Molecular epidemiologic analyses of bacteria of the genera Pasteurella and Mannheimia to establish valid diagnostic tools based on Multiplex polymerase chain reactions

Deckblatt-Impressum Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung Literaturübersicht Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Anhang Danksagung Lebenslauf SelbständigkeitserklärungIn der vorliegenden experimentellen Arbeit wurde ein großes Kollektiv von Stämmen der wichtigen Tierseuchenerreger aus der Gattung Pasteurella- und Mannheimia unterschiedli-cher Wirtstiere und Erkrankungskomplexe mittels phäno- und genotypischer Methoden cha-rakterisiert. Ausgangspunkt der molekularen Analysen war es, die etablierten unzureichenden phänotypischen Identifizierungstests durch unzweifelhafte molekulare Methoden zu ersetzen. Somit sollte die Basis für zukünftige molekulare epidemiologische Arbeiten geschaffen wer-den, mit denen endlich das Vorkommen und die pathogenetische Bedeutung dieser wichtigen Tierseuchenerreger valide analysiert werden kann Unseres Wissens wurde mit 289 P. multocida ssp.-, 25 P. sensu stricto- und 104 M. species-Isolaten erstmals ein derart umfangreiches repräsentatives Kollektiv an Stämmen untersucht, wodurch die Etablierung aussagefähiger diagnostischer Werkzeuge gelang. Pasteurella ssp. und spp. wurden mittels PCR und DNS-DNS-Hybridisierung auf das Vor-handensein von 16 virulenzassoziierten Genen [psl, ompH, oma87, ptf, pfha, nanB, nanH, toxA, tbpA, tonB, hgbA, hgbB, sodA, sodC, iga, escv], einem Spezies-spezifischen- [kmt] und fünf Kapsel- Genen [capA, capB, capD, capE, capF], M. haemolytica und sonstige Mannhei-mia- Spezies auf das Vorhandensein von 12 virulenzassoziierten Genen [lktA, pomA, gcp, trBA, tonB, irp, sodA, sodC, escv, iga, adh] hin untersucht. Bei den P. multocida ssp.-Isolaten gelang fast regelmäßig der Nachweis von Genen, die für äußere Membranproteine (psl, ompH, oma87), Kolonisationsfaktoren (ptf, nanB, nanH), Superoxid-Dismutasen (sodC, sodA) und Eisenakquirierungssysteme (tonB, hgbA, hgbB) kodieren. Mit einer niedrigeren Prävalenz kamen dagegen das offensichtlich für Isolate ruminaler Wirte spezifische Eisenakqurierungs-Gen tbpA (31,5 %), das für ein filamentöses Hämagglutinin kodierende pfha (37,0 %) sowie das Dermonekrotoxin-kodierende toxA (12,5 %) vor. P. sensu stricto- Isolate besaßen z. T. keines der untersuchten Gene bzw. je nach Spezies ompH, oma87, psl und tonB und konnten auf diese Weise genotypisch von P. multo-cida ssp. differenziert werden. 86,9 % der P. multocida ssp. wiesen eine Kombination der drei Adhäsionsgene pft, nanH und nanB auf; 39 % dieser Stämme besaßen zusätzlich pfhA, das auffallend selten bei Kapseltyp D-Stämmen (4,8 %) vorkam. Die gleichzeitige Expression von Fimbrien, Sialidasen sowie dem Hämagglutinin in P. multocida ssp. stellt eine wichtige Voraussetzung für eine erfolgreiche Etablierung des Bakteriums in verschiedenen Wirtsorga-nismen bzw. deren Geweben dar und könnte ein Grund für das außerordentlich breite Wirts- spektrum dieser Pasteurellen sein. Auch die häufig vorkommende Kombination der Eisenak-quirierungs-Gene hgbA, hgbB und tonB (76,5 %), sowie zusätzlich von tbpA (20,4 %) sollte P. multocida ssp. einen entscheidenden Wachstumsvorteil in unterschiedlichen Wirtstieren ermöglichen. Diese Ergebnisse geben somit entscheidende Anhaltspunkte für zukünftige ex-perimentelle Untersuchungen zur Bedeutung von Adhäsions- und Eisenakquierierungsgenen in der Pathogenese unterschiedlicher Pasteurella-bedingter Erkrankungskomplexe. Weiterhin sollte die Eignung der entsprechenden Genprodukte als mögliche Kandidaten für Impfstoffe geprüft werden. Von den 89 M. haemolytica-Stämmen konnten aufgrund der Biotypisierung 66 der Biogruppe 1 zugeordnet werden, während 23 Stämme anderen Biogruppen angehörten bzw. sechs ein derart abweichendes Biochemieprofil aufwiesen, dass sie nur durch anschließende Sequenzanalyse ihrer 16S-rRNS-Gene als M. varigena (5) bzw. M. ruminalis (1) identifiziert werden konnten. Mit Ausnahme der Gene lktA, pomA und trBA, die fast regelmäßig bei den M. haemolytica-Stämmen vorkamen, war bei anderen untersuchten Faktoren, wie tonB, irp, gcp und sodA eine deutlich höhere Prävalenz bei den pathogenetisch bedeutenderen Biogrup-pe 1-Stämmen im Vergleich zu denen anderer Biogruppen vorhanden. Auch diese neuen Da-ten zu den erst kürzlich identifizierten Genen geben Anlass, deren Bedeutung für den Infek¬tions¬prozess zukünftig stärker zu überprüfen. Auf der Grundlage dieser Analyse-Daten konnten zwei Multiplex-PCRen entwickelt werden, die einen Verzicht auf den konventionellen und teilweise unzuverlässigen Nachweis von P. multocida ssp. und M. haemolytica auf der Basis ihrer phänotypischen Merkmalsaus-prägungen ermöglichen. Aufgrund der in dieser Arbeit bestätigten regionalen Bedeutung von P. multocida ssp. multocida Kapseltyp A- und D-Stämmen sowie der tierseuchenrechtlichen Relevanz des toxA-Gens wurde der Nachweis dieser drei Gene neben dem für das M. haemolytica lktA und pomA in die erste Multiplex-PCR zur Differenzierung der beiden bakteriellen Spezies integriert, die beispielsweise im Rahmen der Enzootischen Bronchomop-neumonie oft als Mischkultur angetroffen werden. Eine zweite Multiplex-PCR, die in Zukunft für umfassende epidemiologische Studien zur Bestimmung des Besiedlungsmusters von P. multocida ssp.-Isolaten bei unterschiedlichen Wirtstieren angewendet werden kann, erlaubt den gleichzeitigen Nachweis der Gene oma87, ptf, pfha, capF, tbpA, hgbA, hgbB, tonB, nanH und nanB. Dies schien erforderlich, da die Prävalenzdaten der bei P. multocida ssp.-Isolaten untersuchten Gene auf eine sich möglicherweise ändernde Epidemiologie bei diesen Stämmen hinweisen. So ergab sich beispielsweise die Frage nach einem horizontalen Gen-Transfer des phagenkodierten toxA nicht nur zwischen Stämmen mit einzelnen Kapseltypen, sondern auch zwischen Stämmen unterschiedlicher Wirtstiere, da toxA entgegen früherer Angaben vermehrt bei Kapseltyp A-Stämmen nicht nur beim Schwein sondern auch beim Wiederkäuer nachge-wiesen werden konnte. Des weiteren deutete die Tatsache, dass bei Isolaten, die von an Rhini-tis Atrophicans erkrankten Schweinen isoliert worden waren, z. T. kein toxA nachgewiesen werden konnte, darauf hin, dass die Tiere mit mehreren P. multocida ssp.-Stämmen infiziert sein können. Dieser epidemiologisch wichtige Befund blieb jedoch aufgrund der begrenzten Möglichkeiten der konventionellen Diagnostik zur Differenzierung der Isolate bislang uner-kannt. Ein weiteres interessantes Ergebnis war der Nachweis von P. multocida-Kapeltyp F-Stämmen bei Rindern und Katzen. Laut ensprechender Literatur kommen diese Stämme nur beim Geflügel vor. Die vorliegenden Daten deuten darauf hin, dass auch hier ein Wandel in der Adaptation von Kapseltypen an bestimmte Wirtstiere erfolgt ist, dessen Ausmaß in Folge¬unter¬suchungen mittels der hier etablierten Multiplex-PCRen eingeschätzt werden kann. Abschließend wurden phylogenetische Untersuchungen auf Basis der DNS-Sequenz¬analyse des variablen ompH von Pasteurellen durchgeführt. Aufgrund des Nachweises von ompH nicht nur bei P. multocida ssp. sondern auch bei verschiedenen P. sensu stricto-Isolaten wurden diese Analysen an 12 Pasteurella-Isolaten vorgenommen. Obwohl es sich bei dem Genprodukt um ein äußeres Membranprotein handelt, das aufgrund seiner Lokalisation einem ständigen Selektionsdruck unterliegt, spiegeln die DNS- Sequenzdaten jene phylogenetischen und taxono¬mischen Einordnungen von Pasteurellaceae-Spezies wider, die auf der Basis der 16S-rRNS erhoben wurden. Die Untersuchung stellt insofern eine sinnvolle Ergänzung ande-rer Methoden zur phylogenetischen Einordnung einzelner Pasteurella-Spezies dar und führt zu einem besseren Verständnis der evolutionären Entwicklung dieser Mikroorganismen.In this experimental thesis, an extensive collection of the important veterinary pathogenic genera Pasteurella and Mannheimia from various hosts and disease complexes were charac-terized with pheno- and genotypical methods. The molecular analyses were necessary to re-place the currently established ambiguous phenotypical identification methods with valid, unambiguous molecular tools. With these tools, future molecular epidemiological studies are enabled, which finally will lead to a valid analysis of the actual prevalence as well as the pathogenic role of these important veterinary pathogens. To our knowledge, with a total of 289 P. multocida ssp.-, 25 P. sensu stricto- as well as 104 M. species, for the first time such a large collection has been analysed, facilitating the establishment of valid diagnostical tools. Via PCR and DNA-DNA-hybridization, Pasteurella ssp. and spp. were investigated for the presence of 16 virulence associated genes [psl, ompH, oma87, ptf, pfha, nanB, nanH, toxA, tbpA, tonB, hgbA, hgbB, sodA, sodC, iga, escv], one spe-cies-specific [kmt] and five capsule genes [capA, capB, capD, capE, capF], while M. haemolytica and additional Mannheimia species were screened for the presence of 12 viru-lence associated genes [lktA, pomA, gcp, trBA, tonB, irp, sodA, sodC, escv, iga, adh]. Nearly all P. multocida ssp. strains showed a combination of genes, coding for outer membrane proteins (psl, ompH, oma87), colonisation factors (ptf, nanB, nanH), superoxide dismutase (sodC, sodA) as well as iron acquisition systems (tonB, hgbA, hgbB). In contrast, tbpA (31.5 %), coding for an iron acquisition system which seems to be specific for strains from ruminants, pfha (37.0 %), coding for filamentous hemagglutinine, as well as toxA (12.5 %), coding for dermonecrotoxin, were found to be less prevalent. Isolates of P. sensu stricto possessed mostly none of these genes, or, depending on the species investigated, ompH, oma87, psl and tonB only. Thus, genotypically these species could be easily differenti-ated from P. multocida ssp.. 86.9 % of the P. multocida ssp. harboured a combination of three genes encoding adhesins, namely ptf, nanH and nanB. In addition, 39.0 % of those strains reacted positive for pfhA, a gene which was rarely seen in strains of capsule type D (4.8 %). The simultaneous expression of fimbriae, sialidases and hemagglutinine in P. multocida ssp. is a fundamental requirement for successful infection of the host and host tissues, respec-tively, and may be one reason for the wide host spectrum of Pasteurella. Additionally, the finding that genes hgbA, hgbB and tonB, involved in iron acquisition, were found in combina-tion in 76.5 % of the strains, as well as the presence of tbpA in 20.4 % of them should theo-retically give P. multocida ssp. an advantage in multiplication in different hosts. Thus, the data generated also give relevant information for future experimental studies, investigating the role of adhesins as well as iron acquisition systems during pathogenesis of different Pas-teurella-associated disease complexes. Furthermore, the suitability of those gene products as vaccine candidates should be investigated. Biotyping of 89 M. haemolytica strains classified 66 strains as members of biogroup 1, while 23 strains belonged to other biogroups. Six strains gave such a variable biochemical profile, that only 16S-rRNA gene sequencing led to their classification as M. varigena (5) and M. ruminalis (1), respectively. With the exception of lktA, pomA and trBA, which were nearly regularly identified in all strains of M. haemolytica, the other genes under investigation (like tonB, irp, gcp, and sodA) were found with higher prevalence in biogroup 1 strains, which are prone to be more virulent. As the latter genes were identified only recently, these new data also give reason to more detailed analyses of their possible role during the infectious process. Based on these extensive analyses, two Multiplex PCRs were established successfully. With these diagnostic tools, the currently used conventional and partly ambiguous phenotypi-cal methods for the identification of P. multocida ssp. und M. haemolytica can be replaced. Due to the confirmation of the regional importance of P. multocida ssp. multocida capsule type A and D strains, as well as the notification requirement of toxA positive strains, primers for the detection of those three genes as well lktA and pomA for the identification of M. haemolytica were integrated into one Multiplex-PCR. As both species may be present in mixed culture in biological samples, for example in cases of enzootic bronchopneumonia, we reasoned a differentiation to be useful. The establishment of a second Multiplex-PCR, ena-bling the simultaneous identification of oma87, ptf, pfha, capF, tbpA, hgbA, hgbB, tonB, nanH as well as nanB, will be useful in future epidemiological studies to identify colonization of various host species with P. multocida ssp.. Especially the recognition of the epidemiologi-cal shift, which seems to have taken place concerning P. multocida ssp., we sensed this tool to be highly useful. Indeed, the question of a horizontal gene transfer of the phage encoded toxA not only between strains of a distinct capsule type, but also between strains isolated from various host animals is thrilling. In contrast to current knowledge, our data revealed that toxA is also found in strains of capsule type A and not only in strains isolated from swine but also from bovines. Furthermore, the fact that several strains were isolated from pigs suffering from atrophic rhinitis, but being negative for toxA, indicates that these animals may be infected with various different P. multocida strains. This epidemiological highly important finding was unknown so far, most probably due to limitations of the currently used conventional diagnos-tic tools. Another interesting finding was the fact, that P. multocida strains of capsule type F could be isolated from cattle as well as from cats. Due to the literature, such strains can only be found in poultry. In contrast, our results indicate a change in the adaption of strains with certain capsules to distinctive hosts. This epidemiological important finding should be inves- tigated more detailed in the future a perspective enabled by the Multiplex- PCRs established in the current work. Finally, phylogenetic analyses were performed by sequencing the variable outer mem-brane protein gene ompH of Pasteurella. Due to the finding that ompH was present not only in P. multocida ssp., but in various P. sensu stricto isolates additionally, a total of 12 strains were included in this investigation. Although the gene product, an outer membrane protein, is under selective pressure, gene sequence analyses are in accordance with results of phyloge-netical and taxonomical studies of Pasteurellaceae species, based on 16S-rRNA sequence data. Thus, analysing ompH is a useful complementation of phylogenetic investigations of Pasteurella species, clarifying the evolutionary development of these microorganisms

Extended-Spectrum Beta-Lactamases Producing E. coli in Wildlife, yet Another Form of Environmental Pollution?

Wildlife is normally not exposed to clinically used antimicrobial agents but can acquire antimicrobial resistant bacteria through contact with humans, domesticated animals and the environment, where water polluted with feces seems to be the most important vector. Escherichia coli, an ubiquitous commensal bacterial species colonizing the intestinal tract of mammals and birds, is also found in the environment. Extended-spectrum beta-lactamases producing E. coli (ESBL-E. coli) represent a major problem in human and veterinary medicine, particular in nosocomial infections. Additionally an onset of community-acquired ESBL-E. coli infections and an emergence in livestock farming has been observed in recent years, suggesting a successful transmission as well as persistence of ESBL-E. coli strains outside clinical settings. Another parallel worldwide phenomenon is the spread of ESBL-E. coli into the environment beyond human and domesticated animal populations, and this seems to be directly influenced by antibiotic practice. This might be a collateral consequence of the community-onset of ESBL-E. coli infections but can result (a) in a subsequent colonization of wild animal populations which can turn into an infectious source or even a reservoir of ESBL-E. coli, (b) in a contribution of wildlife to the spread and transmission of ESBL-E. coli into fragile environmental niches, (c) in new putative infection cycles between wildlife, domesticated animals and humans, and (d) in problems in the medical treatment of wildlife. This review aims to summarize the current knowledge on ESBL-E. coli in wildlife, in turn underlining the need for more large scale investigations, in particular sentinel studies to monitor the impact of multiresistant bacteria on wildlife

Adhesive threads of extraintestinal pathogenic Escherichia coli

The ability to adhere to host surfaces is by far the most vital step in the successful colonization by microbial pathogens. Colonization begins with the attachment of the bacterium to receptors expressed by cells forming the lining of the mucosa. Long hair like extracellular appendages called fimbriae, produced by most Gram-negative pathogens, mediate specific attachment to the epithelial cell surface. Associated with the fimbriae is a protein called an adhesin, which directs high-affinity binding to specific cell surface components. In the last couple of years, an enormous amount of research has been undertaken that deals with understanding how bacterial pathogens adhere to host cells. E. coli in all probability is one of the best studied free-living organisms. A group of E. coli called Extraintestinal pathogenic E. coli (ExPEC) including both human and animal pathogens like Uropathogenic E. coli (UPEC), Newborn meningitic E. coli (NMEC) and Avian pathogenic E. coli (APEC), have been found to harbour many fimbriae including Type 1 fimbriae, P fimbriae, curli fibres, S fimbriae, F1C fimbriae, Dr fimbriae, afimbrial adhesins, temperature-sensitive haemagglutinin and many novel adhesin gene clusters that have not yet been characterized. Each of these adhesins is unique due to the recognition of an adhesin-specific receptor, though as a group these adhesins share common genomic organization. A newly identified putative adhesin temporarily termed ExPEC Adhesin I, encoded by gene yqi, has been recently found to play a significant role in the pathogenesis of APEC infection, thus making it an interesting candidate for future research. The aim of this review is to describe the role of ExPEC adhesins during extraintestinal infections known till date, and to suggest the idea of investigating their potential role in the colonization of the host gut which is said to be a reservoir for ExPEC

Molecular study on Pasteurella multocida and Mannheimia granulomatis from Kenyan Camels (Camelus dromedarius)

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

Does Reductive Evolution Correlate with Habitat and Pathotype?

IbeA (invasion of brain endothelium), which is located on a genomic island termed GimA, is involved in the pathogenesis of several extraintestinal pathogenic E. coli (ExPEC) pathotypes, including newborn meningitic E. coli (NMEC) and avian pathogenic E. coli (APEC). To unravel the phylogeny of GimA and to investigate its island character, the putative insertion locus of GimA was determined via Long Range PCR and DNA-DNA hybridization in 410 E. coli isolates, including APEC, NMEC, uropathogenic (UPEC), septicemia-associated E. coli (SEPEC), and human and animal fecal isolates as well as in 72 strains of the E. coli reference (ECOR) collection. In addition to a complete GimA (~20.3 kb) and a locus lacking GimA we found a third pattern containing a 342 bp remnant of GimA in this strain collection. The presence of GimA was almost exclusively detected in strains belonging to phylogenetic group B2. In addition, the complete GimA was significantly more frequent in APEC and NMEC strains while the GimA remnant showed a higher association with UPEC strains. A detailed analysis of the ibeA sequences revealed the phylogeny of this gene to be consistent with that obtained by Multi Locus Sequence Typing of the strains. Although common criteria for genomic islands are partially fulfilled, GimA rather seems to be an ancestral part of phylogenetic group B2, and it would therefore be more appropriate to term this genomic region GimA locus instead of genomic island. The existence of two other patterns reflects a genomic rearrangement in a reductive evolution-like manner

Genome Sequence of AvianEscherichia coliStrain IHIT25637, an Extraintestinal PathogenicE. coliStrain of ST131 Encoding Colistin Resistance Determinant MCR-1

Sequence type 131 (ST131) is one of the predominant Escherichia coli lineages among extraintestinal pathogenic E. coli (ExPEC) that causes a variety of diseases in humans and animals and frequently shows multidrug resistance. Here, we report the first genome sequence of an ST131-ExPEC strain from poultry carrying the plasmid-encoded colistin resistance gene mcr-1

ESBL-plasmids carrying toxin-antitoxin systems can be “cured” of wild-type Escherichia coli using a heat technique

Plasmid-encoded extended-spectrum beta-lactamase (ESBL)-enzymes are frequently produced by Escherichia coli. Several ESBL-plasmids contain genes for toxin- antitoxin (TA) systems, which assure the maintenance of plasmids in bacteria and prevent the cells from "post-segregational killing". These systems limit options to "cure" plasmids of ESBL-wild-type strains due to the death of the bacterial cells. A helpful tool to understand the role of ESBL-plasmids in the dissemination of pandemic multi-resistant E. coli are ESBL- plasmid-"cured"-variants (PCVs) and their comparison to ESBL-wild-type strains. The purpose of this study was to construct PCVs of ESBL-wild-type E. coli strains despite the presence of genes for TA systems. Using enhanced temperatures and brain-heart-infusion broth it was possible to construct viable PCVs of wild-type ESBL-E. coli strains. The occurrence of TA system- genes including hok/sok, srnB/C, vagC/D, pemI/K on ESBL-plasmids of replicon types FIA or FIB was demonstrated by bioinformatic analyses. The loss of the plasmid and the genetic identity of PCV and corresponding wild-type strain was confirmed via different methods including plasmid-profile-analysis, pulsed- field gel electrophoresis and bioinformatics using generated whole genome data of the strains. This short report describes the successful construction of viable PCVs of ESBL-wild-type E. coli strains. The results are hence surprising due to the fact that all "cured" ESBL-plasmids contained at least one complete toxin-antitoxin system, whose loss would normally mean the death of bacterial cells

Genome sequence of OXA-23 producing Acinetobacter baumannii IHIT7853, a carbapenem-resistant strain from a cat belonging to international clone IC1

Background: Multidrug resistance in Acinetobacter baumannii has dramatically increased in recent years worldwide. Thus, last-line antibiotics like carbapenems are increasingly being used which in turn further augments selection pressure for resistant strains. Resistance to carbapenems in A. baumannii is frequently mediated by carbapenemases, particularly OXA-23 and OXA-58. Carbapenemase-producing bacteria are mainly described in human patients and the intestinal tract represents a common source for such pathogens. In this study, we sequenced and analyzed the genome of A. baumannii IHIT7853, a carbapenem-resistant, OXA-23 producing strain isolated from cystitis in a cat in 2000 in Germany. Results: Phylogenetic analysis revealed that IHIT7853 belonged to the globally distributed international clone IC1 and MLST type ST1/ST231 (Pasteur/Oxford MLST scheme). A phylogenetic tree based on the maximum common genome of 18 A. baumannii isolates placed IHIT7853 close to human clinical isolates, such as the multidrug-resistant (MDR) outbreak strain AYE that was isolated from a patient with pneumonia and cystitis in 2001 in France. The OXA-23 plasmid sequence could be determined as 53,995 bp in size, possessing resistance genes strA and strB in addition to bla OXA-23. Conclusions: The analysis of the genome of IHIT7853 reveals that companion animals carry MDR A. baumannii that resemble relevant clonal lineages involved in severe infections in humans. As urinary tract infections are often caused by bacteria that reside in the intestinal tract, future studies should unveil, if the animal gut serves as a source for MDR A. baumannii

Phylogenetic diversity, antimicrobial susceptibility and virulence gene profiles of Brachyspira hyodysenteriae isolates from pigs in Germany

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1_RS02885, BHWA1_RS09085, BHWA1_RS04705, and BHWA1_RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s–2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1_RS02885, BHWA1_RS09085, and BHWA1_RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates

A combinational approach of multilocus sequence typing and other molecular typing methods in unravelling the epidemiology of Erysipelothrix rhusiopathiae strains from poultry and mammals

Erysipelothrix rhusiopathiae infections re-emerged as a matter of great concern particularly in the poultry industry. In contrast to porcine isolates, molecular epidemiological traits of avian E. rhusiopathiae isolates are less well known. Thus, we aimed to (i) develop a multilocus sequence typing (MLST) scheme for E. rhusiopathiae, (ii) study the congruence of strain grouping based on pulsed-field gel electrophoresis (PFGE) and MLST, (iii) determine the diversity of the dominant immunogenic protein SpaA, and (iv) examine the distribution of genes putatively linked with virulence among field isolates from poultry (120), swine (24) and other hosts (21), including humans (3). Using seven housekeeping genes for MLST analysis we determined 72 sequence types (STs) among 165 isolates. This indicated an overall high diversity, though 34.5% of all isolates belonged to a single predominant ST-complex, STC9, which grouped strains from birds and mammals, including humans, together. PFGE revealed 58 different clusters and congruence with the sequence-based MLST-method was not common. Based on polymorphisms in the N-terminal hyper-variable region of SpaA the isolates were classified into five groups, which followed the phylogenetic background of the strains. More than 90% of the isolates harboured all 16 putative virulence genes tested and only intI, encoding an internalin-like protein, showed infrequent distribution. MLST data determined E. rhusiopathiae as weakly clonal species with limited host specificity. A common evolutionary origin of isolates as well as shared SpaA variants and virulence genotypes obtained from avian and mammalian hosts indicates common reservoirs, pathogenic pathways and immunogenic properties of the pathogen