OBJECTIVE:Antibiotic resistance in Gram-negative bacteria has become an increasing problem in the treatment of critically ill patients, particularly in central nervous system infections. In clinical practice, MICs and minimal bactericidal concentrations are usually determined in standard test media and not in the respective body fluids. METHODS:MICs and minimal bactericidal concentrations were tested in human cerebrospinal fluid (CSF), Mueller-Hinton broth (MHB), and artificial cerebrospinal fluid by microdilution for 24 h at 37°C in room air supplemented with 5% CO2. RESULTS:Cefotaxime, ceftazidime, and ciprofloxacin inhibited the growth of Pseudomonas aeruginosa, cefotaxime, ceftazidime and amikacin inhibited the growth of Morganella morganii, and amikacin inhibited the growth of Acinetobacter baumannii in the nutrient-deficient CSF medium at lower concentrations than the MICs determined in MHB. The differences in MIC between MHB and artificial cerebrospinal fluid were less pronounced than between MHB and human CSF. CONCLUSIONS:In central nervous system infections, when antimicrobial susceptibility testing in standard media does not result in adequate therapeutic options, we suggest testing the antimicrobial susceptibility of the causative bacterium in human CSF.

Breakpoints to define the susceptibility of pathogens to antibiotics are becoming increasingly popular as guidance for antimicrobial therapy. Some breakpoints consider divergent concentrations of antibiotics in different compartments, others list one breakpoint for infections at all sites. Compared to the determination of exact minimal inhibitory concentrations (MICs) and relation of these MICs to concentrations achievable in ventricular CSF, the exclusive use of breakpoints for central nervous system (CNS) infections is a setback. The lack of the determination of exact MICs may be a risk, particularly when no clinical breakpoints for CNS infections have been defined. Moreover, the current practice of MIC determination of β-lactam/β-lactamase inhibitor combinations with fixed β-lactamase inhibitor concentrations ignores that often these β-lactamase inhibitor concentrations are not attained in CSF with established antibiotic regimens. In CNS infections, we strongly recommend the exact determination of MICs and their interpretation in relation to the true antibiotic concentrations in the infected compartment.

Background: In hospital- and community-acquired central nervous system infections, resistant Gram-positive bacteria are an increasing therapeutic challenge. The present approach does not attempt to identify rapidly bactericidal therapies for susceptible pathogens but aims to improve methods to find antibiotic regimens for multi-resistant pathogens that are effective in vivo in spite of reduced in vitro susceptibility in culture media. Methods: Antibiotic susceptibility was tested in cerebrospinal fluid (CSF) and Mueller-Hinton broth (Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis) or brain-heart infusion (Streptococcus pneumoniae). Results: Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were either lower in CSF than in broth or equal in CSF and broth. The difference between MICs in CSF and broth was prominent with gentamicin, levofloxacin, linezolid (staphylococci), and vancomycin (staphylococci and pneumococcus), whereas it was absent with ampicillin (E. faecalis), penicillin G (S. pneumoniae), linezolid (enterococcus and pneumococcus), and vancomycin (enterococcus). In no case was the MIC or MBC higher in CSF than in broth. Conclusions: Several antibiotics possess an antibacterial effect in CSF at lower concentrations than the MICs determined in broth, i.e., MICs in broth underestimate in situ susceptibility in CSF.

OBJECTIVES:To reduce infections with Clostridioides difficile (CDI) in geriatric patients by interventions easily implementable in standard clinical care. METHODS:Prevalence and incidence of CDI between January 2015 and February 2020 were analysed (n = 25,311 patients). Pre-intervention status was assessed from April 2016 to March 2017 (n = 4,922). Between May 2017 and August 2019, a monocentric interventional crossover study (n = 4,655) was conducted including standard care and three interventions: (A) sporicidal cleaning of hospital wards, (B) probiotics and (C) improvement in personal hygiene for CDI patients. This was followed by a multicentric comparison of the interventional bundle (A + B + C) between September 2019 and February 2020 (n = 2,593) with the pre-intervention phase. In 98 CDI cases and matched controls individual risk factors for the development of CDI were compared. RESULTS:Time series analyses of CDI cases revealed a reduction in the prevalence of CDI in all three participating centres prior to the multicentric intervention phase. In the monocentric phase, no effect of individual interventions on CDI prevalence was identified. However, an aggregated analysis of CDI cases comparing the pre-intervention and the multicentric phase revealed a significant reduction in CDI prevalence. Risk factors for the development of CDI included use of antibiotics, anticoagulants, previous stay in long-term care facilities, prior hospital admissions, cardiac and renal failure, malnutrition and anaemia. CONCLUSIONS:The observed reduction in CDI may be attributed to heightened awareness of the study objectives and specific staff training. Individual interventions did not appear to reduce CDI prevalence. A further randomised trial would be necessary to confirm whether the bundle of interventions is truly effective.

The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus , the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

ObjectivesCarbapenemase-producing Klebsiella pneumoniae pose an increasing risk for healthcare facilities worldwide. A continuous monitoring of ST distribution and its association with resistance and virulence genes is required for early detection of successful K. pneumoniae lineages. In this study, we used WGS to characterize MDR blaOXA-48-positive K. pneumoniae isolated from inpatients at the University Medical Center Göttingen, Germany, between March 2013 and August 2014.MethodsClosed genomes for 16 isolates of carbapenemase-producing K. pneumoniae were generated by single molecule real-time technology using the PacBio RSII platform.ResultsEight of the 16 isolates showed identical XbaI macrorestriction patterns and shared the same MLST, ST147. The eight ST147 isolates differed by only 1-25 SNPs of their core genome, indicating a clonal origin. Most of the eight ST147 isolates carried four plasmids with sizes of 246.8, 96.1, 63.6 and 61.0 kb and a novel linear plasmid prophage, named pKO2, of 54.6 kb. The blaOXA-48 gene was located on a 63.6 kb IncL plasmid and is part of composite transposon Tn1999.2. The ST147 isolates expressed the yersinabactin system as a major virulence factor. The comparative whole-genome analysis revealed several rearrangements of mobile genetic elements and losses of chromosomal and plasmidic regions in the ST147 isolates.ConclusionsSingle molecule real-time sequencing allowed monitoring of the genetic and epigenetic microevolution of MDR OXA-48-producing K. pneumoniae and revealed in addition to SNPs, complex rearrangements of genetic elements.

Zusammenfassung Multiresistente Keime spielen im klinischen Alltag eine zunehmend bedeutsame Rolle. Dieses besonders auf Intensivstationen bzw. in Risikobereichen. Oftmals herrschen Unklarheiten bezüglich diagnostischer Screening-Indikationen und Strategien zur Vermeidung von Übertragungen mittels Hygiene- und Isolierungsmaßnahmen. Wir geben einen orientierenden Überblick über die zur Zeit gängigen Empfehlungen und bewerten diese für Methicillin-resistente Staphylococcusaureus-Stämme (MRSA) und multiresistente gramnegative Bakterien (MRGN).

Kernaussagen Zu den Indikationen zur systemischen Gabe von Antimykotika besonders bei nicht-hämatologischen Patienten und auf Intensivstation bestehen oftmals Unklarheiten. Eine sinnvolle Prophylaxe bzw. risiko- oder diagnostik-orientierte Therapie könnte somit im Einzelfall unterlassen und diagnostische Maßnahmen zu spät eingeleitet werden. Auf der anderen Seite sollte ein unnötiger Antimykotika-Einsatz, wie z. B. die reflexartige Gabe bei Nachweis von Candida aus Bronchialsekreten, vermieden werden. Kommt es hingegen zu einem Nachweis von Pilzen aus Blutkulturen, so ist ein den gängigen Empfehlungen folgendes Therapieschema anzustreben. Dieser Artikel versucht hier Hilfestellung zu geben, indem er das korrekte Prozedere in den genannten Themengebieten anhand aktueller Leitlinien und neuerer Literatur zusammenfasst.

This chapter discusses animal models of toxoplasmosis with special regard to pharmacological applications, and thereby tries to update existing reviews. All mammals can be infected with toxoplasmosis. However, different animal species differ markedly in their resistance to Toxoplasma infection. In addition, the outcome of infection is dependent not only on the animal species but also on the animal strain. The genetic background seems to be of importance since, after infection with Toxoplasma, striking differences in susceptibility of various strains of inbred and outbred mice are observed. The situation becomes even more complex because these differences are not uniform with respect to the strains but are also a function of the mode of inoculation. In addition to host factors, the outcome of a challenge with Toxoplasma is largely influenced by the nature of the infectious agent itself, and one of the most common characteristics of many Toxoplasma strains is the variation in virulence. Depending on the time before animals succumb to infection or the percentage of animals that do succumb, highly virulent, moderately virulent, and less virulent strains have been characterized.

Although innate immunity primarily combats systemic infections of opportunistic fungi such as Aspergillus and Candida spp., acquired and protective immunoreactions were observed long ago in animal trials following sublethal systemic infections caused by viable fungi or after challenging animals with inactivated fungal cells. Based on these observations, fungal antigens should exist which mediate such protective immunoreactions and have in part already been identified. In this context, this review focuses primarily on the various approaches that have been used to identify protection-mediating Aspergillus-antigens and their rationale. Emphasis is placed on screening methods that have exploited genetic or proteomic approaches on the basis of the corresponding fungal genome projects. Thereby, a survey and description is given of the antigens so far known to be capable of inducing immune responses that protect animals against acquiring lethal systemic aspergillosis.

72 Since the mid-1990s, a steady increase in the occurrence of itraconazole resistant Aspergillus 73 fumigatus isolates has been observed in clinical contexts leading to therapeutic failure in the 74 treatment of aspergillosis. This increase has been predominantly linked to a single allele of 75 the cyp51A gene, termed ‘TR/L98H’ which is thought to have arisen through the use of 76 agricultural azoles. 77 Here, we investigated the current epidemiology of triazole resistant A. fumigatus and 78 underlying cyp51A-mutations in clinical samples in Germany. 79 From a total of 527 samples, 17 (3.2%) showed elevated MIC0 values for at least one of the 80 three substances (itraconazole, voriconazole, and posaconazole) tested. The highest 81 prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among 82 resistant isolates, the TR/L98H mutation in cyp51A was most prevalent, but also isolates with 83 the G54W, M220I, and the novel F219C were found. The isolate with the G54W substitution 84 was highly resistant to both itraconazole and posaconazole, while all others showed high 85 level resistance only to itraconazole. For the remaining six isolates no mutations in cyp51A 86 were found, indicating the presence of other mechanisms. With the exception of the strains 87 carrying the F219C and M220I substitutions, many itraconazole resistant strains also showed 88 cross resistance to voriconazole and posaconazole with moderately increased MIC0 values. 89 In conclusion, the prevalence of azole resistant A. fumigatus is lower in our clinical test set 90 than previously reported for other countries. Although the TR/L98H mutation frequently 91 occurs among triazole resistant strains in Germany, it is not the only resistance mechanism 92

ABSTRACT Since the mid-1990s, a steady increase in the occurrence of itraconazole-resistant Aspergillus fumigatus isolates has been observed in clinical contexts, leading to therapeutic failure in the treatment of aspergillosis. This increase has been predominantly linked to a single allele of the cyp51A gene, termed TR/L98H, which is thought to have arisen through the use of agricultural azoles. Here, we investigated the current epidemiology of triazole-resistant A. fumigatus and underlying cyp51A mutations in clinical samples in Germany. From a total of 527 samples, 17 (3.2%) showed elevated MIC0 values (the lowest concentrations with no visible growth) for at least one of the three substances (itraconazole, voriconazole, and posaconazole) tested. The highest prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among resistant isolates, the TR/L98H mutation in cyp51A was the most prevalent, but isolates with the G54W and M220I substitutions and the novel F219C substitution were also found. The isolate with the G54W substitution was highly resistant to both itraconazole and posaconazole, while all others showed high-level resistance only to itraconazole. For the remaining six isolates, no mutations in cyp51A were found, indicating the presence of other mechanisms. With the exception of the strains carrying the F219C and M220I substitutions, many itraconazole-resistant strains also showed cross-resistance to voriconazole and posaconazole with moderately increased MIC0 values. In conclusion, the prevalence of azole-resistant A. fumigatus in our clinical test set is lower than that previously reported for other countries. Although the TR/L98H mutation frequently occurs among triazole-resistant strains in Germany, it is not the only resistance mechanism present.

Background: Invasive Candida infections following abdominal surgery represent a significant medical problem. This study initiates a benchmarking project to pinpoint the current role and epidemiology of candidemia in this patient group in German hospitals.Material and Methods: During the year 2010 data derived from 47704 abdominal surgery cases in hospitals from Germany were analysed in order to determine benchmarking incidences for candidemia.Results and Conclusion: In 20.3% of all recognised bloodstream infections Candida spp. were identified as the responsible organisms. If related to all abdominal surgery cases analysed in this study, a candidemia-benchmarking incidence of 0.15% (95% CI: 0.10-0.21%) was determined. In patients who required intensive care after surgery the incidence of candidemia was found to be 0.89% (95% CI: 0.57-1.38%). The incidence increased to 3.13% (95% CI: 2.09-4.66%) in patients who received blood culture diagnosis. The German National Reference Centre of Systemic Mycosis provides hospital specific data for participants of this study to enable benchmarking and infection control (www.nrz-mykosen.de/gastrointestinalchirurgie).

Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml-1. Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml-1. Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed.