings support the hypothesis that the iguana became infected in this region or in a breeding facility through contact to other infected animals. CONCLUSIONS: The present case highlights the risk of importation of the highly pathogenic and zoonotic B. pseudomallei into non-endemic regions through animal trade. Therefore, veterinarians treating animals from these areas and physicians examining patients owning such animals should include melioidosis in differential diagnosis whenever specific symptoms appear. Furthermore, veterinary authorities responsible for supervision of traders and pet shops should be aware of this risk of zoonotic transmission.

Development and validation of a chemiluminescent western blot assay for glanders (Burkholderia mallei) serodetection.

Glanders, caused by Burkholderia mallei, is a zoonotic disease of equids. Serologic testing for glanders is required by disease-free countries before international movement of equids. The World Organisation for Animal Health Terrestrial Manual recommends the complement fixation test (CFT) for clearance of individual animals for movement, but the CFT is prone to false-positive results. A colorimetric western blot (WB) assay was developed and validated to resolve false-positive CFT results; however, that assay is relatively time-consuming, and the interpretation is subjective. We present here a procedurally similar chemiluminescent WB assay that performs comparably to the validated colorimetric WB assay and offers noticeable benefits of decreased time-to-result and greater ease of interpretation.

European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

Seroprevalence and risk factors associated with brucellosis as a professional hazard in Pakistan.

The present study was conducted to determine the seroprevalence and identify risk factors associated with brucellosis in humans at high risk in the Potohar plateau of northeastern Pakistan. A total of 262 serum samples were collected from persons of different occupational groups: veterinary personnel, milkers, abattoir workers, livestock farmers, and others (drivers, security guards, housewives). Data related to gender, age, occupation, contact with animals, brucellosis-related symptoms, consumption of raw milk, and geographical region were collected. The Rose Bengal plate test and the serum agglutination test were performed to determine the seroprevalence of brucellosis. The overall seroprevalence was found to be 6.9% (95% confidence interval [CI]: 4.1, 10.6). Real-time polymerase chain reaction assay showed that all cases were affected by Brucella abortus. Individuals who consumed raw milk had higher odds of brucellosis seropositivity. This is the first report of human brucellosis related to B. abortus in high-risk professionals from Pakistan by the combined use of serological and molecular methods.

Raman spectroscopy as a potential tool for detection of Brucella spp. in milk.

Detection of Brucella, causing brucellosis, is very challenging, since the applied techniques are mostly time-demanding and not standardized. While the common detection system relies on the cultivation of the bacteria, further classical typing up to the biotype level is mostly based on phenotypic or genotypic characteristics. The results of genotyping do not always fit the existing taxonomy, and misidentifications between genetically closely related genera cannot be avoided. This situation gets even worse, when detection from complex matrices, such as milk, is necessary. For these reasons, the availability of a method that allows early and reliable identification of possible Brucella isolates for both clinical and epidemiological reasons would be extremely useful. We evaluated micro-Raman spectroscopy in combination with chemometric analysis to identify Brucella from agar plates and directly from milk: prior to these studies, the samples were inactivated via formaldehyde treatment to ensure a higher working safety. The single-cell Raman spectra of different Brucella, Escherichia, Ochrobactrum, Pseudomonas, and Yersinia spp. were measured to create two independent databases for detection in media and milk. Identification accuracies of 92% for Brucella from medium and 94% for Brucella from milk were obtained while analyzing the single-cell Raman spectra via support vector machine. Even the identification of the other genera yielded sufficient results, with accuracies of >90%. In summary, micro-Raman spectroscopy is a promising alternative for detecting Brucella. The measurements we performed at the single-cell level thus allow fast identification within a few hours without a demanding process for sample preparation.

Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing.

BACKGROUND: Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. RESULTS: A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. CONCLUSIONS: Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

Sequence-based detection and typing procedures for Burkholderia mallei: Assessment and prospects.

Although glanders has been eradicated in most of the developed world, the disease still persists in various countries such as Brazil, India, Pakistan, Bangladesh, Nepal, Iran, Bahrain, UAE and Turkey. It is one of the notifiable diseases listed by the World Organization for Animal Health. Occurrence of glanders imposes restriction on equestrian events and restricts equine movement, thus causing economic losses to equine industry. The genetic diversity and global distribution of the causing agent, Burkholderia (B.) mallei, have not been assessed in detail and are complicated by the high clonality of this organism. Among the identification and typing methods, PCR-based methods for distinguishing B. mallei from its close relative B. pseudomallei as well as genotyping using tandem repeat regions (MLVA) are established. The advent and continuous advancement of the sequencing techniques and the reconstruction of closed genomes enable the development of genome guided epidemiological tools. For achieving a higher genomic resolution, genotyping methods based on whole genome sequencing data can be employed, like genome-wide single nucleotide polymorphisms. One of the limitations in obtaining complete genomic sequences for further molecular characterization of B. mallei is its high GC content. In this review, we aim to provide an overview of the widely used detection and typing methods for B. mallei and illustrate gaps that still require development. The genomic features of Burkholderia, their high homology and clonality will be first described from a comparative genomics perspective. Then, the commonly used molecular detection (PCR systems) and typing systems (e.g., multilocus sequence typing, variable number of tandem repeat analysis) will be presented and put in perspective with recently developed genomic methods. Also, the increasing availability of B. mallei genomic sequences and evolution of the sequencing methods offers exciting prospects for further refinement of B. mallei typing, that could overcome the difficulties presently encountered with this particular bacterium.

Sequencing-Based Genotyping of Pakistani Burkholderia mallei Strains: A Useful Way for Investigating Glanders Outbreaks.

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.

Molecular Typing of Ukrainian Bacillus anthracis Strains by Combining Whole-Genome Sequencing Techniques.

Anthrax is a recurrent zoonosis in the Ukraine with outbreaks occurring repeatedly in certain areas. For determining whether several Bacillus anthracis genotypes are circulating in this region, four strains from various sources isolated from different regions of the Ukraine were investigated. By combining long- and short-read next-generation sequencing techniques, highly accurate genomes were reconstructed, enabling detailed in silico genotyping. Thus, the strains could be assigned to the Tsiankovskii subgroup of the "TransEurAsia" clade, which is commonly found in this region. Their high genetic similarity suggests that the four strains are members of the endemic population whose progenitor was once introduced in the Ukraine and bordering regions. This study provides information on B. anthracis strains from a region where there is little knowledge of the local population, thereby adding to the picture of global B. anthracis genotype distribution. We also emphasize the importance of surveillance and prevention methods regarding anthrax outbreaks, as other studies predicted a higher number of cases in the future due to global warming.

Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method.

Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller-Hinton without additives for the AST of B. melitensis.

Rapid identification of Burkholderia pseudomallei and Burkholderia mallei by fluorescence in situ hybridization (FISH) from culture and paraffin-embedded tissue samples.

We evaluated newly developed probes for rapid identification of Burkholderia (B.) pseudomallei and B. mallei and differentiation from B. thailandensis by fluorescence in situ hybridization (FISH). FISH correctly identified 100% of the tested B. pseudomallei (11), B. mallei (11), and B. thailandensis (1) strains, excluded 100% of all tested negative controls (61), and allowed demonstration of B. pseudomallei infection in a paraffin-embedded spleen tissue sample of an experimentally infected mouse.

Assessment of two isolation techniques for bacteria in milk towards their compatibility with Raman spectroscopy.

The identification of single microorganism in food samples by conventional plating techniques or molecular genetic methods requires a time consuming enrichment step. Raman spectroscopy in combination with a suitable extraction method however offers the possibility to rapidly identify bacteria on a single cell level. Here we evaluate the two well-known bacteria extraction methods from milk: "buoyant density centrifugation" and "enzymatic milk clearing" towards their recovery efficiency and their compatibility with Raman spectroscopy for a rapid identification of microorganisms in milk. The achieved recovery yields are slightly better compared to those which are already applied for food investigations, where a loss of one order of magnitude is usually reached. For example, buoyant density centrifugation allows collecting up to 35% of the milk-spiked microorganisms. To prove the suitability of the isolation techniques for use in combination with the spectroscopic approach, a small Raman database has been created by recording Raman spectra of well-known contaminants in dairy products. Two subspecies of Escherichia coli and three different Pseudomonas species, which were inoculated to UHT (ultra-high-temperature processed) milk and afterwards extracted by the two techniques mentioned above, were analysed. At a first glance, grave spectral artefacts caused by the matrix itself or especially by the extraction techniques were not obvious. But via chemometric analysis, it could be shown that these factors noticeably influence the identification rates: while the samples prepared via milk clearing did not provide sufficient identification results, buoyant density centrifugation allows an identification of the investigated species with an overall accuracy of 91% in combination with linear discriminant analysis.

Use of a Western blot technique for the serodiagnosis of glanders.

BACKGROUND: The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. RESULTS: The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. CONCLUSIONS: The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.