Ornithobacterium rhinotracheale: MALDI-TOF MS and Whole Genome Sequencing Confirm That Serotypes K, L and M Deviate from Well-Known Reference Strains and Numerous Field Isolates.

Ornithobacterium rhinotracheale is one of the most important bacterial agents of respiratory diseases in poultry. For correct identification and characterization of this fastidious bacterium, reliable diagnostic tools are essential. Still, phenotypic tests are used to identify O. rhinotracheale and serotyping is the most common method for characterization, despite known drawbacks and disadvantages such as divergent results, cross-reactivity between strains, or the non-typeability of strains. The intention of the present study was to evaluate MALDI-TOF MS and whole genome sequencing for the identification and characterization of O. rhinotracheale. For this purpose, a selection of 59 well-defined reference strains and 47 field strains derived from outbreaks on Austrian turkey farms were investigated by MALDI-TOF MS. The field strains originated from different geographical areas in Austria with some of the isolates derived from multiple outbreaks on farms within a year, or recurrent outbreaks over several years. MALDI-TOF MS proved a suitable method for identification of O. rhinotracheale to genus or species level except for 3 strains representing serotypes M, K and F. Phylogenetic analysis showed that most strains grouped within one cluster even though they were comprised of different serotypes, while serotypes F, K, and M clearly formed a different cluster. All field isolates from turkey farms clustered together, independent of the origin of the isolates, e.g., geographical area, multiple outbreaks within a year or recurrent outbreaks over several years. Whole genome sequencing of serotype M, K and F strains confirmed the extraordinary status and deviation from known fully-sequenced strains due to a lack of sequence similarity. This was further confirmed by alignments of single genes (16S-RNA and rpoB) and multilocus sequence typing although the demarcation was less obvious. Altogether, the results indicate that these three serotypes belong to a different species than O. rhinotracheale, and might even be members of multiple new species.

Sequencing of five poultry strains elucidates phylogenetic relationships and divergence in virulence genes in Morganella morganii.

BACKGROUND: M. morganii is a bacterium frequently associated with urinary infections in humans. While many human strains are sequenced, only the genomes of few poultry strains are available. Here, we performed a detailed characterization of five highly resistant Morganella morganii strains isolated in association with Escherichia coli from diseased domestic Austrian poultry flocks, namely geese, turkeys and chicken layers. Additionally, we sequenced the genomes of these strains by NGS and analyzed phylogenetic clustering, resistance and virulence genes in the context of host-specificity. RESULTS: Two strains were identified to be Extended Spectrum Beta Lactamase (ESBL) and one as AmpC beta-lactamases (AMP-C) phenotype, while two were ESBL negative. By integrating the genome sequences of these five poultry strains with all the available M. morganii genomes, we constructed a phylogenetic tree that clearly separates the Morganella genus into two clusters (M1 and M2), which approximately reflect the proposed subspecies classification (morganii and sibonii). Additionally, we found no association between phylogenetic structure and host, suggesting interspecies transmission. All five poultry strains contained genes for resistance to aminocoumarins, beta-lactams, colistin, elfamycins, fluoroquinolones, phenicol, rifampin and tetracycline. A comparative genomics analysis of virulence genes showed acquisition of novel virulence genes involved in secretion system and adherence in cluster M2. We showed that some of these genes were acquired by horizontal gene transfer from closely related Morganellaceae species and propose that novel virulence genes could be responsible for expansion of tissue tropism in M. morganii. Finally, we detected variability in copy number and high sequence divergence in toxin genes and provided evidence for positive selection in insecticidal toxins genes, likely reflecting host-related adaptations. CONCLUSIONS: In summary, this study describes i) the first isolation and characterization of M. morganii from goose and turkey, ii) a large-scale genetic analysis of M. morganii and an attempt to generate a global picture of the M. morganii intraspecific phylogenetic structure.

Identification of biofilm hotspots in a meat processing environment: Detection of spoilage bacteria in multi-species biofilms.

Biofilms are comprised of microorganisms embedded in a self-produced matrix that normally adhere to a surface. In the food processing environment they are suggested to be a source of contamination leading to food spoilage or the transmission of food-borne pathogens. To date, research has mainly focused on the presence of (biofilm-forming) bacteria within food processing environments, without measuring the associated biofilm matrix components. Here, we assessed the presence of biofilms within a meat processing environment, processing pork, poultry and beef, by the detection of microorganisms and at least two biofilm matrix components. Sampling included 47 food contact surfaces and 61 non-food contact surfaces from eleven rooms within an Austrian meat processing plant, either during operation or after cleaning and disinfection. The 108 samples were analysed for the presence of microorganisms by cultivation and targeted quantitative real-time PCR based on 16S rRNA. Furthermore, the presence of the major matrix components carbohydrates, extracellular DNA and proteins was evaluated. Overall, we identified ten biofilm hotspots, among them seven of which were sampled during operation and three after cleaning and disinfection. Five biofilms were detected on food contact surfaces (cutters and associated equipment and a screw conveyor) and five on non-food contact surfaces (drains and water hoses) resulting in 9.3 % of the sites being classified as biofilm positive. From these biofilm positive samples, we cultivated bacteria of 29 different genera. The most prevalent bacteria belonged to the genera Brochothrix (present in 80 % of biofilms), Pseudomonas and Psychrobacter (isolated from 70 % biofilms). From each biofilm we isolated bacteria from four to twelve different genera, indicating the presence of multi-species biofilms. This work ultimately determined the presence of multi-species biofilms within the meat processing environment, thereby identifying various sources of potential contamination. Especially the identification of biofilms in water hoses and associated parts highlights the need of a frequent monitoring at these sites. The knowledge gained about the presence and composition of biofilms (i.e. chemical and microbiological) will help to prevent and reduce biofilm formation within food processing environments.

MALDI-TOF mass spectrometry confirms difficulties in separating species of the Avibacterium genus.

In the present study a well-characterized strain collection (n = 33) of Avibacterium species was investigated by matrix-assisted laser desorption ionization-time-of flight mass spectrometry (MALDI-TOF MS). The robustness of the currently available reference database (Bruker Biotyper 3.0) was tested to determine the degree of identification of these strains. Reproducible signal patterns were obtained from all strains. However, identification of most strains was only possible at genus level. Furthermore, two strains could not be identified by this method. Based on their protein spectra profiles, a MALDI main spectra dendrogram was created to determine their relationship. Most strains were closely related-for example, 26 strains formed cluster 1 including the type strains of Avibacterium volantium, Avibacterium gallinarum, Avibacterium endocarditidis and Avibacterium avium-while Avibacterium paragallinarum biovars 1 and 2 formed cluster 2 and, finally, strain 55000 remained on its own. The present MALDI-TOF MS results confirm recent findings that only certain isolates of Av. paragallinarum represent a well-defined species within the genus Avibacterium, making a taxonomic revision essential. To improve identification of Avibacterium at species level by MALDI-TOF MS, relevant reference strains were included in the newly created database and results are presented. In conclusion, Av. paragallinarum can be identified by MALDI/Biotyper and not the other species of the genus.

Assessing pathogenicity of Gallibacterium anatis in a natural infection model: the respiratory and reproductive tracts of chickens are targets for bacterial colonization.

Two separate bird trials were performed to establish a reliable route of infection for Gallibacterium anatis in chickens, comparing intranasal (i.n.) and intravenous (i.v.) applications. Additionally, three mutually divergent isolates from three geographical locations, as shown by MALDI-TOF-MS and partial rpoB gene sequence analysis, were compared. In the first trial, birds were infected with one of the selected isolates by the i.v. or i.n. route. Subsequently, birds were killed 3, 12 and 24 h post infection following i.v. infection while at 3, 7 and 10 days post infection (dpi) in the case of i.n. infection along with birds of the control group. As a result, i.n. infection showed prominent and consistent bacterial tissue distribution in different organs persisting until 10 dpi, which was a striking contrast to the i.v. infection route. Likewise, histopathology revealed mild to severe tracheal lesions following i.n. infection. The second trial was set up to confirm both the achieved results and the robustness of i.n. infection but with an extended observation period, until 28 dpi In agreement with the preceding trial, identical results for bacteriological and histopathological examinations were obtained with persistency of bacteria until 28 dpi Comparing the three different isolates from Mexico, China and Austria, the Mexican isolate showed a somewhat higher pathogenicity than the other strains. Consequently, pathogenesis of G. anatis strains was studied in chickens elucidating i.n. infection as the most reliable route characterized by a long-lasting bacteraemia, targeting the respiratory and reproductive tract.

MALDI-TOF mass spectrometry confirms clonal lineages of Gallibacterium anatis between chicken flocks.

Gallibacterium anatis has been suggested to have a causal role in the salpingitis/peritonitis complex in chickens, beside its isolation from the respiratory tract. As G. anatis strains from different flocks were compared by MALDI-TOF MS proteomic phenotyping it could be demonstrated that in most flocks one clonal lineage was present. This finding is also reflected by data achieved when isolates from different organs within a bird generally belong to the same clonal lineage. In addition, it was also confirmed by two independent experiments, as well as, two MALDI instruments. Altogether, proteomic phenotyping indicates that the nature of a chicken flock may play a certain role in particular clone type selection of G. anatis.

Identification of Gallibacterium species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry evaluated by multilocus sequence analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) whole-cell fingerprinting was used for characterization of 66 reference strains of Gallibacterium. The 4 recognised Gallibacterium species and Gallibacterium genomospecies 1 yielded reproducible and unique mass spectrum profiles, which were confirmed with Bruker Biotyper reference database version 3. The reproducibility of MALDI-TOF MS results were evaluated varying the age and storage of the cultures investigated. Reliable species identification was possible for up to 8 days of storage at 4°C and less reliable if the bacteria were stored at room temperature (20°C). However, if the strains were grown longer than 48h at 37°C under microaerobic atmosphere, poor identification results were obtained, due to changes in protein profile. The MALDI-TOF MS results of all 66 strains demonstrated 87.9% concordance with results based upon biochemical/physiological characterization. In addition, diversities outlined by MALDI-TOF MS were verified by sequencing the rpoB (n=43), 16S rRNA (n=28), infB (n=14), and recN (n=14) genes (multilocus sequence analysis, MLSA). In addition, discrepancies were observed between some of the genes sequenced. Results obtained demonstrated that MALDI-TOF MS fingerprinting represents a fast and reliable method for identification and differentiation of the 4 recognised Gallibacterium species and possible a fifth species Gallibacterium genomospecies 1, with applications in clinical diagnostics.

Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ionization time of flight mass spectrometry analysis.

Rapid and reliable identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, has become increasingly important, since many of them are now recognized as human and/or animal pathogens. Matrix-associated laser desorption/ionization-time of flight (MALDI-TOF) MS has been shown to be a rapid and sensitive method for characterization of micro-organisms. In this study, we therefore established a reference database of selected Arcobacter, Helicobacter and Campylobacter species for MALDI-TOF MS identification. Besides the species with significance as food-borne pathogens - Arcobacter butzleri, Helicobacter pullorum, Campylobacter jejuni and Campylobacter coli - several other members of these genera were included in the reference library to determine the species specificity of the designed MALDI Biotyper reference database library. Strains that made up the reference database library were grown on Columbia agar, and yielded reproducible and unique mass spectra profiles, which were compared with the Bruker Biotyper database, version 2. The database was used to identify 144 clinical isolates using whole spectral profiles. Furthermore, reproducibility of MALDI-TOF MS results was evaluated with respect to age and/or storage of bacteria and different growth media. It was found that correct identification could be obtained even if the bacteria were stored at room temperature or at 4 degrees C up to 9 days before being tested. In addition, bacteria were correctly identified when grown on Campylosel agar; however, they were not when grown on modified charcoal cefoperazone deoxycholate agar. These results indicate that MALDI-TOF MS fingerprinting is a fast and reliable method for the identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, with applications in clinical diagnostics.