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Additional file 1: Fig. S1. Distribution of 1,198 isolates and enrichment analysis: A) The plot represents the proportion of E. coli isolates isolated from hosts in four countries. The number above each plot indicates the total number of isolates per host. B) Phylogroups enriched with different hosts (Pearson residual > 0 represents positive correlation indicating the enrichment of certain host-species in distinct clusters at p-value < 2.2e−16). Fig. S2. Core genome phylogeny of E. coli isolates from our collection (n=1,198) and reference strains (n=146) from the ECOR collection, RefSeq and cryptic clades annotated with their phylogroups (phylogroups were determined by ClermonTyper v. 1.3). Fig S3. Core-genome phylogeny and Accessory-genome clustering of 1,198 E. coli isolates showing different BAPS clusters. Each BAPS on accessory genome clustering consist of the same strains as on the core-genome phylogeny. Fig. S4. Minimum-spanning tree of MLST profiles of 1,198 E. coli isolates. Left: the number of isolates constituting an ST; Right: the proportion of hosts in each ST. Fig. S5. Genetic surroundings of the human-associated nan gene cluster in the genomes of all isolates in which it was identified.

A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration.

A total of 114 Staphylococcus isolates from various infections of companion animals, including 43 feline Staphylococcus aureus, 19 canine S. aureus, 11 feline Staphylococcus pseudintermedius and 41 canine S. pseudintermedius were investigated for (i) their susceptibility to 24 antimicrobial agents and three combinations of antimicrobial agents by broth microdilution following CLSI recommendations and (ii) the corresponding resistance genes. In addition, the isolates were tested for their susceptibility to the four biocides benzalkonium chloride, chlorhexidine, polyhexanide and octenidine by a recently developed biocide susceptibility testing protocol. Penicillin resistance via blaZ was the dominant resistance property in all four groups of isolates ranging between 76.7 and 90.9%. About one quarter of the isolates (25.4%) proved to be methicillin-resistant and carried the genes mecA or mecC. Macrolide resistance was the second most prevalent resistance property (27.2%) and all isolates harbored the resistance genes erm(A), erm(B), erm(C), erm(T) or msr(A), alone or in combinations. Fluoroquinolone resistance was detected in 21.1% of all isolates tested, whereas tetracycline resistance via tet(K) and/or tet(M) occurred in 19.3% of the isolates. Resistance to last resort antimicrobial agents in human medicine was seen only in single isolates, if at all. The minimal inhibitory concentrations (MICs) of the four biocides showed unimodal distributions and were very similar for the four groups of staphylococci. Because of the large number of (multi)resistant isolates, antimicrobial susceptibility testing of feline and canine S. aureus and S. pseudintermedius isolates is highly recommended before the start of an antimicrobial chemotherapy. Moreover, no hints towards the development of biocide resistance were detected.

Background: Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated. Methods: In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences. Results: Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels. Conclusions: The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.

Im Rahmen der vorliegenden Studie wurden 382 porcine P. multocida aus dem Respirationstrakt erkrankter sowie klinisch gesunder Schweine mit Hilfe molekularbiologischer Techniken untersucht, um Informationen zur Phylogenie der Spezies und zur Diversität und Verteilung virulenzassoziierter Gene in porcinen P. multocida aus Deutschland zu erhalten. Die Ergebnisse der Ribotypisierung (13 Ribotypmuster) und der MLST (11 Sequenztypen) zeigten eine geringe Diversität und somit auch eine begrenzte Anzahl an Phylotypen in der aktuellen P. multocida-Population in deutschen Schweinebeständen. Bestimmte Muster virulenzassoziierter Gene waren mit bestimmten Phylotypen assoziiert. Die fehlende Assoziation zwischen Genotypen und vorberichtlichen Informationen zum Gesundheitsstatus weisen auf die Bedeutung von wirts- und umweltassoziierten Faktoren für die Ausbildung porciner Pasteurellosen hin. Die Ergebnisse von MLST und Ribotypisierung zeigten signifikante Assoziationen zueinander. Die wenigen Unterschiede können durch die Schwierigkeiten der Validierung der Ribotypisierungsergebnisse oder die Nutzung suboptimaler Gensequenzen begründet liegen. Dieser wichtige Punkt sollte durch zukünftige, umfassende Untersuchung jener Stämme, für die inkongruente Ergebnisse ermittelt wurden, beleuchtet werden. Die in früheren Studien beschriebenen Assoziationen der Kapseltypen an die Habitate oberer Respirationstrakt (capD) und unterer Respirationstrakt (capA) konnten auch in der vorliegenden Arbeit nachgewiesen werden. Signifikanzen in der Verteilung der Kapseltypen A (assoziiert an Ribotypmuster IA-7 und IIA-1) und D (assoziiert an IA-1) sowie einzelner virulenzassoziierter Gene (pfhaB, hgbB) konnten mittels Ribotypisierung gezeigt werden. Das Verteilungsmuster der Kapseltypen vor dem phylogenetischen Hintergrund deutet auf eine homoplastische Evolution von Stämmen des Kapseltyps A, während Stämme des Kapseltyps D nur einem ST-Komplex angehören, was einen klonalen Hintergrund nahelegt. Auch unter Berücksichtigung der in der MLST-Datenbank hinterlegten Daten (Stand: November 2010) weiterer P. multocida bestätigt sich die Tendenz der Verteilung der Kapseltypen. Die Gene exbB/tonB, oma87, ptfA, nanB, nanH, hgbA und tbpA waren in allen resp. Keinem (tbpA) untersuchten Isolat(en) nachweisbar. Das Virulenzgenmuster der porcinen P. multocida unterschied sich in den Genen toxA (3,4 % der Isolate positiv), pfhaB (20,4 % positiv) sowie hgbB (84,8 % positiv). Nach unserem Wissensstand konnte durch die MLST toxA-positiver Stämme erstmals ein Hinweis auf eine klonale Verbreitung dieser Stämme erbracht werden, unabhängig von der phagenkodierten Natur des toxA. Diese Daten sprechen gegen eine horizontale Verbreitung des phagenkodierten Gens. Sowohl die Ergebnisse der Ribotypisierung als auch der MLST zeigten eine negative Assoziation zwischen pfhaB- und hgbB-positiven Isolaten. Die in der vorliegenden Studie erhobenen Daten ermöglichen eine gezieltere Auswahl zukünftiger Impfstämme anhand des Phylotyps. Darüberhinaus favorisieren wir, neben der Berücksichtigung der lange bekannten Kapseltypen und des Dermonekrotoxins, welches in Form von Toxinfragmenten oder Toxoiden verwendet wird, die Aufnahme des PfhaB in die Vakzinen, da dieses Protein Ähnlichkeiten zum Protein Fha von B. pertussis aufweist. Hintergrund ist die Tatsache, dass Fha als ein effektives Immunogen beim Menschen bekannt ist, wodurch künftige Studien zur Wirksamkeit PfhaB-haltiger Vakzinen beim Schwein angeregt werden., In this study 382 porcine P. multocida isolates from the respiratory tract of diseased and clinically healthy swine were characterized using molecular techniques to unravel the phylogeny of the species as well as the diversity and distribution of virulence associated genes in porcine P. multocida from Germany. The results of ribotyping (13 ribotype patterns) and MLST (11 sequence types) revealed a limited diversity, thus a limited number of phylotypes of the current P. multocida population in german pig livestock. Certain virulence gene patterns were associated with certain phylotypes. The missing association between distinctive genotypes and clinical outcome indicate the high influence of host-associated and environmental-associated factors for the development of pasteurellosis in pigs. MLST and ribotyping showed significant associations. Minor differences are assumably based on the difficulties to validate band patterns gained by ribotyping or a suboptimal choosing of gene sequences used in the MLST scheme. This important issue should be addressed in future studies by more deeply analysing those strains that gave incongruent results. Former studies described associations between capsular types to the upper (capA) resp. lower (capD) respiratory tract, which could be confirmed in this work. In addition, significant associations between capsular type A-positive isolates and ribotypes IA-7 and IIA-1 and capsular type D and ribotype IA-1 as well as specific virulence associated genes were found. The distribution of capsular types in the phylogenetic background points towards a homoplastic evolution of capA-positive isolates, while isolates of capsular type D cluster in only one ST-complex, indicating a clonal background. This tendency is supported by the data currently (november 2010) present in the online available MLST-database. The genes exbB/tonB, oma87, ptfA, nanB, nanH, hgbA, and tbpA were present in all resp. none (tbpA) isolates tested. Differences were only obvious in the distribution of toxA (3,4 %), pfhaB (20,4 %), and hgbB (84,8 %). Due to our knowledge, the investigation of the toxApositive isolates using MLST for the first time led to the conclusion that these isolates spread clonally, despite the phage-coded nature of the toxA gene. These data argue against a horizontal spread of toxA. Furthermore, the results of ribotyping as well as the results of MLST both revealed a negative association between pfhaB- and hgbB-positive strains. The data gained in this study give the opportunity for a more sound selection of new vaccine strains by considering the phylotype of the P. multocida strains. Thus, besides considering the well known capsular type and toxin, used in the form of toxin-fragment or toxoid, weenvision an inclusion of PfhaB into the vaccine, as this protein has similarities to the Fha protein of B. pertussis. As Fha is known to be an effective immunogen in humans, this fact is fostering future studies to proof the efficacy of vaccines containing PfhaB to prevent porcine pasteurellosis.