Wolfgang Witte

Publications of Wolfgang Witte

• Sharing More than Friendship - Nasal Colonization with Coagulase-Positive Staphylococci (CPS) and Co-Habitation Aspects of Dogs and Their Owners.

  Authors: Birgit Walther, Julia Hermes, Christiane Cuny, Lothar H Wieler, Szilvia Vincze, Yassmin Abou Elnaga, Ivonne Stamm, Peter A Kopp, Barbara Kohn, Wolfgang Witte, Andreas Jansen, Franz J Conraths, Torsten Semmler, Tim Eckmanns, Antina Lübke-Becker

  PloS one. 01/2012; 7(4):e35197.

  Since the relationship between dogs and their owners has changed, and dogs moved from being working dogs to family members in post-industrial countries, we hypothesized that zoonotic transmission ofSince the relationship between dogs and their owners has changed, and dogs moved from being working dogs to family members in post-industrial countries, we hypothesized that zoonotic transmission of opportunistic pathogens like coagulase positive staphylococci (CPS) is likely between dogs and their owners. CPS- nasal carriage, different aspects of human-to-dog relationship as well as potential interspecies transmission risk factors were investigated by offering nasal swabs and a questionnaire to dog owners (108) and their dogs (108) at a dog show in 2009. S. aureus was found in swabs of 20 (18.5%) humans and two dogs (1.8%), and spa types which correspond to well known human S. aureus lineages dominated (e.g. CC45, CC30 and CC22). Multilocus sequence typing (MLST) of the two canine strains revealed ST72 and ST2065 (single locus variant of ST34). Fifteen dogs (13.9%) and six owners (5.6%) harboured S. pseudintermedius, including one mecA-positive human isolate (MRSP). Pulsed field gel electrophoresis (PFGE) revealed that one dog/owner pair harboured indistinguishable S. pseudintermedius- isolates of ST33. Ten (48%) of the 21 S. pseudintermedius-isolates showed resistance towards more than one antimicrobial class. 88.9% of the dog owners reported to allow at least one dog into the house, 68.5% allow the dog(s) to rest on the sofa, 39.8% allow their dogs to come onto the bed, 93.5% let them lick their hands and 52.8% let them lick their face. Bivariate analysis of putative risk factors revealed that dog owners who keep more than two dogs have a significantly higher chance of being colonized with S. pseudintermedius than those who keep 1-2 dogs (p<0.05). In conclusion, CPS transmission between dog owners and their dogs is possible. Further investigation regarding interspecies transmission and the diverse adaptive pathways influencing the epidemiology of CPS (including MRSA and MRSP) in different hosts is needed.

• Single-Nucleotide Polymorphism Genotyping Identifies a Locally Endemic Clone of Methicillin-Resistant Staphylococcus aureus.

  Authors: Ulrich Nübel, Andreas Nitsche, Franziska Layer, Birgit Strommenger, Wolfgang Witte

  PloS one. 01/2012; 7(3):e32698.

  We developed, tested, and applied a TaqMan real-time PCR assay for interrogation of three single-nucleotide polymorphisms that differentiate a clade (termed 't003-X') within the radiation ofWe developed, tested, and applied a TaqMan real-time PCR assay for interrogation of three single-nucleotide polymorphisms that differentiate a clade (termed 't003-X') within the radiation of methicillin-resistant Staphylococcus aureus (MRSA) ST225. The TaqMan assay achieved 98% typeability and results were fully concordant with DNA sequencing. By applying this assay to 305 ST225 isolates from an international collection, we demonstrate that clade t003-X is endemic in a single acute-care hospital in Germany at least since 2006, where it has caused a substantial proportion of infections. The strain was also detected in another hospital located 16 kilometers away. Strikingly, however, clade t003-X was not found in 62 other hospitals throughout Germany nor among isolates from other countries, and, hence, displayed a very restricted geographical distribution. Consequently, our results show that SNP-typing may be useful to identify and track MRSA clones that are specific to individual healthcare institutions. In contrast, the spatial dissemination pattern observed here had not been resolved by other typing procedures, including multilocus sequence typing (MLST), spa typing, DNA macrorestriction, and multilocus variable-number tandem repeat analysis (MLVA).

• Absence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex CC398 as a Nasal Colonizer of Pigs Raised in an Alternative System.

  Authors: Christiane Cuny, Alexander W Friedrich, Wolfgang Witte

  Applied and environmental microbiology. 12/2011; 78(4):1296-7.

  Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adheringLivestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adhering to an alternative system. LA-MRSA ST398 was not detected in nasal swabs from 178 pigs or from 89 humans working and living on these farms.

• From types to trees: reconstructing the spatial spread of Staphylococcus aureus based on DNA variation.

  Authors: Ulrich Nübel, Birgit Strommenger, Franziska Layer, Wolfgang Witte

  International journal of medical microbiology : IJMM. 12/2011; 301(8):614-8.

  Tracing the spatial spread of pathogens is a key objective of molecular infectious disease epidemiology. Accordingly, a wide range of genotyping approaches have been used to monitor the disseminationTracing the spatial spread of pathogens is a key objective of molecular infectious disease epidemiology. Accordingly, a wide range of genotyping approaches have been used to monitor the dissemination of Staphylococcus aureus strains, from localized outbreaks to global spread. We provide a critical review of available methods, revealing that molecular markers currently in use for typing S. aureus acquire changes so slowly that they monitor evolutionary change over timescales that are largely irrelevant to epidemiology. Moreover, the more variable markers frequently do not reflect the pathogen's evolutionary history and, hence, provide potentially misleading information about spread. More recent work has demonstrated that staphylococcal evolution proceeds sufficiently fast that the dynamics of S. aureus spatial spread can be elucidated at great detail on the basis of genome-wide single-nucleotide polymorphisms.

• Comparison of direct cultivation on a selective solid medium, polymerase chain reaction from an enrichment broth, and the BD GeneOhm™ VanR Assay for identification of vancomycin-resistant enterococci in screening specimens.

  Authors: Guido Werner, Annerose Serr, Sabine Schütt, Christian Schneider, Ingo Klare, Wolfgang Witte, Constanze Wendt

  Diagnostic microbiology and infectious disease. 08/2011; 70(4):512-21.

  Fast and reliable diagnostics of vancomycin-resistant enterococci (VRE) is an important prerequisite for containing VRE transmission rates and controlling VRE outbreaks among hospital patients. TheFast and reliable diagnostics of vancomycin-resistant enterococci (VRE) is an important prerequisite for containing VRE transmission rates and controlling VRE outbreaks among hospital patients. The BD GeneOhm™ VanR Assay (Becton Dickinson Diagnostics, Erembodegem, Belgium) is a real-time polymerase chain reaction (PCR) assay for screening perianal/rectal samples for the presence of vanA or vanB genes that can be associated with VRE. A set of 51 reference strains (vanA-G genotypes) were correctly identified. Performance of the assay was evaluated and compared with culture-based methods and subsequent PCR analysis in 2 university hospitals with a different VRE prevalence. A total of 1786 samples were analyzed. With the use of the BD GeneOhm™ VanR Assay, 88 of 102 vanA-positive specimens, 62 of 67 vanB-positive specimens, 3 of 4 vanA- and vanB-positive specimens, and 1403 of 1613 negative specimens were correctly identified. The overall sensitivity was 93.1%; the specificity was 87.0% mainly due to false-positive vanB results. Results did not differ between study institutions.

• Emergence and spread of cfr-mediated multiresistance in staphylococci: an interdisciplinary challenge.

  Authors: Wolfgang Witte, Christiane Cuny

  Future microbiology. 08/2011; 6(8):925-31.

  In staphylococci, methylation of A2503 of 23S rRNA leads to resistance against several classes of antibiotics (oxazolidinones, phenicols, streptogramin compounds, lincosamidins and pleuromutilins).In staphylococci, methylation of A2503 of 23S rRNA leads to resistance against several classes of antibiotics (oxazolidinones, phenicols, streptogramin compounds, lincosamidins and pleuromutilins). The corresponding resistance gene cfr is located on plasmid(s) and is transferable within and between staphylococcal species including Staphylococcus aureus. It first emerged in coagulase-negative staphylococci, later in Central Europe also in S. aureus ST9 and in methicillin-resistant S. aureus ST398, which have their main reservoir in pigs, and meanwhile also in nosocomial coagulase-negative staphylococci from Southern Europe and the USA, and furthermore in nosocomial methicillin-resistant S. aureus in Spain. Timely detection and targeted prevention of further dissemination in both human and veterinary medicine is warranted for preserving the activity linezolid as an important antibiotic for treatment of staphylococcal infections.

• IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium.

  Authors: Guido Werner, Carola Fleige, Uta Geringer, Willem van Schaik, Ingo Klare, Wolfgang Witte

  BMC infectious diseases. 03/2011; 11:80.

  Hospital strains of Enterococcus faecium could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, theseHospital strains of Enterococcus faecium could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, these techniques are laborious, time-consuming and cost-intensive. Our aim was to identify hospital E. faecium strains and differentiate them from colonizing and animal variants by a simple, inexpensive and reliable PCR-based screening assay. We describe here performance and predictive value of a single PCR detecting the insertion element, IS16, to identify hospital E. faecium isolates within a collection of 260 strains of hospital, animal and human commensal origins. Specific primers were selected amplifying a 547-bp fragment of IS16. Presence of IS16 was determined by PCR screenings among the 260 E. faecium isolates. Distribution of IS16 was compared with a prevalence of commonly used markers for hospital strains, esp and hylEfm. All isolates were typed by MLST and partly by PFGE. Location of IS16 was analysed by Southern hybridization of plasmid and chromosomal DNA. IS16 was exclusively distributed only among 155 invasive strains belonging to the clonal complex of hospital-associated strains ("CC17"; 28 MLST types) and various vancomycin resistance genotypes (vanA/B/negative). The five invasive IS16-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS16 was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS16-positive human commensal isolates revealed MLST types belonging to the clonal complex of hospital-associated strains (CC17). The values predicting a hospital-associated strain ("CC17") deduced from presence and absence of IS16 was 100% and thus superior to screening for the presence of esp (66%) and/or hylEfm (46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS16. This simple screening assay for insertion element IS16 is capable of differentiating hospital-associated from human commensal, livestock- and food-associated E. faecium strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given E. faecium isolate identified within the nosocomial setting.

• Rare occurrence of methicillin-resistant Staphylococcus aureus CC130 with a novel mecA homologue in humans in Germany.

  Authors: Christiane Cuny, Franziska Layer, Birgit Strommenger, Wolfgang Witte

  PloS one. 01/2011; 6(9):e24360.

  MRSA CC130 containing the mecA homologue mecA(LGA251) were reported from the UK and from Denmark so far from cattle and humans. Here we report on 11 MRSA CC130 among a sample of 12691 isolates ofMRSA CC130 containing the mecA homologue mecA(LGA251) were reported from the UK and from Denmark so far from cattle and humans. Here we report on 11 MRSA CC130 among a sample of 12691 isolates of human origin collected from January 2006 until June 2011. MRSA CC130 grew insufficiently on chromogernic agar plates for detection of MRSA; the agglutination test for presence of PBP2a was negative. We designed primers for specific detection of mecA(LGA251) as well as for concomitant detection of both, mec(LGA251) and mecA. As already described, the isolates exhibited spa-types t843, t1736, and t1773. The ccrA homologue indicated the presence SCCmec(XI). When subjected to further characterization by means of a commercially available microarray the isolates were negative for sak chp, and scn, and as expected positive for hla, untruncated hlb, and hld. They furthermore contained edinB, aur, slpA, slpB, slpE. From genes coding for surface and cell wall associated products the ica-operon, cap8, clfA, clfF, ebpS, fnbA, fnbB, sdrC were detected but not cna. The isolates were negative for enterotoxin genes and tst, as well as for eta, and etb; agr-type was III.

• Intra- and interspecies genomic transfer of the Enterococcus faecalis pathogenicity island.

  Authors: Jenny A Laverde Gomez, Antoni P A Hendrickx, Rob J Willems, Janetta Top, Irina Sava, Johannes Huebner, Wolfgang Witte, Guido Werner

  PloS one. 01/2011; 6(4):e16720.

  Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibioticEnterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.

• Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria.

  Authors: Adebayo O Shittu, Kenneth Okon, Solayide Adesida, Omotayo Oyedara, Wolfgang Witte, Birgit Strommenger, Franziska Layer, Ulrich Nübel

  BMC microbiology. 01/2011; 11:92.

  Staphylococcus aureus is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of S. aureus infections,Staphylococcus aureus is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of S. aureus infections, it is crucial to understand the trends in the antibiotic-resistance patterns. In addition, the occurrence and changes in types of S. aureus, clonal identities, and their geographic spread is essential for the establishment of adequate infection control programmes. In this study, 68 S. aureus isolates obtained from clinical and non-clinical sources in Nigeria between January and April 2009 were characterized using phenotypic and molecular methods. All the S. aureus isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline. Sixteen percent of the isolates were resistant to oxacillin, while 55% and 72% of isolates were resistant to tetracycline and trimethoprim/sulphamethoxazole (cotrimoxazole), respectively (Table 1). There was excellent correlation between the broth microdilution assay and detection of antibiotic resistance genes by the multiplex PCR, in the determination of S. aureus resistance to erythromycin, gentamicin, methicillin and tetracycline. A total of 28 spa types were identified in the study, and the predominant spa type among the methicillin-susceptible S. aureus (MSSA) isolates was t084 (13 isolates). The t037-ST241-SCCmecIII type was the only clone identified in Maiduguri (North-East Nigeria) while in South-West Nigeria, diversity among the MRSA isolates (t451-ST8-SCCmecV; t008-ST94-SCCmecIV; t002-ST5-SCCmecV; t064-ST8-SCCmecV) was observed. The toxin genes seh and etd were detected in isolates affiliated with clonal complexes CC1, CC80 and sequence type ST25, respectively. The proportion of PVL-positive isolates among MSSA was high (40%). Most of the PVL-positive MSSA isolates were obtained from wound infections and associated with clonal complexes CC1, CC30, CC121 and with sequence type ST152. The use of phenotypic and molecular methods provided useful information on antibiotic resistance and molecular diversity of S. aureus in Nigeria. The high proportion of PVL-positive MSSA isolates affiliated to various clonal complexes and detected in all the health institutions is a major concern, both as a source of severe infections and as a potential reservoir that could lead to the emergence of PVL-positive MRSA. This study presents the first baseline information on the nature of the antibiotic resistance genes from S. aureus isolates in Nigeria. There is the need to curtail the spread and establishment of MRSA and PVL-positive MSSA clones in Nigerian health care institutions.

• Host range of enterococcal vanA plasmids among Gram-positive intestinal bacteria.

  Authors: Guido Werner, Ana R Freitas, Teresa M Coque, Johanna E Sollid, Camilla Lester, Anette M Hammerum, Lourdes Garcia-Migura, Lars B Jensen, Maria V Francia, Wolfgang Witte, R J Willems, Arnfinn Sundsfjord

  The Journal of antimicrobial chemotherapy. 12/2010; 66(2):273-82.

  The most prevalent type of acquired glycopeptide resistance is encoded by the vanA transposon Tn1546 located mainly on transferable plasmids in Enterococcus faecium. The limited occurrence in otherThe most prevalent type of acquired glycopeptide resistance is encoded by the vanA transposon Tn1546 located mainly on transferable plasmids in Enterococcus faecium. The limited occurrence in other species could be due to the lack of inter-species transferability and/or stability of Tn1546-containing plasmids in other species. We investigated the in vitro transferability of 14 pre-characterized vanA-containing plasmids hosted by E. faecium (n = 9), Enterococcus faecalis (n = 4) and Enterococcus raffinosus (n = 1) into several enterococcal, lactobacterial, lactococcal and bifidobacterial recipients. A filter-mating protocol was harmonized using procedures of seven partner laboratories. Donor strains were mated with three E. faecium recipients, three E. faecalis recipients, a Lactobacillus acidophilus recipient, a Lactococcus lactis recipient and two Bifidobacterium recipients. Transfer rates were calculated per donor and recipient. Transconjugants were confirmed by determining their phenotypic and genotypic properties. Stability of plasmids in the new host was assessed in long-term growth experiments. In total, 282 enterococcal matings and 73 inter-genus matings were performed and evaluated. In summary, intra-species transfer was far more frequent than inter-species transfer, if that was detectable at all. All recipients of the same species behaved similarly. Inter-genus transfer was shown for broad host range control plasmids (pIP501/pAMβ1) only. Acquired resistance plasmids remained stable in the new host. Intra-species transfer of enterococcal vanA plasmids was far more frequent than transfer across species or genus barriers and may thus explain the preferred prevalence of vanA-containing plasmids among E. faecium. A reservoir of vanA plasmids in non-enterococcal intestinal colonizers does not seem to be reasonable.

• Identification of specific genes in Staphylococcus aureus strains associated with bovine mastitis.

  Authors: Svitlana Kozytska, Dennis Stauss, Marie-Christin Pawlik, Selma Hensen, Martin Eckart, Wilma Ziebuhr, Wolfgang Witte, Knut Ohlsen

  Veterinary microbiology. 10/2010; 145(3-4):360-5.

  Staphylococcus aureus is a common cause of bovine mastitis that is responsible for the main economic loss to the dairy industry. For identification of putative, bovine-specific molecular marker aStaphylococcus aureus is a common cause of bovine mastitis that is responsible for the main economic loss to the dairy industry. For identification of putative, bovine-specific molecular marker a genome comparison between bovine S. aureus strain RF122 and 52 previously sequenced S. aureus isolates associated with human infections using genome viewer, annotation tool Artemis Comparison Tool (ACT), KEGG and NCBI BLAST databases was carried out. This led to the identification of 16 unique RF122 gene sequences that may be used as molecular marker to distinguish bovine from human strains. The distribution of these genes was analyzed in a collection of bovine mastitis strains from the Netherlands and human clinical isolates by PCR and Southern blotting. Only four genes within the pathogenicity island SaPIbov3 (sab1890, sab1891, sab1892, sab1893) were present in the majority of isolates from cattle but were absent from human clinical S. aureus isolates. These results suggest that there is no gene/ORF uniformly shared by all bovine S. aureus strains that could be uniformly used as a diagnostic marker gene.

• A multiresistance megaplasmid pLG1 bearing a hylEfm genomic island in hospital Enterococcus faecium isolates.

  Authors: Jenny A Laverde Gomez, Willem van Schaik, Ana R Freitas, Teresa M Coque, Keith E Weaver, Maria Victoria Francia, Wolfgang Witte, Guido Werner

  International journal of medical microbiology : IJMM. 10/2010; 301(2):165-75.

  Enterococcus faecium is considered to be a nosocomial pathogen with increasing medical importance. The putative virulence factor, hyl(Efm), encoding a putative hyaluronidase, is enriched among theEnterococcus faecium is considered to be a nosocomial pathogen with increasing medical importance. The putative virulence factor, hyl(Efm), encoding a putative hyaluronidase, is enriched among the hospital-associated polyclonal subpopulation of E. faecium.. The hyl(Efm) gene is described to be part of a genomic island and was recently identified to be plasmid-located. Here, we present a description of the structure, localization, and distribution of the putative pathogenicity factor hyl(Efm) and its putative island among 39 clinical isolates and elucidate the composition and host range of pLG1, a hyl(Efm) multiresistance plasmid of approximately 281.02kb. The hyl(Efm) gene was located within a 17,824-bp element highly similar to the putative genomic island (GI) structure that had been previously described. This genomic region was conserved among 39 hyl(Efm)-positive strains with variation in a specific region downstream of hyl(Efm) in 18 strains. The putative hyl(Efm) was located on large plasmids (150-350kb) in 37 strains. pLG1 could be horizontally transferred into four different E. faecium recipient strains (n=4) but not into E. faecalis (n=3). Sequencing of pLG1 resolved putative plasmid replication, conjugation, and maintenance determinants as well as a pilin gene cluster, carbon uptake and utilization genes, heavy metal and antibiotic resistance clusters. The hyl(Efm) transferable plasmid pLG1 bears additional putative pathogenicity factors and antibiotic resistance genes. These findings suggest horizontal gene transfer of virulence factors and antibiotic resistance gene clusters by a single genetic event (conjugative transfer) which might be triggered by heavy antibiotic use common in health care units where E. faecium is increasingly prevalent.

• A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus.

  Authors: Ulrich Nübel, Janina Dordel, Kevin Kurt, Birgit Strommenger, Henrik Westh, Sanjay K Shukla, Helena Zemlicková, Raphaël Leblois, Thierry Wirth, Thibaut Jombart, François Balloux, Wolfgang Witte

  PLoS pathogens. 01/2010; 6(4):e1000855.

  Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a keyDue to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics.

• High-level ciprofloxacin resistance among hospital-adapted Enterococcus faecium (CC17).

  Authors: Guido Werner, Carola Fleige, Birgit Ewert, Jenny A Laverde Gomez, Ingo Klare, Wolfgang Witte

  International journal of antimicrobial agents. 12/2009;

  Hospital-adapted Enterococcus faecium differ from their colonising variants in humans and animals by additional genomic content. Molecular typing based on multilocus sequence typing (MLST) allowsHospital-adapted Enterococcus faecium differ from their colonising variants in humans and animals by additional genomic content. Molecular typing based on multilocus sequence typing (MLST) allows allocation of isolates to specific clonal complexes (CCs), such as CC17 for hospital-adapted strains. Acquired ampicillin resistance is a specific feature of these hospital isolates, especially in Europe. A few recent reports have described acquired high-level ciprofloxacin resistance as a supposed feature of hospital-adapted E. faecium strains. In the present retrospective analysis, ciprofloxacin minimum inhibitory concentrations (MICs) of 609 clinical isolates from German hospital patients (1997-2007) were determined and a breakpoint for high-level resistance was deduced (>16mg/L). Acquired high-level ciprofloxacin resistance was distributed among isolates of 26 different MLST types (all CC17), indicating a wide prevalence of this acquired resistance trait among the hospital-adapted E. faecium population. High-level ciprofloxacin resistance was linked to gyrA and parC mutations in 98 investigated isolates. Eleven different allele types or combinations thereof were identified. Their allocation to specific MLST and pulsed-field gel electrophoresis (PFGE) types revealed differences in the emergence and spread of corresponding mutations and strains.

• Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species.

  Authors: Christiane Cuny, Alexander Friedrich, Svetlana Kozytska, Franziska Layer, Ulrich Nübel, Knut Ohlsen, Birgit Strommenger, Birgit Walther, Lothar Wieler, Wolfgang Witte

  International journal of medical microbiology : IJMM. 12/2009;

  The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in animals such as horses, pet animals and productive livestock has raised questions of a probable human origin and in more generalThe emergence of methicillin-resistant Staphylococcus aureus (MRSA) in animals such as horses, pet animals and productive livestock has raised questions of a probable human origin and in more general of host specificity of S. aureus. Particular clonal lineages are obviously specific for humans (e.g. ST15, ST25, ST45) and other for ruminants (e.g. ST151). MRSA associated with veterinary nosocomial infections (e.g. ST8 and ST254 in horses, ST22 in small animals) very likely have their origin in health care facilities. MRSA ST398 which became first known from widespread colonization in industrially raised pigs seems to have a limited host specificity and is able to colonize and to cause infections in various hosts. Mechanisms of host adaptation and their genomic background are poorly understood so far.

• Is there an association between nosocomial infection rates and bacterial cross transmissions?*

  Authors: Axel Kola, Frank Schwab, Sina Bärwolff, Tim Eckmanns, Klaus Weist, Elke Dinger, Ingo Klare, Wolfgang Witte, Henning Ruden, Petra Gastmeier

  Critical care medicine. 09/2009;

  OBJECTIVE:: Surveillance data of nosocomial infection rates are increasingly used for public reporting and interhospital comparisons. Approximately 15% of nosocomial infections on intensive careOBJECTIVE:: Surveillance data of nosocomial infection rates are increasingly used for public reporting and interhospital comparisons. Approximately 15% of nosocomial infections on intensive care units are the result of patient-to-patient transmissions of the causative organisms. These exogenous infections could be prevented by adherence to basic infection control measures. The association between bacterial cross transmissions and nosocomial infection rates was analyzed. DESIGN:: Prospective cohort study during 24 months. SETTING:: Eleven intensive care units from two university hospitals. PATIENTS:: All inpatients. INTERVENTIONS:: None. MEASUREMENTS AND MAIN RESULTS:: Primary isolates of six indicator organisms (Acinetobacter baumannii, Enterococcus faecalis and E. faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) cultured from clinical samples or methicillin-resistant S. aureus surveillance testing of all inpatients were genotyped. Indistinguishable isolates in >/=2 patients defined potential episodes of transmissions. Surveillance of nosocomial infection rates was performed according to the German nosocomial infection surveillance system, Krankenhaus Infektions Surveillance System (KISS). Transmission events and nosocomial infection rates were pooled by intensive care unit to calculate Spearman's rank-correlation test. During 100,781 patient days, 100,829 microbiological specimens from 24,362 patients were sampled (average investigation density: 1.0 sample per patient and day) and 3419 primary indicator organisms were cultured. Altogether, 462 transmissions (incidence density of 4.6 transmissions per 1000 patient days; range, 1.4-8.4 days) and 1216 nosocomial infections (incidence density of 12.1 per 1000 patient days; range, 6.2-16.6 days) were discerned. Correlation analysis was unable to reveal any association between the incidence of cross transmissions and nosocomial infections, duration of hospitalization, or device use. CONCLUSIONS:: Differences in nosocomial infection rates between study intensive care units are not explained solely by cross transmissions. Other factors, like the severity of the patient's underlying diseases, the patient's endogenous flora, or invasive procedures, likely have a dominant effect on the magnitude of nosocomial infection rates.

• Multiplexed genotyping of methicillin-resistant Staphylococcus aureus isolates by use of padlock probes and tag microarrays.

  Authors: Kevin Kurt, Anders Alderborn, Mats Nilsson, Birgit Strommenger, Wolfgang Witte, Ulrich Nübel

  Journal of clinical microbiology. 02/2009;

  We developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay usesWe developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay uses oligonucleotide padlock probes whose two ends are joined through ligation when they hybridize to matching target DNA. Circularized probes are subsequently amplified by PCR with common primers and analysed by using a microarray equipped with universal tag probes. Our set of padlock probes includes oligonucleotides targeting diagnostic regions in mecA, ccrB, and ccrC genes of the SCCmec cassette in methicillin resistant S. aureus (MRSA). These probes determine the presence and type of SCCmec cassettes (i. e., SCCmec types I to VI). Additional oligonucleotides interrogate a number of highly informative single nucleotide polymorphisms retrieved from a multilocus sequence typing (MLST) database. These latter probes enable the exploration of isolates' phylogenetic affiliation to clonal lineages of MRSA as revealed by MLST. The described assay enables multiplexed genotyping of MRSA, based on a single-tube reaction. On a set of clinical isolates of MRSA and methicillin-susceptible S. aureus (n=66), 100% typability and 100% accuracy were achieved. The current assay as reported here provides valuable genotypic information that may usefully complement existing genotyping procedures. Moreover, the assay is easily extendable by incorporating additional padlock probes and will be valuable to quickly and cost-effectively probe large numbers of polymorphisms at different genomic locations, such as those ascertained through currently ongoing mutation discovery and genome re-sequencing projects.

• Nasal colonization of humans with methicillin-resistant Staphylococcus aureus (MRSA) CC398 with and without exposure to pigs.

  Authors: Christiane Cuny, Rolf Nathaus, Franziska Layer, Birgit Strommenger, Doris Altmann, Wolfgang Witte

  PloS one. 02/2009; 4(8):e6800.

  BACKGROUND: Studies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure toBACKGROUND: Studies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure to colonized animals. The study presented here addresses the question of further transmission to non exposed humans. METHODS: After selecting 47 farms with colonized pigs in different regions of Germany we sampled the nares of 113 humans working daily with pigs and of their 116 non exposed family members. The same was performed in 18 veterinarians attending pig farms and in 44 of their non exposed family members. For investigating transmission beyond families we samples the nares of 462 pupils attending a secondary school in a high density pig farming area. MRSA were detected by direct culture on selective agar. The isolates were typed by means of spa-sequence typing and classification of SCCmec elements. For attribution of spa sequence types to clonal lineages as defined by multi locus sequence typing we used the BURP algorithm. Antibiotic susceptibility testing was performed by microbroth dilution assay. RESULTS: At the farms investigated 86% of humans exposed and only 4.3% of their family members were found to carry MRSA exhibiting spa-types corresponding to clonal complex CC398. Nasal colonization was also found in 45% of veterinarians caring for pig farms and in 9% of their non exposed family members. Multivariate analysis revealed that antibiotic usage prior to sampling beard no risk with respect to colonization. From 462 pupils only 3 were found colonized, all 3 were living on pig farms. CONCLUSION: These results indicate that so far the dissemination of MRSA CC398 to non exposed humans is infrequent and probably does not reach beyond familial communities.

• Methicillin-Resistant and -Susceptible Staphylococcus aureus of Clonal Lineages ST398 and ST9 From Swine Carry the Multidrug Resistance Gene cfr.

  Authors: Corinna Kehrenberg, Christiane Cuny, Birgit Strommenger, Stefan Schwarz, Wolfgang Witte

  Antimicrobial agents and chemotherapy. 01/2009;

  Methicillin-resistant Staphylococcus aureus clonal lineage ST398 and methicillin-susceptible lineage ST9 have their main reservoir in swine, but can colonize and cause infections in humans. TheMethicillin-resistant Staphylococcus aureus clonal lineage ST398 and methicillin-susceptible lineage ST9 have their main reservoir in swine, but can colonize and cause infections in humans. The phenicol/lincosamide/-oxazolidinone/pleuromutilin/streptogramin A multidrug resistance gene cfr was detected in isolates of both clonal lineages, rendering a spread to humans with exposure to swine farming possible.