The influence of drug distribution and drug-target binding on target occupancy: The rate-limiting step approximation.

The influence of drug-target binding kinetics on target occupancy can be influenced by drug distribution and diffusion around the target, often referred to as "rebinding" or "diffusion-limited binding". This gives rise to a decreased decline of the drug-target complex concentration as a result of a locally higher drug concentration that arises around the target, which leads to prolonged target exposure to the drug. This phenomenon has been approximated by the steady-state approximation, assuming a steady-state concentration around the target. Recently, a rate-limiting step approximation of drug distribution and drug-target binding has been published. However, a comparison between both approaches has not been made so far. In this study, the rate-limiting step approximation has been rewritten into the same mathematical format as the steady-state approximation in order to compare the performance of both approaches for the investigation of the influence of drug-target binding kinetics on target occupancy. While both approximations clearly indicated the importance of kon and high target concentrations, it was shown that the rate-limiting step approximation is more accurate than the steady-state approximation, especially when dissociation is fast compared to association and distribution out of the binding compartment. It is therefore concluded that the new rate-limiting step approximation is to be preferred for assessing the influence of binding kinetics on local target site concentrations and target occupancy.

Assessment of Interspecies Differences in Drug-Induced QTc Interval Prolongation in Cynomolgus Monkeys, Dogs and Humans.

BACKGROUND AND PURPOSE: The selection of the most suitable animal species and subsequent translation of the concentration-effect relationship to humans are critical steps for accurate assessment of the pro-arrhythmic risk of candidate molecules. The objective of this investigation was to assess quantitatively the differences in the QTc prolonging effects of moxifloxacin between cynomolgus monkeys, dogs and humans. The impact of interspecies differences is also illustrated for a new candidate molecule. EXPERIMENTAL APPROACH: Pharmacokinetic data and ECG recordings from pre-clinical protocols in monkeys and dogs and from a phase I trial in healthy subjects were identified for the purpose of this analysis. A previously established Bayesian model describing the combined effect of heart rate, circadian variation and drug effect on the QT interval was used to describe the pharmacokinetic-pharmacodynamic relationships. The probability of a ≥ 10 ms increase in QT was derived as measure of the pro-arrhythmic effect. KEY RESULTS: For moxifloxacin, the concentrations associated with a 50% probability of QT prolongation ≥ 10 ms (Cp50) varied from 20.3 to 6.4 and 2.6 µM in dogs, monkeys and humans, respectively. For NCE05, these values were 0.4 µM vs 2.0 µM for monkeys and humans, respectively. CONCLUSIONS AND IMPLICATIONS: Our findings reveal significant interspecies differences in the QT-prolonging effect of moxifloxacin. In addition to the dissimilarity in pharmacokinetics across species, it is likely that differences in pharmacodynamics also play an important role. It appears that, regardless of the animal model used, a translation function is needed to predict concentration-effect relationships in humans.

State-wide surveillance of antibiotic resistance patterns and spa types of methicillin-resistant Staphylococcus aureus from blood cultures in North Rhine-Westphalia, 2011-2013.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of bacteraemia. We aimed to obtain a complete picture of severe MRSA infections by characterizing all MRSA isolates from bloodstream infections in the largest German federal state (North Rhine-Westphalia, 18 million inhabitants) using S. aureus protein A (spa) sequence-typing and antimicrobial susceptibility testing. MRSA isolates (n = 1952) were collected prospectively (2011-2013) and spa-typed. Among 181 different spa types, t003 (n = 746 isolates; 38.2%) and t032 (n = 594; 30.4%) were predominant. Analysis of the geographical occurrence of spa clonal complexes (spa-CCs) and spa types revealed divergent distribution between federal state districts for spa-CCs 003 (p < 0.001; including t003, p < 0.001 and t264, p < 0.001), 008 (p 0.021), 011 (p 0.002), 032 (p < 0.001; including t022, p 0.014 and t032, p < 0.001) and spa type t2807 (p < 0.001). MICs of antimicrobial substances were tested using broth microdilution. Of all isolates, 96% were resistant to fluoroquinolones, 78% to erythromycin, 70% to clindamycin, 4% to gentamicin, 2% to rifampicin, 0.4% to daptomycin, 0.1% to linezolid and 0% to vancomycin, respectively. Vancomycin MICs of 2 mg/L involved 0.5% of the isolates. In conclusion, the detection of regional molecular clusters added valuable information for epidemiological case tracing and allowed conclusions to be reached on the importance of newly emerging MRSA reservoirs, such as livestock (spa-CC011), for MRSA bacteraemia in some parts of the federal state. Susceptibility testing revealed broad resistance to substances used for oral treatment, but demonstrated that those antibiotics that are mostly applied for treatment of MRSA bacteraemia and important combination partners were highly susceptible.

Systematic literature analysis and review of targeted preventive measures to limit healthcare-associated infections by meticillin-resistant Staphylococcus aureus.

Meticillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections in Europe. Many examples have demonstrated that the spread of MRSA within healthcare settings can be reduced by targeted infection control measures. The aim of this systematic literature analysis and review was to summarise the evidence for the use of bacterial cultures for active surveillance the benefit of rapid screening tests, as well as the use of decolonisation therapies and different types of isolation measures. We included 83 studies published between 2000 and 2012. Although the studies reported good evidence supporting the role of active surveillance followed by decolonisation therapy, the effectiveness of single-room isolation was mostly shown in non-controlled studies, which should inspire further research regarding this issue. Overall, this review highlighted that when planning the implementation of preventive interventions, there is a need to consider the prevalence of MRSA, the incidence of infections, the competing effect of standard control measures (e.g. hand hygiene) and the likelihood of transmission in the respective settings of implementation.

Phenotyping of Staphylococcus aureus reveals a new virulent ST398 lineage.

Staphylococcus aureus sequence type (ST)398, which is commonly found as a colonization strain in pig farming, is emerging more frequently as the cause of human infections. In this study, we analysed ST398 of porcine and human origin at the genetic, protein and immunogenic levels. Although genetic analysis of the genes encoding the major virulence factors revealed the presence of the same genes in all strains studied, the results demonstrated spa type crossing alterations in adhesion abilities in addition to a strongly enhanced lysis activity directly linked to impaired clearance attributable to polymorphonuclear leukocytes (PMNs). This change in virulence pattern indicates high heterogenicity in the ST398 pool that is not based on a different genetic make-up, but probably on variations in the genetic regulation systems. These modifications, which are tightly connected to pathogenicity, cannot be detected by conventional diagnostic methods.

[Current data and trends on methicillin-resistant Staphylococcus aureus (MRSA)]. / Aktuelle Daten und Trends zu Methicillin-resistenten Staphylococcus aureus (MRSA).

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a problem in hospital settings worldwide. The National Reference Centre for Staphylococci performs molecular typing on a representative sample set of MRSA isolates from German hospitals for assessing long-term trends thus following the dynamics of emergence and spread of MRSA clones. The article focuses on recent data concerning antibiotic resistance and epidemic MRSA in nosocomial settings and also reflects the impact of community-acquired MRSA and MRSA from zoonotic reservoirs. Identifying common and newly emerging clones is an on-going challenge in the changing epidemiology of MRSA and prevention of further spread needs molecular surveillance.

[Vancomycin-resistant enterococci (VRE). Recent results and trends in development of antibiotic resistance]. / Vancomycin-resistente Enterokokken (VRE). Aktuelle Daten und Trends zur Resistenzentwicklung.

Enterococci (mainly E. faecalis, E. faecium) are important nosocomial pathogens predominantly affecting older and/or immunocompromised patients. The bacteria possess a broad spectrum of intrinsic and acquired antibiotic resistance properties. Among these, the transferrable glycopeptide resistance of the vanA and vanB genotypes in vancomycin-resistant enterococci (VRE; reservoir: E. faecium) as well as resistance to last resort antibiotics (e.g. linezolid and tigecycline) are of special concern. Enterococci (including VRE) are easily transferred in hospitals; however, colonizations are far more frequent than infections. Resistance frequencies for vancomycin in clinical E. faecium isolates have remained at a relatively constant level of 8-15% (but with local or regional variations) in recent years whereas frequencies for teicoplanin resistance have shown a slight decrease. Glycopeptide resistance trends correlate with a spread of hospital-associated E. faecium strains carrying the vanA and, with rising frequency in recent years, the vanB gene cluster, the latter being associated with teicoplanin susceptibility. This increased occurrence of vanB-positive E. faecium strains may be caused by an increased use of antibiotics selecting enterococci and VRE as well as due to methodological reasons (e.g. reduced EUCAST MIC-breakpoints for glycopeptides; increased use and sensitive performance of chromogenic VRE agars, increased use of molecular diagnostic assays).

High prevalence of extended-spectrum-ß-lactamase-producing Enterobacteriaceae in organic and conventional retail chicken meat, Germany.

OBJECTIVES: To determine the prevalence of extended-spectrum ß-lactamase (ESBL) production in Enterobacteriaceae in retail chicken meat in Germany. METHODS: A total of 399 chicken meat samples from nine supermarket chains, four organic food stores and one butcher's shop in two geographically distinct regions (Berlin and Greifswald) were screened for ESBL production using selective agar. Phenotypic ESBL isolates were tested for bla(TEM), bla(CTX-M) and bla(SHV) genes using PCR and DNA sequencing. Antibiotic coresistances were determined and strain typing was performed using PCR-based phylogenetic grouping and XbaI-PFGE. RESULTS: A total of 185 confirmed ESBL isolates were obtained from 175 samples (43.9%) from all tested sources. The majority of isolates were Escherichia coli producing ESBL types SHV-12 (n = 82), CTX-M-1 (n = 77) and TEM-52 (n = 16). No differences could be observed in the prevalence of ESBL producers between organic and conventional samples. 73.0% of the ESBL producers showed coresistance to tetracycline, 35.7% to co-trimoxazole and 7.6% to ciprofloxacin. Strain typing of selected E. coli isolates from Berlin revealed identical macrorestriction patterns for several isolates from samples taken from the same stores. CONCLUSIONS: This is the first comprehensive study from Germany showing a high prevalence of TEM-, CTX-M- and SHV-type ESBLs in Enterobacteriaceae isolated from retail chicken meat. The high rate of coresistance to different classes of antibiotics in the ESBL producers might reflect the common veterinary usage of these and related substances. There is an urgent need to further evaluate the role of poultry in the transmission of highly resistant ESBL-producing bacteria in humans.

Evaluation of adding a second marker to overcome Staphylococcus aureus spa typing homoplasies.

The utility of sequencing a second highly variable locus in addition to the spa gene (e.g., double-locus sequence typing [DLST]) was investigated to overcome limitations of a Staphylococcus aureus single-locus typing method. Although adding a second locus seemed to increase discriminatory power, it was not sufficient to definitively infer evolutionary relationships within a single multilocus sequence type (ST-5).

[The influenza pandemic 1968-1970: crisis management in separated Germany - "Vodka and Raspberry Tea"]. / Die Grippepandemie 1968-1970: Strategien der Krisenbewältigung im getrennten Deutschland. "Wodka und Himbeertee".

The Hong Kong Flu in the years 1968-1970 challenged both German health care systems. This article intends to analyse the patterns of reaction to the pandemic. Both German states faced the threat according to their respective ideological orientation. This applied to the two parts of Berlin - West and East - as well. In the GDR the control of influenza was centrally organized. When the pandemic passed away an influenza guiding document ("Führungsdokument") was made obligatory for the fight against the plague. In the FRG hospital treatment maintained predominance while the outpatient sector was administrated by physicians in private practice. In West- Berlin outpatient clinics were declined by the Association of Physicians ("Kassenärztliche Vereinigung"). In 1970 a first concept of surveillance was presented on the level of the state in West Germany. In the years 1968-1970 vaccinations were not common in both German states. The essay is based on the analysis of archival sources, monographs, scientific and newspaper articles.

[Pain and anesthesiology : aspects of the development of modern pain therapy in the twentieth century]. / Schmerz und Anästhesiologie : Aspekte der Entwicklung der modernen Schmerztherapie im 20. Jahrhundert.

The connection between the development of anesthesiology and pain therapy in the twentieth century is close. The optimistic idea to overcome pain by using general anesthesia derives from the nineteenth century. Treatment of nonsurgical pain remained in the background for a long time and innovations in pain medicine did not improve the insufficient care for patients with postoperative pain. Therapy of chronic pain was mainly surgical and the extreme of this surgical approach was psychosurgery. In the years following World War II leucotomy and lobotomy were established as methods to separate the psychological processing of pain from the experience of pain. Meanwhile, the French "pain surgeon" René Leriche elaborated a theory of pain where chronic pain was no longer seen as a symptom but as a "douleur-maladie", a pain disease. His theory was considered on various occasions but did not gain acceptance before the 1950s. Research in anesthesiology, such as that conducted by the American scientist Henry Beecher separated psyche and physiology with respect to pathological pain. This was contrasted by the approach of clinical anesthesia to pain therapy, which was based on regional anesthesia. The first "pain clinics" were "nerve block clinics". John Bonica, a regional anesthesiologist, extended the framework of pain therapy by introducing multidisciplinary teamwork into the therapy of chronic pain. From today's viewpoint his 1953 monograph The Management of Chronic Pain is a milestone in the development of modern pain therapy. However, Bonica's work did not attain major importance until 1960 when he was appointed to a newly established chair. Gradually, chronic pain was recognized as an independent illness and differentiated as such from acute pain. In 1965 the gate control theory by Melzack and Wall offered a possible explanation for the mechanisms of chronic pain. By the end of the 1970s the spectrum was extended to the biopsychosocial approach which was foremost developed by the American psychiatrist George Engel, defined chronic pain as an illness rather than a disease. Concurrently, the radical behaviorism of the late 1960s affected both the therapy of chronic and of acute pain. Based on this theory, patient-controlled analgesia (PCA) was introduced in the 1970s and 1980s. Acute pain services (APS) in hospitals, were developed beginning in the 1980s using the continuous release of opioids. Regional anesthesia played a greater role than general anesthesia in developing pain therapy in the twentieth century and paved the way for pain therapy. The restriction to nerve blocks in pain centers was overcome by the expansion of theoretical foundations beyond the framework of anesthesiology. Impulses from psychology and psychosomatic medicine were crucial. The evolution of cancer pain therapy was distinct from non-cancer pain therapy.

Recognition of Clostridium difficile PCR-ribotypes 001, 027 and 126/078 using an extended MALDI-TOF MS system.

During the last decade, Clostridium difficile infection (CDI) increased markedly inside as well as outside of hospitals. In association with the occurrence of new hypervirulent C. difficile strains, CDI became more important. Until now typing of C. difficile strains has been enabled by PCR-ribotyping. However, this method is restricted to specialized laboratories combined with high maintenance cost. Therefore, we tested MALDI-TOF mass spectrometry for typing of C. difficile to provide a fast method for surveillance of CDI. Using a standard set of 25 different C. difficile PCR ribotypes a database was made by different mass spectra recorded in the SARAMIS software (AnagnosTec, Zossen, Germany). The database was validated with 355 C. difficile strains belonging to 29 different PCR ribotypes collected prospectively from all submitted feces samples in 2009. The most frequent PCR ribotypes were type 001 (70%), 027 (4.8%) and 078/126 (4.7%). All three types were recognized by MALDI-TOF MS. We conclude that an extended MALDI-TOF system was capable to recognize specific markers for ribotypes 001, 027 and 078/126 allowing an effective identification of these strains.

[Anesthesia and Angelman syndrome]. / Anästhesie beim Angelman-Syndrom.

Angelman syndrome (AS) is a rare neurodevelopmental disorder with an incidence of 1:10,000-1:40,000 caused by deficient genetic imprinting in the chromosomal segment 15q11-q13. Experimental data suggest that the gamma-aminobutyric acid A (GABA(A)) receptor as well as the N-methyl-D-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors may be affected by this condition. The first description of the syndrome goes back to 1965 when the British pediatrician Harry Angelman (1915-1996) recognized similar clinical features in three children. Angelman's description of puppet children was changed to happy puppet syndrome 2 years later before this euphemistic denotation was replaced by the concept Angelman syndrome over the years. Angelman syndrome is characterized by ataxia, jerky movements especially hand flapping, a seizure disorder with a characteristic electroencephalogram (EEG), severe learning difficulties, a happy disposition, lack of verbal communication and dysmorphic facial features. Most hospitalizations are caused by epilepsy and the most common indications for surgical procedures are in dental medicine. The first anesthesiology case report to be published dates back to 2001. A total of 13 cases have now been published and in 11 cases the age was registered (mean age 11.6 years, standard deviation 11.7 and 2 outliers aged 27 and 40 years). In this paper, the published case reports are contrasted with 15 cases of anesthesia in 6 patients with AS who underwent surgery during 14 years of routine operations at a Berlin anesthesiology clinic (mean age 15.9 years, standard deviation 4.2 with no outliers). Besides neurosurgical and orthopedic operations most were dental interventions. Summarized, these cases of anesthesia and the results of the published case reports allow the formulation of guidelines for administration of anesthesia in AS cases but do not permit conclusions on which method of anesthesia is the safest for AS patients. For the preoperative consultation and anesthetization, communication with the patients requires the aid of parents or other relatives. Water and reflecting surfaces may be used to gain contact with AS patients. Patients with AS feel pain like any other person although they are frequently smiling and laughing and this has to be considered especially in major surgery (e.g. scoliosis surgery). The most important life-threatening complication is bradycardia due to vagal hypertonia which can lead to asystole with delayed response to atropine. None of the Berlin patients had severe bradycardia but the complication has to be taken into consideration. The use of drugs to ensure complete reversal of neuromuscular relaxation should be avoided because anticholinergic agents could cause bradycardia. The use of sugammadex in cases of AS has not been tested. To avoid elevation of the vagal tone, the indications for laparascopy have to be considered very carefully. There is no evidence that any drug or hypnotic may be more appropriate or advantageous. Balanced anesthesia and total intravenous anesthesia are possible but the duration of drug effect has to be taken into account. If ketamine is used the side-effects of the drug (psychomimetic reactions, muscular rigidity) should be prevented by the consistent administration of propofol, midazolam or thiopental. Usually AS patients are agitated so that regional anesthesia techniques are difficult to administer. If regional anesthesia does have considerable advantages over general anesthesia in a particular case, peripheral regional anesthesia should be preferred, especially because scoliosis is often present. There is no evidence that AS patients cause more intubation problems but because of facial dysmorphia accurate evaluation is needed in advance. This is even more important for older AS patients because the dysmorphia tends to accelerate during the course of life. Although epilepsy is the primary feature of AS, not every EEG alteration indicates the presence of epilepsy. The advantage in using neuromonitoring for measuring the depth of anesthesia is limited. Administration of anticonvulsants must be continued if they were used preoperatively.

Nosocomial Staphylococcal scalded skin syndrome caused by intra-articular injection.

BACKGROUND: The pathogenic role of nasal carriage as a source for cutaneous and soft-tissue Staphylococcus aureus (SA) infections, and Staphylococcal scalded skin syndrome (SSSS) in particular, is unclear. OBSERVATION: We herein describe a nosocomial outbreak of SSSS in three orthopaedic patients who received intra-articular injections by a single orthopaedic surgeon. Bacteriological samples from the index patients and medical personnel involved in their care were assessed by phage typing, polymerase chain reaction for exfoliative toxin genes, SmaI macro-restriction analysis and molecular spa-typing. These studies first revealed SA cultural growth in synovial fluid of all three patients as well as nasal mucosa of one medical assistant. Moreover, all SA isolates had the same phage typing and antibiotic susceptibilities and were positive for exfoliative toxin ETa by polymerase chain reaction. SmaI macro-restriction and spa-typing further confirmed all proband isolates to be identical. CONCLUSION: These findings provide evidence that SA nasal colonization of otherwise healthy carriers is a risk factor for SA infections, including SSSS, in predisposed individuals.

Methicillin-resistant Staphylococcus aureus (MRSA): burden of disease and control challenges in Europe.

Methicillin-resistant Staphylococcus aureus (MRSA) isa major cause of healthcare- and community-associated infections worldwide. Within the healthcare setting alone, MRSA infections are estimated to affect more than 150,000 patients annually in the European Union (EU), resulting in attributable extra in-hospital costs of EUR 380 million for EU healthcare systems. Pan-European surveillance data on bloodstream infections show marked variability among EU Member States in the proportion of S. aureus that are methicillin-resistant, ranging from less than 1% to more than 50%. In the past five years, the MRSA bacteraemia rates have decreased significantly in 10 EU countries with higher endemic rates of MRSA infections. In addition to healthcare-associated infections, new MRSA strains have recently emerged as community and livestock-associated human pathogens in most EU Member States. The prevention and control of MRSA have therefore been identified as public health priorities in the EU. In this review, we describe the current burden of MRSA infections in healthcare and community settings across Europe and outline the main threats caused by recent changes in the epidemiology of MRSA. Thereby, we aim at identifying unmet needs of surveillance, prevention and control of MRSA in Europe.

[Epidemiology of severe Clostridium difficile infections in Hesse, Germany in 2008-2009]. / Schwer verlaufende Clostridium-difficile-Infektionen in Hessen 2008 - 2009.

BACKGROUND AND OBJECTIVE: Clostridium difficile is a major cause of health care associated infections in industrialized countries. During the past decade, the incidence and clinical severity of C. difficile infections (CDI) have increased markedly. This increase has been associated with the emergence of a possibly highly virulent strain, the C. difficile PCR ribotype 027. We investigated the emergence of severe CDI and the associated PCR ribotypes in Hesse, Germany. PATIENTS AND METHODS: We conducted a retrospective analysis of clinical information and ribotyping results of all cases of severe CDI that were reported to the Hesse State Health Office or sent to our microbiologic diagnostic laboratory for detection and molecular typing of C. difficile in severe cases of CDI from 01/2008 to 12/2009. The data of a of 88 patients and 50 isolates were analysed. RESULTS: 89% of patients were at least 65 years old; the mean age was 77 years. The clinical outcome was known in 85 patients. 27% had died within 30 days of the diagnosis of CDI. Ribotyping results were available in 39 and 11 patients from 2008 and 2009, respectively. The isolates were assigned to nine different ribotypes. RT 027 and RT 001 were the most frequent ribotypes with 31 and 10 isolates, respectively. All other ribotypes were isolated once or twice. CONCLUSION: Our data indicate that C. difficile RT 027 and RT 001 are prevalent in Hesse and are often associated with severe or notifiable CDI. The high prevalence of RT 027 among the reported CDI cases does not indicate a generally high prevalence of the latter strain in Hesse, because detection of RT 027 was a case definition criterion, a fact that may cause a bias in the reported data. Further investigation would help to improve our understanding of the molecular epidemiology of severe CDI and to improve the prevention strategies.

[Emergence of clostridium difficile ribotype 027 in Germany: epidemiological and clinical characteristics]. / Clostridium-difficile-Ribotyp 027: Epidemiologie und Klinik des erstmaligen endemischen Auftretens in Deutschland.

INTRODUCTION: In September 2007 an increase of severe Clostridium difficile-associated infections (CDI) was noticed in a hospital in the city of Trier, Germany. It was assumed that a new, possibly hypervirulent strain (PCR ribotype 027) was related to these events. An outbreak investigation was initiated by the local health authorities and the Robert Koch Institute to describe the epidemiology of the possible outbreak and to identify and control the possible sources. METHODS: In addition to retrospective case-finding of severe CDI and ribotype 027 infections by analysis of patient documents and certificates of death, an active surveillance system for severe CDI and ribotype 027 infections was established in the 6 hospitals of the affected region. In all suspected cases, a test for toxin A/B and a stool culture for C. difficile were conducted simultaneously. Bacterial isolates were further characterised by PCR ribotyping. Data on the course of disease, case fatality, and possible risk factors for CDI-related deaths were assessed using a standardised questionnaire. Environmental investigations were done. RESULTS: By 31 January 2008, 27 cases of severe CDI and 21 cases with C. difficile ribotype 027 infections were found in the area under investigation. Active surveillance found 76 of 399 (19 %) patients positive for C. difficile. In 20 patients, PCR ribotyp 027 could be proven. In total, 9 deaths occurred (19 %). An existing immunosupressive therapy (OR 35.8; 95 % CI 2.8 - 464.5) was related to case fatality in the multivariate analysis. Severe cases of CDI were also observed in non-ribotype 027 infections. In the screening of hospital personnel (n = 161), 6 % were found positive for toxin A/B. DISCUSSION: This investigation demonstrated the endemicity of C. difficile PCR ribotype 027 in Germany for the first time. As a consequence from this study, severe CDI became a reportable disease in Germany at the end of 2007. In addition to hygienic measures, the critical use of antibiotics is an important measure to prevent a further increase of CDI.

A novel IS26 structure surrounds blaCTX-M genes in different plasmids from German clinical Escherichia coli isolates.

This report focuses on the molecular characterization of 22 extended-spectrum beta-lactamase-producing Escherichia coli isolates collected in a German university hospital during a period of 9 months in 2006. Relationship analysis of clinical isolates was done via PFGE, multilocus sequence typing, plasmid profiling and additionally PCR for bla(ESBL) detection and determination of phylogroups. After conjugal transfer, plasmid isolation and subsequent PCR for bla(ESBL) detection and determination of incompatibility groups were performed. Using one-primer walking, up to 3600 bp upstream and downstream of different bla(CTX-M) genes could be sequenced. beta-Lactamases found were TEM-1 (n=14), SHV-5 (n=1) and a wide variety of CTX-M types (n=21), i.e. CTX-M-15 (n=12), CTX-M-1 (n=4), CTX-M-14 (n=2), CTX-M-9 (n=1), CTX-M-3 (n=1) and one new type, CTX-M-65 (n=1). In 18 isolates, bla(ESBL) genes were located on conjugative plasmids of sizes between 40 and 180 kbp belonging to incompatibility groups FII (n=9), N (n=5) and I1 (n=4). bla(CTX-M) was found to be associated with the common elements ISEcp1, IS26 and IS903-D, but with unusual spacer sequences for ISEcp1 in two isolates. These insertion sequences, connected to bla(CTX-M) as well as other genes, were located between two IS26 elements in a configuration that has not yet been described. The results reveal the emergence of bla(ESBL), predominantly bla(CTX-M), located on different plasmids harboured by genotypically different E. coli strains. The identical gene arrangement in the bla(CTX-M) neighbourhood in plasmids of different incompatibility groups indicates a main role of IS26 in distribution of mobile resistance elements between different plasmids.