219 results on '"Torsten Semmler"'
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2. Occurrence of Mobile Colistin Resistance Genes mcr-1–mcr-10 including Novel mcr Gene Variants in Different Pathotypes of Porcine Escherichia coli Isolates Collected in Germany from 2000 to 2021
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Lisa Göpel, Ellen Prenger-Berninghoff, Silver A. Wolf, Torsten Semmler, Rolf Bauerfeind, and Christa Ewers
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Escherichia coli ,pathotype ,mobile colistin resistance ,mcr-1 ,mcr-4 ,mcr-5 ,Microbiology ,QR1-502 - Abstract
In the European Union, gastrointestinal disease in pigs is the main indication for the use of colistin, but large-scale epidemiologic data concerning the frequency of mobile colistin resistance (mcr) genes in pig-associated pathotypes of Escherichia coli (E. coli) are lacking. Multiplex polymerase chain reactions were used to detect virulence-associated genes (VAGs) and mcr-1–mcr-10 genes in 10,573 porcine E. coli isolates collected in Germany from July 2000 to December 2021. Whole genome sequencing was performed on 220 representative mcr-positive E. coli strains. The total frequency of mcr genes was 10.2%, the most frequent being mcr-1 (8.4%) and mcr-4 (1.6%). All other mcr genes were rarely identified (mcr-2, mcr-3, mcr-5) or absent (mcr-6 to mcr-10). The highest frequencies of mcr genes were found in enterotoxigenic and shiga toxin-encoding E. coli (ETEC/STEC hybrid) and in edema disease E. coli (EDEC) strains (21.9% and 17.7%, respectively). We report three novel mcr variants, mcr-1.36, mcr-4.8, and mcr-5.5. In 39 attaching and effacing E. coli (AEEC) isolates analyzed in our study, the eae subtype β1 was the most prevalent (71.8%). Constant surveillance for the presence of mcr genes in various sectors should consider the different frequency of mcr-positive isolates in pathogenic E. coli.
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- 2023
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3. Nanopore adaptive sampling effectively enriches bacterial plasmids
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Jens-Uwe Ulrich, Lennard Epping, Tanja Pilz, Birgit Walther, Kerstin Stingl, Torsten Semmler, and Bernhard Y. Renard
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adaptive sampling ,readuntil ,nanopore sequencing ,plasmid ,bacteria ,enrichment ,Microbiology ,QR1-502 - Abstract
ABSTRACTBacterial plasmids play a major role in the spread of antibiotic resistance genes. However, their characterization via DNA sequencing suffers from the low abundance of plasmid DNA in those samples. Although sample preparation methods can enrich the proportion of plasmid DNA before sequencing, these methods are expensive and laborious, and they might introduce a bias by enriching only for specific plasmid DNA sequences. Nanopore adaptive sampling could overcome these issues by rejecting uninteresting DNA molecules during the sequencing process. In this study, we assess the application of adaptive sampling for the enrichment of low-abundant plasmids in known bacterial isolates using two different adaptive sampling tools. We show that a significant enrichment can be achieved even on expired flow cells. By applying adaptive sampling, we also improve the quality of de novo plasmid assemblies and reduce the sequencing time. However, our experiments also highlight issues with adaptive sampling if target and non-target sequences span similar regions.IMPORTANCEAntimicrobial resistance causes millions of deaths every year. Mobile genetic elements like bacterial plasmids are key drivers for the dissemination of antimicrobial resistance genes. This makes the characterization of plasmids via DNA sequencing an important tool for clinical microbiologists. Since plasmids are often underrepresented in bacterial samples, plasmid sequencing can be challenging and laborious. To accelerate the sequencing process, we evaluate nanopore adaptive sampling as an in silico method for the enrichment of low-abundant plasmids. Our results show the potential of this cost-efficient method for future plasmid research but also indicate issues that arise from using reference sequences.
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- 2024
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4. Genomic insights into virulence, antimicrobial resistance, and adaptation acumen of Escherichia coli isolated from an urban environment
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Poorvi Saini, Viraj Bandsode, Anuradha Singh, Suresh Kumar Mendem, Torsten Semmler, Munirul Alam, and Niyaz Ahmed
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Escherichia coli ,genome analysis ,virulence ,antimicrobial resistance ,environment ,Microbiology ,QR1-502 - Abstract
ABSTRACT Populations of common commensal bacteria such as Escherichia coli undergo genetic changes by the acquisition of certain virulence and antimicrobial resistance (AMR) encoding genetic elements leading to the emergence of pathogenic strains capable of surviving in the previously uninhabited or protected niches. These bacteria are also reported to be prevalent in the environment where they survive by adopting various recombination strategies to counter microflora of the soil and water, under constant selection pressure(s). In this study, we performed molecular characterization, phenotypic AMR analysis, and whole genome sequencing (WGS) of E. coli (n = 37) isolated from soil and surface water representing the urban and peri-urban areas. The primary aim of this study was to understand the genetic architecture and pathogenic acumen exhibited by environmental E. coli. WGS-based analysis entailing resistome and virulome profiling indicated the presence of various virulence (adherence, iron uptake, and toxins) and AMR encoding genes, including blaNDM-5 in the environmental isolates. A majority of our isolates belonged to phylogroup B1 (73%). A few isolates in our collection were of sequence type(s) (ST) 58 and 224 that could have emerged recently as clonal lineages and might pose risk of infection/transmission. Mobile genetic elements (MGEs) such as plasmids (predominantly) of the IncF family, prophages, pipolins, and insertion elements such as IS1 and IS5 were also observed to exist, which may presumably aid in the propagation of genes encoding resistance against antimicrobial drugs. The observed high prevalence of MGEs associated with multidrug resistance in pathogenic E. coli isolates belonging to the phylogroup B1 underscores the need for extended surveillance to keep track of and prevent the transmission of the bacterium to certain vulnerable human and animal populations.IMPORTANCEEvolutionary patterns of E. coli bacteria convey that they evolve into highly pathogenic forms by acquiring fitness advantages, such as AMR, and various virulence factors through the horizontal gene transfer (HGT)-mediated acquisition of MGEs. However, limited research on the genetic profiles of environmental E. coli, particularly from India, hinders our understanding of their transition to pathogenic forms and impedes the adoption of a comprehensive approach to address the connection between environmentally dwelling E. coli populations and human and veterinary public health. This study focuses on high-resolution genomic analysis of the environmental E. coli isolates aiming to understand the genetic similarities and differences among isolates from different environmental niches and uncover the survival strategies employed by these bacteria to thrive in their surroundings. Our approach involved molecular characterization of environmental samples using PCR-based DNA fingerprinting and subsequent WGS analysis. This multidisciplinary approach is likely to provide valuable insights into the understanding of any potential spill-over to human and animal populations and locales. Investigating these environmental isolates has significant potential for developing epidemiological strategies against transmission and understanding niche-specific evolutionary patterns.
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- 2024
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5. Unraveling the evolutionary dynamics of toxin-antitoxin systems in diverse genetic lineages of Escherichia coli including the high-risk clonal complexes
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Anuradha Singh, Aditya Kumar Lankapalli, Suresh Kumar Mendem, Torsten Semmler, and Niyaz Ahmed
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Escherichia coli ,toxin-antitoxin (TA) systems ,sequence type (ST) ,antimicrobial resistance ,virulence ,adaptation ,Microbiology ,QR1-502 - Abstract
ABSTRACTEscherichia coli is a highly versatile microorganism with a unique ability to survive and persist in varied niches of the human and animal hosts and the environment. While commensal strains of E. coli play a crucial role in preserving and maintaining the balance and function of their community within the gut microflora, pathogenic strains are often implicated in a wide range of infections and outbreaks, posing a serious threat to public health systems. With the increasing burden of highly virulent and antimicrobial-resistant E. coli infections, it is imperative to understand the host-defense mechanisms of bacteria from all possible dimensions. Mobile genetic elements (MGE)-mediated acquisition of genetic material, chromosomal reduction, and genome optimization are three important events that play a significant role in bacterial evolution and enable them to survive in diverse environmental niches. Toxin-antitoxin (TA) systems are genetic elements that help in the maintenance of MGEs and are often associated with stress response phenotypes, including antimicrobial resistance. In this study, large-scale comparative genomics of 950 genomes spanning 19 different sequence types (STs) (eight phylogroups) revealed ST-wide prevalence patterns of TA systems with a median of 23 toxin groups per strain. Our analyses revealed significant genomic reduction in the members of phylogroup B2 (ST131, ST95, ST73, ST12, and ST127) and phylogroup C (ST410) as evident from a diminished toxin repertoire amidst abundant orphan antitoxins. Moreover, our observations also enabled crucial insights into the copy number of toxin groups, the genetic organization of TA operons, and their association with other genetic coordinates (antimicrobial resistance encoding genes/virulence genes/mobile genetic elements). By unraveling the association of the genetic coordinates/STs with the toxin groups, this study significantly boosts our understanding of the functional implications of TA systems in different evolutionary contexts entailing pathogenic Escherichia.IMPORTANCELarge-scale genomic studies of E. coli provide an invaluable opportunity to understand how genomic fine-tuning contributes to the transition of bacterial lifestyle from being commensals to mutualists or pathogens. Within this context, through machine learning-based studies, it appears that TA systems play an important role in the classification of high-risk clonal lineages and could be attributed to their epidemiological success. Due to these profound indications and assumptions, we attempted to provide unique insights into the ordered world of TA systems at the population level by investigating the diversity and evolutionary patterns of TA genes across 19 different STs of E. coli. Further in-depth analysis of ST-specific TA structures and associated genetic coordinates holds the potential to elucidate the functional implications of TA systems in bacterial cell survival and persistence, by and large.
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- 2024
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6. Genome-wide association reveals host-specific genomic traits in Escherichia coli
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Sumeet K. Tiwari, Boas C. L. van der Putten, Thilo M. Fuchs, Trung N. Vinh, Martin Bootsma, Rik Oldenkamp, Roberto La Ragione, Sebastien Matamoros, Ngo T. Hoa, Christian Berens, Joy Leng, Julio Álvarez, Marta Ferrandis-Vila, Jenny M. Ritchie, Angelika Fruth, Stefan Schwarz, Lucas Domínguez, María Ugarte-Ruiz, Astrid Bethe, Charlotte Huber, Vanessa Johanns, Ivonne Stamm, Lothar H. Wieler, Christa Ewers, Amanda Fivian-Hughes, Herbert Schmidt, Christian Menge, Torsten Semmler, and Constance Schultsz
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Escherichia coli ,GWAS ,Host-specificity ,Sialic acid ,Biology (General) ,QH301-705.5 - Abstract
Abstract Background Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. Results We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. Conclusions This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.
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- 2023
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7. Repeated Occurrence of Mobile Colistin Resistance Gene-Carrying Plasmids in Pathogenic Escherichia coli from German Pig Farms
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Lisa Göpel, Ellen Prenger-Berninghoff, Silver A. Wolf, Torsten Semmler, Rolf Bauerfeind, and Christa Ewers
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Escherichia coli ,ETEC ,EDEC ,mobile colistin resistance ,mcr-1 ,plasmid ,Biology (General) ,QH301-705.5 - Abstract
The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time.
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- 2024
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8. Antibiotic prophylaxis and hospitalization of horses subjected to median laparotomy: gut microbiota trajectories and abundance increase of Escherichia
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Anne Kauter, Julian Brombach, Antina Lübke-Becker, Dania Kannapin, Corinna Bang, Sören Franzenburg, Sabita D. Stoeckle, Alexander Mellmann, Natalie Scherff, Robin Köck, Sebastian Guenther, Lothar H. Wieler, Heidrun Gehlen, Torsten Semmler, Silver A. Wolf, and Birgit Walther
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horse ,microbiome ,gastrointestinal tract ,microbiota ,16S rRNA gene sequencing ,hospitalization ,Microbiology ,QR1-502 - Abstract
IntroductionHorse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum β-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited.MethodsWe collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1–V2 gene sequencing.ResultsWe included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1.ConclusionColic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses.
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- 2023
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9. ESBL-Type and AmpC-Type Beta-Lactamases in Third Generation Cephalosporin-Resistant Enterobacterales Isolated from Animal Feces in Madagascar
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Ulrich Schotte, Julian Ehlers, Johanna Nieter, Raphaël Rakotozandrindrainy, Silver A. Wolf, Torsten Semmler, Hagen Frickmann, Sven Poppert, and Christa Ewers
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cephalosporin resistance ,fosfomycin ,epidemiology ,feces ,animals ,Madagascar ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
Third generation cephalosporin-resistant (3GCR) Enterobacterales are known to be prevalent in Madagascar, with high colonization or infection rates in particular in Madagascan patients. Extended spectrum beta-lactamases (ESBLs) have been reported to be the predominant underlying resistance mechanism in human isolates. So far, little is known on antimicrobial resistance and its molecular determinants in Enterobacterales and other bacteria causing enteric colonization of Madagascan wild animals. To address this topic, swabs from 49 animal stool droppings were collected in the Madagascan Tsimanapesotsa National Park and assessed by cultural growth of bacterial microorganisms on elective media. In addition to 7 Acinetobacter spp., a total of 31 Enterobacterales growing on elective agar for Enterobacterales could be isolated and subjected to whole genome sequencing. Enterobacter spp. was the most frequently isolated genus, and AmpC-type beta-lactamases were the quantitatively dominating molecular resistance mechanism. In contrast, the blaCTX-M-15 gene, which has repeatedly been associated with 3GC-resistance in Madagascan Enterobacterales from humans, was detected in a single Escherichia coli isolate only. The identification of the fosfomycin-resistance gene fosA in a high proportion of isolates is concerning, as fosfomycin is increasingly used to treat infections caused by multidrug-resistant bacteria. In conclusion, the proof-of-principle assessment indicated a high colonization rate of resistant bacteria in stool droppings of Madagascan wild animals with a particular focus on 3GCR Enterobacterales. Future studies should confirm these preliminary results in a more systematic way and assess the molecular relationship of animal and human isolates to identify potential routes of transmission.
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- 2024
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10. Using unique ORFan genes as strain-specific identifiers for Escherichia coli
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Marta Ferrandis-Vila, Sumeet K. Tiwari, Svenja Mamerow, Torsten Semmler, HECTOR consortium, Christian Menge, and Christian Berens
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ORFan gene ,Strain identification ,Multiplex PCR ,qPCR ,E. coli ,Host-specificity ,Microbiology ,QR1-502 - Abstract
Abstract Background Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. Results Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. Conclusions The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.
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- 2022
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11. Genotypic Characterization of Uropathogenic Escherichia coli from Companion Animals: Predominance of ST372 in Dogs and Human-Related ST73 in Cats
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Sophie Aurich, Silver Anthony Wolf, Ellen Prenger-Berninghoff, Lakshmipriya Thrukonda, Torsten Semmler, and Christa Ewers
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ExPEC ,UPEC ,dog ,cat ,antimicrobial resistance genes ,phylogroup B2 ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Extraintestinal pathogenic Escherichia coli (ExPEC) account for over 80% and 60% of bacterial urinary tract infections (UTIs) in humans and animals, respectively. As shared uropathogenic E. coli (UPEC) strains have been previously reported among humans and pets, our study aimed to characterize E. coli lineages among UTI isolates from dogs and cats and to assess their overlaps with human UPEC lineages. We analysed 315 non-duplicate E. coli isolates from the UT of dogs (198) and cats (117) collected in central Germany in 2019 and 2020 utilizing whole genome sequencing and in silico methods. Phylogroup B2 (77.8%), dog-associated sequence type (ST) 372 (18.1%), and human-associated ST73 (16.6%), were predominant. Other STs included ST12 (8.6%), ST141 (5.1%), ST127 (4.8%), and ST131 (3.5%). Among these, 58.4% were assigned to the ExPEC group and 51.1% to the UPEC group based on their virulence associated gene (VAG) profile (ExPEC, presence of ≥VAGs: papAH and/or papC, sfa/focG, afaD/draBC, kpsMTII, and iutA; UPEC, additionally cnf1 or hlyD). Extended-spectrum cephalosporin (ESC) resistance mediated by extended-spectrum β-lactamases (ESBL) and AmpC-β-lactamase was identified in 1.9% of the isolates, along with one carbapenemase-producing isolate and one isolate carrying a mcr gene. Low occurrence of ESC-resistant or multidrug-resistant (MDR) isolates (2.9%) in the two most frequently detected STs implies that E. coli isolated from UTIs of companion animals are to a lesser extent associated with resistance, but possess virulence-associated genes enabling efficient UT colonization and carriage. Detection of human-related pandemic lineages suggests interspecies transmission and underscores the importance of monitoring companion animals.
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- 2023
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12. Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients
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Astrid Lewin, Elisabeth Kamal, Torsten Semmler, Katja Winter, Sandra Kaiser, Hubert Schäfer, Lei Mao, Patience Eschenhagen, Claudia Grehn, Jennifer Bender, and Carsten Schwarz
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mycobacterium abscessus ,mycobacteroides abscessus ,nontuberculous mycobacteria ,lung infection ,cystic fibrosis ,genomics ,population structure ,gene mutations ,evolution ,transmission ,Infectious and parasitic diseases ,RC109-216 - Abstract
Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.
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- 2021
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13. Occurrence of mcr-1 and mcr-2 colistin resistance genes in porcine Escherichia coli isolates (2010–2020) and genomic characterization of mcr-2-positive E. coli
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Christa Ewers, Lisa Göpel, Ellen Prenger-Berninghoff, Torsten Semmler, Katharina Kerner, and Rolf Bauerfeind
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mobile colistin resistance ,mcr-2 ,Escherichia coli ,swine ,pathotype ,plasmid ,Microbiology ,QR1-502 - Abstract
IntroductionThe global emergence of plasmid-mediated colistin resistance is threatening the efficacy of colistin as one of the last treatment options against multi-drug resistant Gram-negative bacteria. To date, ten mcr-genes (mcr-1 to mcr-10) were reported. While mcr-1 has disseminated globally, the occurrence of mcr-2 was reported scarcely.Methods and resultsWe determined the occurrence of mcr-1 and mcr-2 genes among Escherichia coli isolates from swine and performed detailed genomic characterization of mcr-2-positive strains. In the years 2010-2017, 7,614 porcine E. coli isolates were obtained from fecal swine samples in Europe and isolates carrying at least one of the virulence associated genes predicting Shiga toxin producing E. coli (STEC), enterotoxigenic E. coli (ETEC) or enteropathogenic E. coli (EPEC) were stored. 793 (10.4%) of these isolates carried the mcr-1 gene. Of 1,477 additional E. coli isolates obtained from sheep blood agar containing 4 mg/L colistin between 2018 and 2020, 36 (2.4%) isolates were mcr-1-positive. In contrast to mcr-1, the mcr-2 gene occurred at a very low frequency (0.13%) among the overall 9,091 isolates. Most mcr-2-positive isolates originated from Belgium (n = 9), one from Spain and two from Germany. They were obtained from six different farms and revealed multilocus sequence types ST10, ST29, ST93, ST100, ST3057 and ST5786. While the originally described mcr-2.1 was predominant, we also detected a new mcr-2 variant in two isolates from Belgium, which was termed mcr-2.8. MCR-2 isolates were mostly classified as ETEC or ETEC-like, while one isolate from Spain represented an atypical enteropathogenic E. coli (aEPEC; eae+). The ST29-aEPEC isolate carried mcr-2 on the chromosome. Another eight isolates carried their mcr-2 gene on IncX4 plasmids that resembled the pKP37-BE MCR-2 plasmid originally described in Belgium in 2015. Three ST100 E. coli isolates from a single farm in Belgium carried the mcr-2.1 gene on a 47-kb self-transmissible IncP type plasmid of a new IncP-1 clade.DiscussionThis is the first report of mcr-2 genes in E. coli isolates from Germany. The detection of a new mcr-2 allele and a novel plasmid backbone suggests the presence of so far undetected mcr-2 variants and mobilizable vehicles.
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- 2022
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14. How to survive pig farming: Mechanism of SCCmec element deletion and metabolic stress adaptation in livestock-associated MRSA
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Charlotte Huber, Silver A. Wolf, Wilma Ziebuhr, Mark A. Holmes, Julia Assmann, Antina Lübke-Becker, Andrea Thürmer, Torsten Semmler, Julian Brombach, Astrid Bethe, Markus Bischoff, Lothar H. Wieler, Lennard Epping, and Birgit Walther
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methicillin resistant Staphylococcus aureus ,livestock associated ,SCCmec ,transcriptome analysis ,recombination ,deletion ,Microbiology ,QR1-502 - Abstract
Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly different compared to the unchallenged controls. We concluded that, from an evolutionary perspective, bacteria may save energy by incidentally dismissing a fully replicative SCCmec element in contrast to the induction of ccr genes on a population scale. Since the genomic SCCmec integration site is a hot-spot of recombination, occasional losses of elements of 16 kb size may restore capacities for the uptake of foreign genetic material. Subsequent spread of resistance, on the other hand, might depend on the autonomous replication machinery of the deleted SCCmec elements that probably enhance chances for reintegration of SCCmec into susceptible genomes by mere multiplication.
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- 2022
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15. Transmission pathways of campylobacter spp. at broiler farms and their environment in Brandenburg, Germany
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Benjamin Reichelt, Vanessa Szott, Lennard Epping, Torsten Semmler, Roswitha Merle, Uwe Roesler, and Anika Friese
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campylobacter ,environment ,transmission ,broiler ,WGS ,Microbiology ,QR1-502 - Abstract
Broiler meat is widely known as an important source of foodborne Campylobacter jejuni and Campylobacter coli infections in humans. In this study, we thoroughly investigated transmission pathways that may contribute to possible Campylobacter contamination inside and outside broiler houses. For this purpose we carried out a comprehensive longitudinal sampling approach, using a semi-quantitative cultivation method to identify and quantify transmissions and reservoirs of Campylobacter spp.. Three german broiler farms in Brandenburg and their surrounding areas were intensively sampled, from April 2018 until September 2020. Consecutive fattening cycles and intervening downtimes after cleaning and disinfection were systematically sampled in summer and winter. To display the potential phylogeny of barn and environmental isolates, whole genome sequencing (WGS) and bioinformatic analyses were performed. Results obtained in this study showed very high Campylobacter prevalence in 51/76 pooled feces (67.1%) and 49/76 boot swabs (64.5%). Average counts between 6.4 to 8.36 log10MPN/g were detected in pooled feces. In addition, levels of 4.7 and 4.1 log10MPN/g were detected in boot swabs and litter, respectively. Samples from the barn interior showed mean Campyloacter values in swabs from drinkers 2.6 log10MPN/g, walls 2.0 log10MPN/g, troughs 1.7 log10MPN/g, boards 1.6 log10MPN/g, ventilations 0.9 log10MPN/g and 0.7 log10MPN/g for air samples. However, Campylobacter was detected only in 7/456 (1.5%) of the environmental samples (water bodies, puddles or water-filled wheel tracks; average of 0.6 log10MPN/g). Furthermore, WGS showed recurring Campylobacter genotypes over several consecutive fattening periods, indicating that Campylobacter genotypes persist in the environment during downtime periods. However, after cleaning and disinfection of the barns, we were unable to identify potential sources in the broiler houses. Interestingly, alternating Campylobacter genotypes were observed after each fattening period, also indicating sources of contamination from the wider environment outside the farm. Therefore, the results of this study suggest that a potential risk of Campylobacter transmission may originate from present environmental sources (litter and water reservoirs). However, the sources of Campylobacter transmission may vary depending on the operation and farm environmental conditions.
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- 2022
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16. Genome-wide insights into population structure and host specificity of Campylobacter jejuni
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Lennard Epping, Birgit Walther, Rosario M. Piro, Marie-Theres Knüver, Charlotte Huber, Andrea Thürmer, Antje Flieger, Angelika Fruth, Nicol Janecko, Lothar H. Wieler, Kerstin Stingl, and Torsten Semmler
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Medicine ,Science - Abstract
Abstract The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the bacterium to adapt to host species' guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.
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- 2021
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17. Genomic Diversity, Antimicrobial Susceptibility, and Biofilm Formation of Clinical Acinetobacter baumannii Isolates from Horses
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Johanna Rühl-Teichner, Lisa Jacobmeyer, Ursula Leidner, Torsten Semmler, and Christa Ewers
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multidrug resistance ,international clones ,biofilm-associated genes ,Biology (General) ,QH301-705.5 - Abstract
Acinetobacter (A.) baumannii is an opportunistic pathogen that causes severe infections in humans and animals, including horses. The occurrence of dominant international clones (ICs), frequent multidrug resistance, and the capability to form biofilms are considered major factors in the successful spread of A. baumannii in human and veterinary clinical environments. Since little is known about A. baumannii isolates from horses, we studied 78 equine A. baumannii isolates obtained from clinical samples between 2008 and 2020 for their antimicrobial resistance (AMR), clonal distribution, biofilm-associated genes (BAGs), and biofilm-forming capability. Based on whole-genome sequence analyses, ICs, multilocus (ML) and core-genome ML sequence types (STs), and AMR genes were determined. Antimicrobial susceptibility testing was performed by microbroth dilution. A crystal violet assay was used for biofilm quantification. Almost 37.2% of the isolates were assigned to IC1 (10.3%), IC2 (20.5%), and IC3 (6.4%). Overall, the isolates revealed high genomic diversity. We identified 51 different STs, including 22 novel STs (ST1723–ST1744), and 34 variants of the intrinsic oxacillinase (OXA), including 8 novel variants (OXA-970 to OXA-977). All isolates were resistant to ampicillin, amoxicillin/clavulanic acid, cephalexin, cefpodoxime, and nitrofurantoin. IC1-IC3 isolates were also resistant to gentamicin, enrofloxacin, marbofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. All isolates were susceptible to imipenem. Thirty-one multidrug-resistant (MDR) isolates mainly accumulated in the IC1-IC3 groups. In general, these isolates showed less biofilm formation (IC1 = 25.0%, IC2 = 18.4%, IC3 = 15.0%) than the group of non-IC1-IC3 isolates (58.4%). Isolates belonging to the same ICs/STs revealed identical BAG patterns. BAG blp1 was absent in all isolates, whereas bfmR and pgaA were present in all isolates. At the level of the IC groups, the AMR status was negatively correlated with the isolates’ ability to form a biofilm. A considerable portion of equine A. baumannii isolates revealed ICs/STs that are globally present in humans. Both an MDR phenotype and the capability to form biofilms might lead to therapeutic failures in equine medicine, particularly due to the limited availability of licensed drugs.
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- 2023
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18. Genetic Organization of Acquired Antimicrobial Resistance Genes and Detection of Resistance-Mediating Mutations in a Gallibacterium anatis Isolate from a Calf Suffering from a Respiratory Tract Infection
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Anne-Kathrin Schink, Dennis Hanke, Torsten Semmler, Nicole Roschanski, and Stefan Schwarz
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Gallibacterium anatis ,antimicrobial resistance ,cattle ,multidrug resistance ,respiratory disease ,whole-genome sequencing ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Gallibacterium (G.) anatis isolates associated with respiratory diseases in calves and harboring acquired antimicrobial resistance genes have been described in Belgium. The aim of this study was to analyze the genetic organization of acquired resistance genes in the G. anatis isolate IMT49310 from a German calf suffering from a respiratory tract infection. The isolate was submitted to antimicrobial susceptibility testing, and a closed genome was obtained by a hybrid assembly of Illumina MiSeq short-reads and MinION long-reads. Isolate IMT49310 showed elevated MIC values for macrolides, aminoglycosides, florfenicol, tetracyclines, and trimethoprim/sulfamethoxazole. The acquired resistance genes catA1, floR, aadA1, aadB, aphA1, strA, tet(M), tet(B), erm(B), and sul2 were identified within three resistance gene regions in the genome, some of which were associated with IS elements, such as ISVsa5-like or IS15DII. Furthermore, nucleotide exchanges within the QRDRs of gyrA and parC, resulting in amino acid exchanges S83F and D87A in GyrA and S80I in ParC, were identified. Even if the role in the pathogenesis of respiratory tract infections in cattle needs to be further investigated, the identification of a G. anatis isolate with reduced susceptibility to regularly used antimicrobial agents in cases of fatal bovine respiratory tract infections is worrisome, and such isolates might also act as a reservoir for antimicrobial resistance genes.
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- 2023
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19. Molecular Features and Antimicrobial Susceptibilities of Streptococcus equi ssp. equi Isolates from Strangles Cases in Indonesia
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Dordia Anindita Rotinsulu, Christa Ewers, Katharina Kerner, Amrozi Amrozi, Retno Damayanti Soejoedono, Torsten Semmler, and Rolf Bauerfeind
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strangles ,Streptococcus equi ,antimicrobial susceptibility ,cgMLST ,seM typing ,MLST ,Veterinary medicine ,SF600-1100 - Abstract
Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains.
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- 2023
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20. Farming Practice Influences Antimicrobial Resistance Burden of Non-Aureus Staphylococci in Pig Husbandries
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Manonmani Soundararajan, Gabriella Marincola, Olivia Liong, Tessa Marciniak, Freya D. R. Wencker, Franka Hofmann, Hannah Schollenbruch, Iris Kobusch, Sabrina Linnemann, Silver A. Wolf, Mustafa Helal, Torsten Semmler, Birgit Walther, Christoph Schoen, Justin Nyasinga, Gunturu Revathi, Marc Boelhauve, and Wilma Ziebuhr
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non-aureus staphylococci ,NAS ,alternative pig farming ,antimicrobial resistance ,one-health approach ,intervention strategies ,Biology (General) ,QH301-705.5 - Abstract
Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.
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- 2022
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21. Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations
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Prasad Thomas, Mostafa Y. Abdel-Glil, Inga Eichhorn, Torsten Semmler, Christiane Werckenthin, Christina Baumbach, Wybke Murmann, Anne Bodenthin-Drauschke, Pia Zimmermann, Ulrich Schotte, Domenico Galante, Durda Slavic, Martin Wagner, Lothar H. Wieler, Heinrich Neubauer, and Christian Seyboldt
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strain typing ,Clostridium chauvoei ,genome analysis ,pangenome SNPs ,CRISPR spacer-typing ,cgMLST ,Microbiology ,QR1-502 - Abstract
Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.
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- 2021
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22. Characterization of Campylobacter spp. Strains Isolated From Wild Birds in Turkey
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Cemil Kürekci, Fatih Sakin, Lennard Epping, Marie-Theres Knüver, Torsten Semmler, and Kerstin Stingl
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C. coli clade ,whole-genome sequencing ,Eurasian coots ,environmental Campylobacter ,diagnostics ,Microbiology ,QR1-502 - Abstract
Turkey is an important stopover site for many migrating birds between Europe, Asia and Africa. Campylobacter spp. are frequently found in wildlife, in particular waterfowl, and distinct strains are disseminated within this reservoir. In this study, 183 wild birds of hunting areas in Turkey were collected and thermophilic Campylobacter spp. from cloacal swabs were isolated at a prevalence of 5.2% from song thrushes (6/116) and 93% from Eurasian coots (41/44). After PCR species differentiation and flaA restriction profiles determination, C. jejuni and C. coli strains were further investigated by whole genome sequencing. PCR target amplification of the ceuE gene, commonly used for C. coli species-identification was inefficient and even hampered in one isolate. A close look on the ceuE sequence revealed that various mismatches in the ceuE oligo annealing sites caused less efficient diagnostic detection. All C. coli isolates belonged to the environmental clade II and clade III, for which thirty-six novel MLST types were identified. Further single nucleotide polymorphism (SNP) analysis showed a high genomic divergence between the C. coli isolates. High variability was also implicated for putative plasmid-located genes detected in 51% of the C. coli isolates. Distinct gene variants in clades II and III C. coli were identified by a k-mer analysis. After substracting k-mers in common with C. coli clade I database, 11 and 35 distinct genes were identified in clades II and III isolates, mainly involved in surface structures and modifications as well as signal transduction, suggesting niche adaptation of C. coli strains in wild birds. All strains were susceptible against (fluoro-)quinolones, erythromycin, tetracycline, gentamicin and only one isolate was resistant against streptomycin, suggesting that the sensitive phenotype was due to absence of selective pressure and niche separation in wild birds in Turkey. We conclude that Campylobacter spp. isolates from wildlife and environmental sources are still scarce in the databases and that there is a need for more studies on thermophilic Campylobacter spp. from different places all over the world in order to complement our understanding on dissemination and adaptation to distinct niches of this global food-borne pathogen.
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- 2021
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23. Staphylococcus aureus nasal colonization among dental health care workers in Northern Germany (StaphDent study)
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Nadine Lerche, Silva Holtfreter, Birgit Walther, Torsten Semmler, Fawaz Al’Sholui, Stephanie J. Dancer, Georg Daeschlein, Nils-Olaf Hübner, Barbara M. Bröker, Roald Papke, Thomas Kohlmann, Romy Baguhl, Ulrike Seifert, and Axel Kramer
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Staphylococcus aureus ,MRSA ,dental practices ,dentist ,spa typing ,epidemiology ,Microbiology ,QR1-502 ,Other systems of medicine ,RZ201-999 - Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) can colonize dental patients and students, however, studies on the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) among dental health care workers (DHCW) including use of personal protective equipment (PPE) are scarce. We conducted an observational study (StaphDent study) to (I) determine the prevalence of MRSA and MSSA colonization in DHCW in the region of Mecklenburg Western-Pomerania, Germany, (II) resolve the S. aureus population structure to gain hints on possible transmission events between co-workers, and (III) clarify use of PPE. Nasal swabs were obtained from dentists (n = 149), dental assistants (n = 297) and other dental practice staff (n = 38). Clonal relatedness of MSSA isolates was investigated using spa typing and, in some cases, whole genome sequencing (WGS). PPE use was assessed by questionnaire. While 22.3% (108/485) of the participants were colonized with MSSA, MRSA was not detected. MSSA prevalence was not associated with size of dental practices, gender, age, or duration of employment. The identified 61 spa types grouped into 17 clonal complexes and four sequence types. Most spa types (n = 47) were identified only once. In ten dental practices one spa type occurred twice. WGS data analysis confirmed a close clonal relationship for 4/10 isolate pairs. PPE was regularly used by most dentists and assistants. To conclude, the failure to recover MRSA from DHCW reflects the low MRSA prevalence in this region. Widespread PPE use suggests adherence to routine hygiene protocols. Compared to other regional HCW MRSA rates the consequent usage of PPE seems to be protective.
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- 2021
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24. Comparison of different technologies for the decipherment of the whole genome sequence of Campylobacter jejuni BfR-CA-14430
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Lennard Epping, Julia C. Golz, Marie-Theres Knüver, Charlotte Huber, Andrea Thürmer, Lothar H. Wieler, Kerstin Stingl, and Torsten Semmler
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Campylobacter jejuni ,Long read sequencing ,Hybrid assemblies ,Assembler comparison ,Antibiotic resistance ,Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Abstract Background Campylobacter jejuni is a zoonotic pathogen that infects the human gut through the food chain mainly by consumption of undercooked chicken meat, raw chicken cross-contaminated ready-to-eat food or by raw milk. In the last decades, C. jejuni has increasingly become the most common bacterial cause for food-born infections in high income countries, costing public health systems billions of euros each year. Currently, different whole genome sequencing techniques such as short-read bridge amplification and long-read single molecule real-time sequencing techniques are applied for in-depth analysis of bacterial species, in particular, Illumina MiSeq, PacBio and MinION. Results In this study, we analyzed a recently isolated C. jejuni strain from chicken meat by short- and long-read data from Illumina, PacBio and MinION sequencing technologies. For comparability, this strain is used in the German PAC-CAMPY research consortium in several studies, including phenotypic analysis of biofilm formation, natural transformation and in vivo colonization models. The complete assembled genome sequence most likely consists of a chromosome of 1,645,980 bp covering 1665 coding sequences as well as a plasmid sequence with 41,772 bp that encodes for 46 genes. Multilocus sequence typing revealed that the strain belongs to the clonal complex CC-21 (ST-44) which is known to be involved in C. jejuni human infections, including outbreaks. Furthermore, we discovered resistance determinants and a point mutation in the DNA gyrase (gyrA) that render the bacterium resistant against ampicillin, tetracycline and (fluoro-)quinolones. Conclusion The comparison of Illumina MiSeq, PacBio and MinION sequencing and analyses with different assembly tools enabled us to reconstruct a complete chromosome as well as a circular plasmid sequence of the C. jejuni strain BfR-CA-14430. Illumina short-read sequencing in combination with either PacBio or MinION can substantially improve the quality of the complete chromosome and epichromosomal elements on the level of mismatches and insertions/deletions, depending on the assembly program used.
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- 2019
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25. The gut microbiome of horses: current research on equine enteral microbiota and future perspectives
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Anne Kauter, Lennard Epping, Torsten Semmler, Esther-Maria Antao, Dania Kannapin, Sabita D. Stoeckle, Heidrun Gehlen, Antina Lübke-Becker, Sebastian Günther, Lothar H. Wieler, and Birgit Walther
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Horse ,Microbiome ,Gastrointestinal tract ,Microbiota ,Disease ,Health ,Veterinary medicine ,SF600-1100 ,Microbiology ,QR1-502 - Abstract
Abstract Understanding the complex interactions of microbial communities including bacteria, archaea, parasites, viruses and fungi of the gastrointestinal tract (GIT) associated with states of either health or disease is still an expanding research field in both, human and veterinary medicine. GIT disorders and their consequences are among the most important diseases of domesticated Equidae, but current gaps of knowledge hinder adequate progress with respect to disease prevention and microbiome-based interventions. Current literature on enteral microbiomes mirrors a vast data and knowledge imbalance, with only few studies tackling archaea, viruses and eukaryotes compared with those addressing the bacterial components. Until recently, culture-dependent methods were used for the identification and description of compositional changes of enteral microorganisms, limiting the outcome to cultivatable bacteria only. Today, next generation sequencing technologies provide access to the entirety of genes (microbiome) associated with the microorganisms of the equine GIT including the mass of uncultured microbiota, or “microbial dark matter”. This review illustrates methods commonly used for enteral microbiome analysis in horses and summarizes key findings reached for bacteria, viruses and fungi so far. Moreover, reasonable possibilities to combine different explorative techniques are described. As a future perspective, knowledge expansion concerning beneficial compositions of microorganisms within the equine GIT creates novel possibilities for early disorder diagnostics as well as innovative therapeutic approaches. In addition, analysis of shotgun metagenomic data enables tracking of certain microorganisms beyond species barriers: transmission events of bacteria including pathogens and opportunists harboring antibiotic resistance factors between different horses but also between humans and horses will reach new levels of depth concerning strain-level distinctions.
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- 2019
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26. Genomic Analysis of Acinetobacter baumannii Isolates Carrying OXA-23 and OXA-58 Genes from Animals Reveals ST1 and ST25 as Major Clonal Lineages
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Lisa Jacobmeyer, Torsten Semmler, Ivonne Stamm, and Christa Ewers
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companion animal ,carbapanemase ,OXA ,international clone ,resistance island ,veterinary ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Acinetobacter baumannii is increasingly being recognized as a relevant pathogen for animals with a putative zoonotic impact. This study aimed at identifying and characterizing carbapenemase-producing A. baumannii from animals. Among 503 A. baumannii, mainly isolated from dogs/cats (75.7%) between 2013 and 2018, 42 isolates from 22 veterinary clinics (VCs) harboured blaOXA-58 (n = 29) or blaOXA-23 (n = 13). The blaOXA-58 gene was located on plasmids (11.4–21.1 kb) within different genetic surroundings (patterns A–D). BlaOXA-23 was embedded in Tn2006 on the chromosome (n = 4; pattern a) or Tn2008 on plasmids (n = 9; 41.2–71.3 kb; patterns b–e). The predominant IC1-ST1P-OXA-58 (66.7%; 96.4% cgMLST complex type (CT)-1808) was disseminated among 11 VCs in Germany. Resistance islands AbaR3-like (n = 15) and AbaR10 (n = 1) have emerged among ST1-isolates since 2016. IC7-ST25P-OXA-23 isolates (21.4%) occurred in seven VCs in Germany, France and Italy and differed in their resistance gene patterns from those of OXA-58 isolates. They were separated into six CTs, basically according to their regional origin. Other STs observed were ST10, ST578 and ST602. In conclusion, OXA-23 and OXA-58 were linked with ST1 and ST25, two globally distributed lineages in humans. The suggested transmission of certain lineages within and among VCs together with the acquisition of AbaR islands hints at a successful dissemination of multidrug-resistant strains in the VC environment.
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- 2022
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27. Progressive Lameness of a Greater One-Horned Rhinoceros (Rhinoceros unicornis) Associated with a Retroperitoneal Abscess and Thrombus Caused by Streptococcus dysgalactiae Subspecies equisimilis
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Anne Elisabeth Reetz, Etienne Aubry, Kinga Teske, Andreas Ochs, Lennard Epping, Torsten Semmler, Antina Lübke-Becker, Marcus Fulde, and Lars Mundhenk
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Greater one-horned rhinoceros ,Rhinoceros unicornis ,lameness ,Streptococcus dysgalactiae subspecies equisimilis ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
In rhinoceroses, lameness is an occasionally seen symptom primarily caused by lesions affecting the feet and interdigital space. A 3-year-old male Greater one-horned rhinoceros developed a progressive, severe movement disorder of the right hind limb with subsequent death. The pathological analysis diagnosed a severe, retroperitoneal abscess and chronic thrombosis of the right iliac artery. Streptococci detected in the abscess were further identified as Streptococcus dysgalactiae subspecies equisimilis by culture and molecular techniques. The identical isolate was also identified in a vaginal swab of the dam. The list of differential diagnoses for lameness in rhinoceroses must be expanded by processes affecting other than the extremities per se.
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- 2022
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28. Genomic Diversity and Virulence Potential of ESBL- and AmpC-β-Lactamase-Producing Escherichia coli Strains From Healthy Food Animals Across Europe
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Christa Ewers, Anno de Jong, Ellen Prenger-Berninghoff, Farid El Garch, Ursula Leidner, Sumeet K. Tiwari, and Torsten Semmler
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Escherichia coli ,livestock ,ESBL ,AmpC ,virulence ,sequence type ,Microbiology ,QR1-502 - Abstract
The role of livestock animals as a putative source of ESBL/pAmpC E. coli for humans is a central issue of research. In a large-scale pan-European surveillance, 2,993 commensal Escherichia spp. isolates were recovered from randomly collected fecal samples of healthy cattle, pigs and chickens in various abattoirs. One-hundred Escherichia spp. isolates (0.5% from cattle, 1.3% pigs, 8.0% chickens) fulfilled the criteria for cefotaxime and ceftazidime non-wildtype (EUCAST). In silico screening of WGS data of 99 isolates (98 E. coli and 1 E. fergusonii) revealed blaSHV–12 (32.3%), blaCTX–M–1 (24.2%), and blaCMY–2 (22.2%) as predominant ESBL/pAmpC types. Other types were blaSHV–2 (1.0%), blaCTX–M–2/–14/–15 (1.0/6.1/1.0%), and blaTEM–52 (5.1%). Six isolates revealed AmpC-promoter mutations (position −42 (C > T) and one carried mcr-1. The majority (91.3%) of ESBL/pAmpC genes were located on plasmids. SHV-12 was mainly (50%) encoded on IncI1α plasmids (pST-3/-26/-95), followed by IncX3 (12.5%) and IncK2 (3.1%). The blaTEM–52 genes were located on IncI1α-pST-36 (60%) and IncX1 plasmids (20%). The dominant plasmid lineage among CTX-M-1 isolates was IncI1α (pST-3/-295/-317) (87.5%), followed by IncN-pST-1 (8.3%). CMY-2 was mostly identified on IncI1α (pST-12/-2) (54.5%) and IncK2 (31.8%) plasmids. Several plasmids revealed high similarity to published plasmids from human and animal Enterobacteriaceae. The isolates were assigned to phylogroups A/C (34.7/7.1%), B1 (27.6%), B2 (3.1%), D/F (9.2/10.2%), E (5.1%), and to E. clades (3.0%). With 51 known and 2 novel MLST types, a wide variety of STs was found, including STs previously observed in human isolates (ST10/38/117/131/648). ESBL/AmpC types or STs were rarely correlated with the geographic origin of the isolates or animal species. Virulence gene typing identified extraintestinal pathogenic E. coli (ExPEC; 2.0%), avian pathogenic E. coli (APEC; 51.5%), and atypical enteropathogenic E. coli (EPEC; 6.1%). In conclusion, the high diversity of STs and phylogenetic groups provides hardly any hint for clonal spread of single lineages but hints toward the dissemination of cephalosporin resistance genes in livestock via distinct, globally successful plasmid lineages. Even though a number of isolates could not be assigned to a distinct pathotype, our finding of combined multidrug-resistance and virulence in this facultative pathogen should be considered an additional threat to public health.
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- 2021
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29. Frequency, Local Dynamics, and Genomic Characteristics of ESBL-Producing Escherichia coli Isolated From Specimens of Hospitalized Horses
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Anne Kauter, Lennard Epping, Fereshteh Ghazisaeedi, Antina Lübke-Becker, Silver A. Wolf, Dania Kannapin, Sabita D. Stoeckle, Torsten Semmler, Sebastian Günther, Heidrun Gehlen, and Birgit Walther
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horse ,ESBL ,Escherichia coli ,antibiotic resistance ,multidrug resistant ,spread ,Microbiology ,QR1-502 - Abstract
Previous research identified veterinary clinics as hotspots with respect to accumulation and spread of multidrug resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (EC). Therefore, promoting the prudent use of antibiotics to decrease selective pressure in that particular clinical environment is preferable to enhance biosecurity for animal patients and hospital staff. Accordingly, this study comparatively investigated the impact of two distinct perioperative antibiotic prophylaxis (PAP) regimens (short-term versus prolonged) on ESBL-EC carriage of horses subjected to colic surgery. While all horses received a combination of penicillin/gentamicin (P/G) as PAP, they were assigned to either the “single-shot group” (SSG) or the conventional “5-day group” (5DG). Fecal samples collected on arrival (t0), on the 3rd (t1) and on the 10th day after surgery (t2) were screened for ESBL-EC. All isolates were further investigated using whole genome sequences. In total, 81 of 98 horses met the inclusion criteria for this study. ESBL-EC identified in samples available at t0, t1 and t2 were 4.8% (SSG) and 9.7% (5DG), 37% (SSG) and 47.2% (5DG) as well as 55.6% (SSG) and 56.8% (5DG), respectively. Regardless of the P/G PAP regimen, horses were 9.12 times (95% CI 2.79–29.7) more likely to carry ESBL-EC at t1 compared to t0 (p < 0.001) and 15.64 times (95% CI 4.57–53.55) more likely to carry ESBL-EC at t2 compared to t0 (p < 0.001). ESBL-EC belonging to sequence type (ST) 10, ST86, ST641, and ST410 were the most prevalent lineages, with blaCTX–M–1 (60%) being the dominant ESBL gene. A close spatio-temporal relationship between isolates sharing a particular ST was revealed by genome analysis, strongly indicating local spread. Consequently, hospitalization itself has a strong impact on ESBL-EC isolation rates in horses, possibly masking differences between distinct PAP regimens. The results of this study reveal accumulation and spread of multi-drug resistant ESBL-EC among horses subjected to colic surgery with different P/G PAP regimens, challenging the local hygiene management system and work-place safety of veterinary staff. Moreover, the predominance of particular ESBL-EC lineages in clinics providing health care for horses needs further investigation.
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- 2021
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30. Evolutionary Dynamics Based on Comparative Genomics of Pathogenic Escherichia coli Lineages Harboring Polyketide Synthase ( pks ) Island
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Arya Suresh, Sabiha Shaik, Ramani Baddam, Amit Ranjan, Shamsul Qumar, Savita Jadhav, Torsten Semmler, Irfan A. Ghazi, Lothar H. Wieler, and Niyaz Ahmed
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Microbiology ,QR1-502 - Abstract
Extraintestinal pathologies caused by highly virulent strains of E. coliE. colipks
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- 2021
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31. Imported Pet Reptiles and Their 'Blind Passengers'—In-Depth Characterization of 80 Acinetobacter Species Isolates
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Franziska Unger, Tobias Eisenberg, Ellen Prenger-Berninghoff, Ursula Leidner, Torsten Semmler, and Christa Ewers
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reptile ,WGS ,Acinetobacter baumannii ,international clone ,OXA ,phylogeny ,Biology (General) ,QH301-705.5 - Abstract
Reptiles are popular pet animals and important food sources, but the trade of this vertebrate class is—besides welfare and conservation—under debate due to zoonotic microbiota. Ninety-two shipments of live reptiles were sampled during border inspections at Europe’s most relevant transshipment point for the live animal trade. Acinetobacter spp. represented one significant fraction of potentially MDR bacteria that were further analyzed following non-selective isolation or selective enrichment from feces, urinate, or skin samples. Taxonomic positions of respective isolates were confirmed by MALDI-TOF MS and whole-genome sequencing analysis (GBDP, dDDH, ANIb, and rMLST). The majority of the 80 isolates represented established species; however, a proportion of potentially novel taxa was found. Antimicrobial properties and genome-resistance gene screening revealed novel and existing resistance mechanisms. Acinetobacter spp. strains were most often resistant to 6–10 substance groups (n = 63) in vitro. Resistance to fluorchinolones (n = 4) and colistin (n = 7), but not to carbapenems, was noted, and novel oxacillinase variants (n = 39) were detected among other genes. Phylogenetic analysis (MLST) assigned few isolates to the known STs (25, 46, 49, 220, and 249) and to a number of novel STs. No correlation was found to indicate that MDR Acinetobacter spp. in reptiles were associated with harvesting mode, e.g., captive-bred, wild-caught, or farmed in natural ecosystems. The community of Acinetobacter spp. in healthy reptiles turned out to be highly variable, with many isolates displaying a MDR phenotype or genotype.
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- 2022
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32. First report of NDM-1 in an Acinetobacter baumannii strain from a pet animal in Europe
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Lisa Jacobmeyer, Ivonne Stamm, Torsten Semmler, and Christa Ewers
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Microbiology ,QR1-502 - Published
- 2021
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33. High-Zinc Supplementation of Weaned Piglets Affects Frequencies of Virulence and Bacteriocin Associated Genes Among Intestinal Escherichia coli Populations
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Vanessa C. Johanns, Lennard Epping, Torsten Semmler, Fereshteh Ghazisaeedi, Antina Lübke-Becker, Yvonne Pfeifer, Inga Eichhorn, Roswitha Merle, Astrid Bethe, Birgit Walther, and Lothar H. Wieler
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E. coli ,zinc ,pig ,virulence associated genes ,bacteriocins ,gut ,Veterinary medicine ,SF600-1100 - Abstract
To prevent economic losses due to post-weaning diarrhea (PWD) in industrial pig production, zinc (Zn) feed additives have been widely used, especially since awareness has risen that the regular application of antibiotics promotes buildup of antimicrobial resistance in both commensal and pathogenic bacteria. In a previous study on 179 Escherichia coli collected from piglets sacrificed at the end of a Zn feeding trial, including isolates obtained from animals of a high-zinc fed group (HZG) and a corresponding control group (CG), we found that the isolate collection exhibited three different levels of tolerance toward zinc, i.e., the minimal inhibitory concentration (MIC) detected was 128, followed by 256 and 512 μg/ml ZnCl2. We further provided evidence that enhanced zinc tolerance in porcine intestinal E. coli populations is clearly linked to excessive zinc feeding. Here we provide insights about the genomic make-up and phylogenetic background of these 179 E. coli genomes. Bayesian analysis of the population structure (BAPS) revealed a lack of association between the actual zinc tolerance level and a particular phylogenetic E. coli cluster or even branch for both, isolates belonging to the HZG and CG. In addition, detection rates for genes and operons associated with virulence (VAG) and bacteriocins (BAG) were lower in isolates originating from the HZG (41 vs. 65% and 22 vs. 35%, p < 0.001 and p = 0.002, resp.). Strikingly, E. coli harboring genes defining distinct pathotypes associated with intestinal disease, i.e., enterotoxigenic, enteropathogenic, and Shiga toxin-producing E. coli (ETEC, EPEC, and STEC) constituted 1% of the isolates belonging to the HZG but 14% of those from the CG. Notably, these pathotypes were positively associated with enhanced zinc tolerance (512 μg/ml ZnCl2 MIC, p < 0.001). Taken together, zinc excess seems to influence carriage rates of VAGs and BAGs in porcine intestinal E. coli populations, and high-zinc feeding is negatively correlated with enteral pathotype occurrences, which might explain earlier observations concerning the relative increase of Enterobacterales considering the overall intestinal microbiota of piglets during zinc feeding trials while PWD rates have decreased.
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- 2020
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34. Comprehensive integrated NGS-based surveillance and contact-network modeling unravels transmission dynamics of vancomycin-resistant enterococci in a high-risk population within a tertiary care hospital.
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Bernd Neumann, Jennifer K Bender, Benjamin F Maier, Alice Wittig, Stephan Fuchs, Dirk Brockmann, Torsten Semmler, Hermann Einsele, Sabrina Kraus, Lothar H Wieler, Ulrich Vogel, and Guido Werner
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Medicine ,Science - Abstract
Vancomycin-resistant E. faecium (VRE) are an important cause of nosocomial infections, which are rapidly transmitted in hospitals. To identify possible transmission routes, we applied combined genomics and contact-network modeling to retrospectively evaluate routine VRE screening data generated by the infection control program of a hemato-oncology unit. Over 1 year, a total of 111 VRE isolates from 111 patients were collected by anal swabs in a tertiary care hospital in Southern Germany. All isolated VRE were whole-genome sequenced, followed by different in-depth bioinformatics analyses including genotyping and determination of phylogenetic relations, aiming to evaluate a standardized workflow. Patient movement data were used to overlay sequencing data to infer transmission events and strain dynamics over time. A predominant clone harboring vanB and exhibiting genotype ST117/CT469 (n = 67) was identified. Our comprehensive combined analyses suggested intra-hospital spread, especially of clone ST117/CT469, despite of extensive screening, single room placement, and contact isolation. A new interactive tool to visualize these complex data was designed. Furthermore, a patient-contact network-modeling approach was developed, which indicates both the periodic import of the clone into the hospital and its spread within the hospital due to patient movements. The analyzed spread of VRE was most likely due to placement of patients in the same room prior to positivity of screening. We successfully demonstrated the added value for this combined strategy to extract well-founded knowledge from interdisciplinary data sources. The combination of patient-contact modeling and high-resolution typing unraveled the transmission dynamics within the hospital department and, additionally, a constant VRE influx over time.
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- 2020
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35. Determination of virulence and fitness genes associated with the pheU, pheV and selC integration sites of LEE-negative food-borne Shiga toxin-producing Escherichia coli strains
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Nadja Saile, Elisabeth Schuh, Torsten Semmler, Inga Eichhorn, Lothar H. Wieler, Andreas Bauwens, and Herbert Schmidt
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STEC ,Food ,pheU ,pheV ,selC integration site ,Genomic island ,Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Abstract Background In the current study, nine foodborne “Locus of Enterocyte Effacement” (LEE)-negative Shiga toxin-producing Escherichia coli (STEC) strains were selected for whole genome sequencing and analysis for yet unknown genetic elements within the already known LEE integration sites selC, pheU and pheV. Foreign DNA ranging in size from 3.4 to 57 kbp was detected and further analyzed. Five STEC strains contained an insertion of foreign DNA adjacent to the selC tRNA gene and five and seven strains contained foreign DNA adjacent to the pheU and pheV tRNA genes, respectively. We characterized the foreign DNA insertion associated with selC (STEC O91:H21 strain 17584/1), pheU (STEC O8:H4 strain RF1a and O55:Hnt strain K30) and pheV (STEC O91:H21 strain 17584/1 and O113:H21 strain TS18/08) as examples. Results In total, 293 open reading frames partially encoding putative virulence factors such as TonB-dependent receptors, DNA helicases, a hemolysin activator protein precursor, antigen 43, anti-restriction protein KlcA, ShiA, and phosphoethanolamine transferases were detected. A virulence type IV toxin-antitoxin system was detected in three strains. Additionally, the ato system was found in one strain. In strain 17584/1 we were able to define a new genomic island which we designated GIselC 17584/1. The island contained integrases and mobile elements in addition to genes for increased fitness and those playing a putative role in pathogenicity. Conclusion The data presented highlight the important role of the three tRNAs selC, pheU, and pheV for the genomic flexibility of E. coli.
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- 2018
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36. ESBL-plasmid carriage in E. coli enhances in vitro bacterial competition fitness and serum resistance in some strains of pandemic sequence types without overall fitness cost
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Amit Ranjan, Julia Scholz, Torsten Semmler, Lothar H. Wieler, Christa Ewers, Stefanie Müller, Derek J. Pickard, Peter Schierack, Karsten Tedin, Niyaz Ahmed, Katharina Schaufler, and Sebastian Guenther
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Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Abstract Background Extended spectrum beta lactamase (ESBL)-producing extraintestinal pathogenic Escherichia coli infections are of global interest because of their clinical and economic impact. The ESBL resistance genes disseminate through plasmids, and are found in successful global lineages such as ST131 and ST648. The carriage of plasmids has been suggested to result in a fitness burden, but recently it was shown that ESBL-plasmids enhanced virulence in pandemic ST131 and ST648 lineages without affecting their fitness. Herein, we investigated the influence of ESBL-plasmids on bacterial competition and serum resistance, both of which are essential characteristics of ExPEC during infections. Methods Triplets of ESBL-plasmid-carrying wildtype (WT), plasmid-cured variant (PCV) and transformant (T) of five ExPEC strains of ST131 and ST648 were used for bacterial competition experiments with colicin-producing commensal E. coli, competitive adhesion experiments and serum survival. In addition, resilience after SDS, acid, osmotic challenges and RNA sequence data were analyzed. Results In all five strains tested, ESBL-plasmid carriage did not negatively influence E. coli fitness in direct bacterial competition with commensal E. coli in vitro. That is, WTs did not show any disadvantages when compared to their isogenic plasmid-free PCV. For one strain we even found the opposite as PCV17433 was out-competed by a commensal strain, which suggests an even protective role of the ESBL-plasmid carried by the WT17433. Similarly, in the serum-resistance experiments, the PCVs of two strains (PCV17433 and PCV17887) were more sensitive to serum, unlike WTs and Ts. The observed inter-strain differences could be explained by the different genetic content of plasmids carried in those strains. Conclusions Overall, we found no compelling evidence for an increased burden resulting from the carriage of ESBL-plasmids in the absence of antimicrobial selection pressure in the strains of pandemic ST131 and ST648; rather, the possession of certain ESBL-plasmids was beneficial for some strains in regarding competition fitness and serum survival.
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- 2018
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37. Re-Emergence and Spread of Haemorrhagic Septicaemia in Germany: The Wolf as a Vector?
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Peter Kutzer, Claudia A. Szentiks, Sabine Bock, Guido Fritsch, Tibor Magyar, Christoph Schulze, Torsten Semmler, and Christa Ewers
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Pasteurella multocida ,wild boar ,domestic animals ,core genome ,MLST ,virulence ,Biology (General) ,QH301-705.5 - Abstract
Since 2010, outbreaks of haemorrhagic septicaemia (HS) caused by Pasteurella (P.) multocida capsular type B (PmB) emerged in Germany. In 2017, we noticed a close spatiotemporal relationship between HS outbreak sites and wolf (Canis lupus) territories. Thus, the main objectives of our study were to investigate the molecular epidemiology of German PmB-HS-isolates and to assess the role of wolves as putative vectors of this pathogen. We collected 83 PmB isolates from HS outbreaks that occurred between 2010 and 2019 and sampled 150 wolves, which were found dead in the years 2017 to 2019, revealing another three PmB isolates. A maximum-likelihood-based phylogeny of the core genomes of 65 PmB-HS-isolates and the three PmB-wolf-isolates showed high relatedness. Furthermore, all belonged to capsular:LPS:MLST genotype B:L2:ST122RIRDC and showed highly similar virulence gene profiles, but clustered separately from 35 global ST122RIRDC strains. Our data revealed that German HS outbreaks were caused by a distinct genomic lineage of PmB-ST122 strains, hinting towards an independent, ongoing epidemiologic event. We demonstrated for the first time, that carnivores, i.e., wolves, might harbour PmB as a part of their oropharyngeal microbiota. Furthermore, the results of our study imply that wolves can carry the pathogen over long distances, indicating a major role of that animal species in the ongoing epidemiological event of HS in Germany.
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- 2021
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38. Rise and Fall of SARS-CoV-2 Lineage A.27 in Germany
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Sébastien Calvignac-Spencer, Matthias Budt, Matthew Huska, Hugues Richard, Luca Leipold, Linus Grabenhenrich, Torsten Semmler, Max von Kleist, Stefan Kröger, Thorsten Wolff, and Martin Hölzer
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public health surveillance ,SARS-CoV-2 variants ,viral genomes ,molecular sequence data ,Microbiology ,QR1-502 - Abstract
Here, we report on the increasing frequency of the SARS-CoV-2 lineage A.27 in Germany during the first months of 2021. Genomic surveillance identified 710 A.27 genomes in Germany as of 2 May 2021, with a vast majority identified in laboratories from a single German state (Baden-Wuerttemberg, n = 572; 80.5%). Baden-Wuerttemberg is located near the border with France, from where most A.27 sequences were entered into public databases until May 2021. The first appearance of this lineage based on sequencing in a laboratory in Baden-Wuerttemberg can be dated to early January ’21. From then on, the relative abundance of A.27 increased until the end of February but has since declined—meanwhile, the abundance of B.1.1.7 increased in the region. The A.27 lineage shows a mutational pattern typical of VOIs/VOCs, including an accumulation of amino acid substitutions in the Spike glycoprotein. Among those, L18F, L452R and N501Y are located in the epitope regions of the N-terminal- (NTD) or receptor binding domain (RBD) and have been suggested to result in immune escape and higher transmissibility. In addition, A.27 does not show the D614G mutation typical for all VOIs/VOCs from the B lineage. Overall, A.27 should continue to be monitored nationally and internationally, even though the observed trend in Germany was initially displaced by B.1.1.7 (Alpha), while now B.1.617.2 (Delta) is on the rise.
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- 2021
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39. Genomic and Functional Characterization of Poultry Escherichia coli From India Revealed Diverse Extended-Spectrum β-Lactamase-Producing Lineages With Shared Virulence Profiles
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Arif Hussain, Sabiha Shaik, Amit Ranjan, Arya Suresh, Nishat Sarker, Torsten Semmler, Lothar H. Wieler, Munirul Alam, Haruo Watanabe, Dipshikha Chakravortty, and Niyaz Ahmed
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poultry Escherichia coli ,genomics ,ESBL ,ExPEC ,India ,Microbiology ,QR1-502 - Abstract
Extended-spectrum β-lactamases (ESBLs) form the most important resistance determinants prevalent worldwide. Data on ESBL-producing Escherichia coli from poultry and livestock are scarce in India. We present data on the functional and genomic characterization of ESBL-producing E. coli obtained from poultry in India. The whole genome sequences of 28 ESBL-producing E. coli were analyzed comprising of 12 broiler chicken E. coli isolates, 11 free-range chicken E. coli isolates, and 5 human extraintestinal pathogenic E. coli. All of the 28 ESBL-producing E. coli isolates were tested for antibiotic susceptibilities, in vitro conjugation, and virulence-associated phenotypic characteristics. A total of 13 sequence types were identified from the poultry E. coli, which included globally successful sequence types such as ST117 (9%), ST131 (4.3%), and ST10 (4.3%). The most common ESBL gene detected in poultry E. coli genomes was blaCTX-M-15 (17%). Also, FIB (73%) and FII (73%) were the most common plasmid replicons identified. Conjugation experiments demonstrated 54 (7/13), 30 (3/10), and 40% (2/5) of broiler, free-range, and human ExPEC E. coli to be able to transfer their ESBL genes, respectively. The in vitro virulence-associated phenotypic tests revealed the broiler, free-range, and human ExPEC isolates to be comparable in biofilm formation, resistance to serum bactericidal activity, adherence, and invasion capabilities. Our overall results showed prevalence of virulence phenotypes among the diverse ESBL-producing E. coli from poultry; while certain E. coli clones from broiler-poultry may indeed have the potential to cause infection in humans.
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- 2019
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40. Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs
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Vanessa C. Johanns, Fereshteh Ghazisaeedi, Lennard Epping, Torsten Semmler, Antina Lübke-Becker, Yvonne Pfeifer, Astrid Bethe, Inga Eichhorn, Roswitha Merle, Birgit Walther, and Lothar H. Wieler
- Subjects
Escherichia coli ,zinc ,antimicrobial resistance ,pig ,heavy metal tolerance ,Microbiology ,QR1-502 - Abstract
Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems.
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- 2019
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41. Sindbis virus polyarthritis outbreak signalled by virus prevalence in the mosquito vectors.
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Jan O Lundström, Jenny C Hesson, Martina L Schäfer, Örjan Östman, Torsten Semmler, Michaël Bekaert, Manfred Weidmann, Åke Lundkvist, and Martin Pfeffer
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Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
Polyarthritis and rash caused by Sindbis virus (SINV), was first recognised in northern Europe about 50 years ago and is known as Ockelbo disease in Sweden and Pogosta disease in Finland. This mosquito-borne virus occurs mainly in tropical and sub-tropical countries, and in northern Europe it is suggested to cause regularly reoccurring outbreaks. Here a seven-year cycle of SINV outbreaks has been referred to in scientific papers, although the hypothesis is based solely on reported human cases. In the search for a more objective outbreak signal, we evaluated mosquito abundance and SINV prevalence in vector mosquitoes from an endemic area in central Sweden. Vector mosquitoes collected in the River Dalälven floodplains during the years before, during, and after the hypothesised 2002 outbreak year were assayed for virus on cell culture. Obtained isolates were partially sequenced, and the nucleotide sequences analysed using Bayesian maximum clade credibility and median joining network analysis. Only one SINV strain was recovered in 2001, and 4 strains in 2003, while 15 strains were recovered in 2002 with significantly increased infection rates in both the enzootic and the bridge-vectors. In 2002, the Maximum Likelihood Estimated infection rates were 10.0/1000 in the enzootic vectors Culex torrentium/pipiens, and 0.62/1000 in the bridge-vector Aedes cinereus, compared to 4.9/1000 and 0.0/1000 in 2001 and 0.0/1000 and 0.32/1000 in 2003 Sequence analysis showed that all isolates belonged to the SINV genotype I (SINV-I). The genetic analysis revealed local maintenance of four SINV-I clades in the River Dalälven floodplains over the years. Our findings suggest that increased SINV-I prevalence in vector mosquitoes constitutes the most valuable outbreak marker for further scrutinising the hypothesized seven-year cycle of SINV-I outbreaks and the mechanisms behind.
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- 2019
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42. Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany
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Peter Klotz, Paul G. Higgins, Andreas R. Schaubmar, Klaus Failing, Ursula Leidner, Harald Seifert, Sandra Scheufen, Torsten Semmler, and Christa Ewers
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ESKAPE ,Acinetobacter baumannii ,antimicrobial susceptibility ,MLST ,cattle ,epidemiology ,Microbiology ,QR1-502 - Abstract
Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans.
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- 2019
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43. Equine Methicillin-Resistant Sequence Type 398 Staphylococcus aureus (MRSA) Harbor Mobile Genetic Elements Promoting Host Adaptation
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Birgit Walther, Katja-Sophia Klein, Ann-Kristin Barton, Torsten Semmler, Charlotte Huber, Roswitha Merle, Karsten Tedin, Franziska Mitrach, Antina Lübke-Becker, and Heidrun Gehlen
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MRSA ,multi-drug resistance ,horses ,one health ,host range ,ST398 ,Microbiology ,QR1-502 - Abstract
Continuing introduction of multi-drug resistant, zoonotic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) in horse clinics challenges the biosafety of employees and animal patients. This study was aimed to determine the occurrence of mobile genetic elements facilitating survival in the early stages of invasive infection in different host species, including humans and horses, in MRSA carried by equine patients admitted to a large horse clinic. A total of 341 equine patients were investigated for carriage of MRSA by hygiene screening directly at hospital admission. MRSA were further investigated by antimicrobial susceptibility testing, whole-genome sequencing and genomic composition, including virulence factors involved in immune evasion and host adaption. From a total of 340 validated specimens from equine nostrils, 3.5% yielded positive results for MRSA. All MRSA were found to be closely related belonging to sequence type (ST) 398_t011 with up to four additional antimicrobial resistances. All MRSA harbored a specific Staphylococcal Pathogenicity Island (SaPIbov5) involved in facilitating survival in ruminant and equine plasma. Moreover, a β-hemolysin (hlb) converting ΦSa3 phage encoding the human-specific Immune Evasion Cluster (IEC) was present in 72% of the isolates. An equid-specific leukotoxin encoded by a further temperate phage (Saeq1) was only rarely detected (22%). Despite the absence of β-hemolysin production for all IEC-positive ST398, a prominent hemolysis zone was demonstrable on sheep blood agar. Thus, IEC might remain undetected among the ST398 lineage, since the presence of IEC is commonly associated with reduction of hemolysis in S. aureus belonging to other genetic backgrounds. Here we describe MRSA-ST398 harboring different mobile genetic elements encoding variants of immune evasion factors and toxins previously shown to contribute to S. aureus invasive diseases in specific host species or ecologic niches. We suggest these combinations contribute to the adaptation of MRSA belonging to ST398 with respect to epidemic spread across different habitats and hosts, and may therefore confer a host “generalist” phenotype.
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- 2018
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44. ESBL-Producing Klebsiella pneumoniae in the Broiler Production Chain and the First Description of ST3128
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Katrin Daehre, Michaela Projahn, Anika Friese, Torsten Semmler, Sebastian Guenther, and Uwe H. Roesler
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extended-spectrum-beta-lactamases ,ESBLs ,Klebsiella pneumoniae ,broiler production ,broiler chicken ,Microbiology ,QR1-502 - Abstract
ESBL-producing Klebsiella pneumoniae (K. pneumoniae) represent an increasing problem both in human and veterinary medicine. As SHV-2 - encoding K. pneumoniae were recently detected in the broiler production we were interested in investigating a possible transmission along the broiler production chain and furthermore, in evaluating their possible impact on human health. Therefore, 41 ESBL-producing K. pneumoniae originating from a parent flock, from the hatcherys' environment during the hatching of that parent flocks' chickens, and from an associated fattening flock were investigated on an Illumina Miseq. Whole genome sequences were analyzed concerning their MLST-type, cgMLST-type, genotypic and phenotypic resistance, plasmid profiles and virulence genes. Irrespective of the origin of isolation all investigated isolates were multi-drug resistant, harbored the same ESBL-gene blaSHV−2, shared the same sequence type (ST3128) and displayed 100% similarity in core genome multilocus sequence typing (cgMLST). In addition, in silico plasmid typing found several Inc/Rep types associated with ESBL-plasmids. Summarizing, identical clones of SHV-2—producing K. pneumoniae were detected in different stages of the industrial broiler production in one out of seven investigated broiler chains. This proves the possibility of pseudo-vertical transmission of multi-resistant human pathogens from parent flocks to hatcheries and fattening flocks. Furthermore, the importance of cross-contamination along the production chain was shown. Although the ESBL-producing K. pneumoniae clone detected here in the broiler production has not been associated with clinical settings so far, our findings present a potential public health threat.
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- 2018
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45. The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4
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Sonam Grover, Tarina Sharma, Yadvir Singh, Sakshi Kohli, Manjunath P., Aditi Singh, Torsten Semmler, Lothar H. Wieler, Karsten Tedin, Nasreen Z. Ehtesham, and Seyed E. Hasnain
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calcium homeostasis ,ER localization signal ,granulomas ,unfolded protein response ,Microbiology ,QR1-502 - Abstract
ABSTRACT The genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism. IMPORTANCE Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease.
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- 2018
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46. Multispecies and Clonal Dissemination of OXA-48 Carbapenemase in Enterobacteriaceae From Companion Animals in Germany, 2009—2016
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Sandra Pulss, Inka Stolle, Ivonne Stamm, Ursula Leidner, Carsten Heydel, Torsten Semmler, Ellen Prenger-Berninghoff, and Christa Ewers
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Escherichia coli ,Klebsiella pneumoniae ,Enterobacter cloacae ,companion animals ,carbapenemase ,nosocomial ,Microbiology ,QR1-502 - Abstract
The increasing spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a serious threat to public health. Recent studies suggested animals as a putative source of such bacteria. We investigated 19,025 Escherichia coli, 1607 Klebsiella spp. and 570 Enterobacter spp. isolated from livestock, companion animal, horse, and pet samples between 2009 and 2016 in our routine diagnostic laboratory for reduced susceptibility to carbapenems (CP) by using meropenem-containing media. Actively screened CP non-susceptible strains as well as 367 archived ESBL/AmpC-β-lactamase-producing Enterobacteriaceae were then tested for the presence of CP genes by PCRs. Among 21,569 isolates, OXA-48 could be identified as the sole carbapenemase type in 137 (0.64%) strains. The blaOXA-48 gene was located on an ∼60-kb IncL plasmid and sequence analysis revealed high similarity to reference plasmid pOXA-48a, which has been involved in the global spread of the blaOXA-48 gene in humans for many years. Klebsiella pneumoniae was the predominant OXA-48 producer (n = 86; 6.6% of all K. pneumoniae isolates), followed by E. cloacae (n = 28; 5.0%), Klebsiella oxytoca (n = 1; 0.3%), and E. coli (n = 22, 0.1%). OXA-48 was not found in livestock, but in dogs (120/3182; 3.8%), cats (13/792; 1.6%), guinea pig (1/43; 2.3%), rat (1/23; 4.3%), mouse (1/180; 0.6%), and one rabbit (1/144; 0.7%). Genotyping identified few major clones among the different enterobacteria species, including sequence types ST11 and ST15 for K. pneumoniae, ST1196 for E. coli, and ST506 and ST78 for E. cloacae, most of which were previously involved in the dissemination of multidrug-resistant strains in humans. The majority of OXA-48 isolates (n = 112) originated from a university veterinary clinic (UVC), while animals from further 16 veterinary institutions were positive. Clonal analyses suggested nosocomial events related to different species and STs in two veterinary clinics and horizontal transfer of the pOXA-48-like plasmid between bacterial species and animals. A systematic monitoring is urgently needed to assess the dissemination of CPE not only in livestock but also in companion animals and veterinary clinics.
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- 2018
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47. Whole Genome Sequence Analysis of CTX-M-15 Producing Klebsiella Isolates Allowed Dissecting a Polyclonal Outbreak Scenario
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Laura Becker, Stephan Fuchs, Yvonne Pfeifer, Torsten Semmler, Tim Eckmanns, Gerit Korr, Dagmar Sissolak, Michael Friedrichs, Edith Zill, Mei-Lin Tung, Christian Dohle, Martin Kaase, Sören Gatermann, Holger Rüssmann, Matthias Steglich, Sebastian Haller, and Guido Werner
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ESBL ,outbreak analysis ,NGS ,strain typing ,CTX-M-15 ,Microbiology ,QR1-502 - Abstract
Extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae pose an important threat of infection with increased morbidity and mortality, especially for immunocompromised patients. Here, we use the rise of multidrug-resistant K. pneumoniae in a German neurorehabilitation center from April 2015 to April 2016 to dissect the benefit of whole genome sequencing (WGS) for outbreak analyses. In total, 53 isolates were obtained from 52 patients and examined using WGS. Two independent analysis strategies (reference-based and -free) revealed the same distinct clusters of two CTX-M-15 producing K. pneumoniae clones (ST15, n = 31; ST405, n = 7) and one CTX-M-15 producing Klebsiella quasipneumoniae strain (ST414, n = 8). Additionally, we determined sequence variations associated with antimicrobial resistance phenotypes in single isolates expressing carbapenem and colistin resistance, respectively. For rapid detection of the major K. pneumoniae outbreak clone (ST15), a selective triplex PCR was deduced from WGS data of the major outbreak strain and K. pneumoniae genome data deposited in central databases. Moreover, we introduce two novel open-source applications supporting reference genome selection (refRank; https://gitlab.com/s.fuchs/refRank) and alignment-based SNP-filtering (SNPfilter; https://gitlab.com/s.fuchs/snpfilter) in NGS analyses.
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- 2018
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48. Clinically Relevant ESBL-Producing K. pneumoniae ST307 and E. coli ST38 in an Urban West African Rat Population
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Katharina Schaufler, Kathrin Nowak, Ariane Düx, Torsten Semmler, Laura Villa, Laye Kourouma, Karim Bangoura, Lothar H. Wieler, Fabian H. Leendertz, and Sebastian Guenther
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ESBL ,rats ,clonal spread ,MLST ,WGS ,one health ,Microbiology ,QR1-502 - Abstract
High-risk ESBL-producing Enterobacteriaceae (ESBL-E) have been described in wild birds and rodents worldwide. Rats are of special interest not only due to their indicator role for environmental pollution with multi-resistant bacteria but also as possible infection source. Data on the presence of high-risk ESBL-E in urban wildlife from Africa remain scarce, however. Twenty-nine animals from three different rat (Rattus) species were captured in the city of Conakry (Guinea, West Africa) in 2015. Rectal swabs were analyzed for ESBL-E using selective media. Species typing and phenotypic antimicrobial resistance analysis to broad-spectrum beta-lactams and other classes of antimicrobials was performed for Enterobacteriaceae-like isolates using the VITEK®2 system (BioMérieux, Germany). Confirmed ESBL-producing E. coli and K. pneumoniae were whole-genome sequenced and resistance genes, phylogenetic background and genes related to bacterial fitness and virulence were analyzed. In total, six of twenty-nine rats (20%) carried ESBL-E (K. pneumoniae and E. coli). All ESBL-producers were multi-drug resistant with blaCTX−M−15 as the dominating ESBL-type. Interestingly, ESBL-associated clonal lineages E. coli ST38 and K. pneumoniae ST307 were found. The ESBL-plasmid in K. pneumoniae ST307 revealed high sequence similarities to pKPN3-307_TypeC, a >200 kbp IncFII plasmid originating from a human clinical ST307 isolate. This was in contrast to the core genome: the rat isolate was distantly related to the human clinical ST307 isolate (27 SNPs/Mbp). In addition, we identified π-fimbrial, capsule 2, and glycogen synthesis clusters in the rodent ST307 isolate, whose involvement in the adaptation to survival outside the host and in human urinary tracts has been suggested. Our results demonstrate the presence of clinically relevant, ESBL-producing K. pneumoniae ST307 and E. coli ST38 clonal lineages in an urban West African rat population. The human community is likely the initial source of ESBL-E however, rats might function as infection source and transmission hub, accelerated by frequent interactions at a human-wildlife interface.
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- 2018
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49. Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Acinetobacter baumannii among horses entering a veterinary teaching hospital: The contemporary 'Trojan Horse'.
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Birgit Walther, Katja-Sophia Klein, Ann-Kristin Barton, Torsten Semmler, Charlotte Huber, Silver Anthony Wolf, Karsten Tedin, Roswitha Merle, Franziska Mitrach, Sebastian Guenther, Antina Lübke-Becker, and Heidrun Gehlen
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Medicine ,Science - Abstract
Pathogens frequently associated with multi-drug resistant (MDR) phenotypes, including extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and Acinetobacter baumannii isolated from horses admitted to horse clinics, pose a risk for animal patients and personnel in horse clinics. To estimate current rates of colonization, a total of 341 equine patients were screened for carriage of zoonotic indicator pathogens at hospital admission. Horses showing clinical signs associated with colic (n = 233) or open wounds (n = 108) were selected for microbiological examination of nostril swabs, faecal samples and wound swabs taken from the open wound group. The results showed alarming carriage rates of Gram-negative MDR pathogens in equine patients: 10.7% (34 of 318) of validated faecal specimens were positive for ESBL-E (94%: ESBL-producing Escherichia coli), with recorded rates of 10.5% for the colic and 11% for the open wound group. 92.7% of the ESBL-producing E. coli were phenotypically resistant to three or more classes of antimicrobials. A. baumannii was rarely detected (0.9%), and all faecal samples investigated were negative for Salmonella, both directly and after two enrichment steps. Screening results for the equine nostril swabs showed detection rates for ESBL-E of 3.4% among colic patients and 0.9% in the open wound group, with an average rate of 2.6% (9/340) for both indications. For all 41 ESBL-producing E. coli isolated, a broad heterogeneity was revealed using pulsed-field gel electrophoresis (PFGE) patterns and whole genome sequencing (WGS) -analysis. However, a predominance of sequence type complex (STC)10 and STC1250 was observed, including several novel STs. The most common genes associated with ESBL-production were identified as blaCTX-M-1 (31/41; 75.6%) and blaSHV-12 (24.4%). The results of this study reveal a disturbingly large fraction of multi-drug resistant and ESBL-producing E. coli among equine patients, posing a clear threat to established hygiene management systems and work-place safety of veterinary staff in horse clinics.
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- 2018
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50. High Prevalence of CTX-M-15-Type ESBL-Producing E. coli from Migratory Avian Species in Pakistan
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Mashkoor Mohsin, Shahbaz Raza, Katharina Schaufler, Nicole Roschanski, Fatima Sarwar, Torsten Semmler, Peter Schierack, and Sebastian Guenther
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antimicrobial resistance ,wild birds ,ESBL-producing E. coli ,genomic epidemiology ,Pakistan ,Microbiology ,QR1-502 - Abstract
The increased presence of clinically relevant multidrug resistant bacteria in natural environments is an emerging challenge for global health care. Little is known regarding the occurrence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) from environmental sentinels in Pakistan. The goal of the current study was to gain insights into the prevalence and phylogenetic relationships of ESBL-E. coli recovered from wild birds in Pakistan during winter migration. After initial screening of fecal samples on selective chromogenic agar, ESBL-E.coli were analyzed phenotypically using the Vitek-2 automated system. Genotypic characterization was performed using whole genome sequencing (WGS) followed by an in-depth in silico analysis. Of 150 birds screened, 26 (17.3%) were fecal carriers of ESBL-E. coli. Of these, 88.4% isolates exhibited multidrug resistance (MDR) phenotypes. Resistance to cefotaxime, ceftazidime, ampicillin, doxycycline, tetracycline and sulfamethoxazole/trimethoprim (CTX-CAZ-AM-DC-TE-SXT) represented the most common pattern of MDR (76.9%). WGS data analysis found blaCTX-M-15 as the predominant ESBL genotype (92.3%). Other genes encoding resistance to sulfonamides (sul1/sul2/sul3), aminoglycosides (strA, strB, aadA1, aadA2, aadA5, aac(3)-IId-like, aac(3)-IVa-like and aph(4)-Ia), trimethoprim (dfrA14 or dfrA17), tetracyclines [tet(A)/tet(B)], and fluoroquinolones (qnrS1) were detected commonly, often encoded on IncF-type plasmids (76.9%). ESBL-E. coli were assigned to 17 different sequence types (STs) of which ST10 and ST7097 (4 isolates each) were the most abundant followed by ST4720, ST93, and ST1139 (2 isolates each). Core-genome phylogeny of the isolates found low numbers (0–29) of single nucleotide polymorphisms (SNPs) in isolates belonged to ST7097 originated from two different locations (Chashma barrage and Rasul barrage). Similar trends were found among isolates belong to ST1139. In addition, WGS-based plasmid typing and S1-digestion found plasmids of the same pMLST type (IncF[F-:A-:B53]) and similar sizes in different bacterial and avian hosts suggesting horizontal gene transfer as another possibility for the spread of ESBL-E. coli in avian wildlife in Pakistan.
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- 2017
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