Analysis of the dynamics of Staphylococcus aureus binding to white blood cells using whole blood assay and geno-to-pheno mapping

Given that binding and internalization of bacteria to host cells promotes infections and invasion, we aimed at characterizing how various S. aureus isolates adhere to and are internalized by different white blood cells. In particular, the role of genetic determinants on the association kinetics should be unveiled. A flow cytometric (FACS) whole blood assay with fluorescently labelled isolates was applied to 56 clinical S. aureus isolates. This phenotypic data was then linked to previously obtained genotyping data (334 genes) with the help of a redescription mining algorithm. Professional phagocytes showed a time-dependent increase of bacterial adhesion and internalization. Isolates showing higher affinity to granulocytes were associated with lower binding to monocytes. In contrast binding activity between S. aureus and lymphocytes could be subdivided into two phases. Preliminary binding (phase 1) was highest directly after co-incubation and was followed by S. aureus detachment or by sustained binding of a small lymphocyte subset (phase 2). Strain-dependent low granulocyte binding was observed for clonal complex 5 (CC5) isolates (MRSA), as compared to CC30 and CC45 (MSSA). S. aureus isolates associated with low granulocyte phagocytosis were characterized by the presence (cap8, can) and the absence (sasG, lukD, isdA, splA, setC) of specific hybridization signals

Molecular epidemiology and antimicrobial resistance of Clostridioides difficile detected in chicken, soil and human samples from Zimbabwe

Background: Clostridioides difficile is the major cause of infectious nosocomial diarrhoea in industrialized nations. Data on the occurrence of C. difficile in Africa, ribotype (RT) distribution, antimicrobial susceptibility patterns and potential zoonotic transmission are scarce. Methods: 80 Zimbabwean C. difficile isolates from different sources (chicken [n = 30], soil [n = 21] and humans [n = 29]) were investigated using ribotyping, toxin gene detection, resistance testing, multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing (WGS). Results: Among chicken isolates, the most common RTs were RT103 (6/30), RT025 (5/30) and RT070 (4/30). Within soil samples, RT025 and RT056 were most common (3/21 each). In contrast, the non-toxigenic RT084 was most frequently found in human isolates (4/29). Toxin genes were detected in only 19/29 human isolates. Susceptibility testing showed no resistance against metronidazole and vancomycin, and resistance against macrolides and rifampicin was scarce (3/80 and 2/80, respectively); however, 26/80 isolates showed moxifloxacin resistance. MLVA and WGS of strains with identical RTs stemming from different sources revealed clustering of RT025 and RT084 isolates from human und non-human samples. Conclusion: No "hypervirulent” strains were found. The detected clusters between human, chicken and soil isolates indicate ongoing transmission between humans and environmental sources and might point towards a zoonotic potential

Aktuelle molekulare Epidemiologie und Antibiotikaresistenzen von Clostridioides difficile in Deutschland im Jahr 2022

Clostridioides (C.) difficile ist der bedeutendste bakte¬rielle Erreger infektiöser nosokomialer Diarrhöen. Wichtigster Risikofaktor für eine Erkrankung ist die Gabe von Antibiotika, die die Darmflora schädigen – insbesondere die „4C“-Antibiotika Clindamycin, Fluorchinolone, Cephalosporine und Aminopenicilline mit Betalaktamase-Inhibitoren. Das Epidemiologische Bulletin 15/2023 gibt einen Überblick über die aktuellen Entwicklungen der C. difficile-Epidemiologie und Resistenzentwicklung in Deutschland. Veränderungen der Erregerepidemiologie und das Erkennen von neuen Erreger- und Resistenzvarianten erfordern eine kontinuierliche Surveillance, die seit 2019 standardisiert in Deutschland am Nationalen Referenzzentrum für C. difficile durchgeführt wird.Peer Reviewe

Simulating the effect of tillage practices with the global ecosystem model LPJmL (version 5.0-tillage)

The effects of tillage on soil properties, crop productivity, and global greenhouse gas emissions have been discussed in the last decades. Global ecosystem models have limited capacity to simulate the various effects of tillage. With respect to the decomposition of soil organic matter, they either assume a constant increase due to tillage or they ignore the effects of tillage. Hence, they do not allow for analysing the effects of tillage and cannot evaluate, for example, reduced tillage or no tillage (referred to here as “no-till”) practises as mitigation practices for climate change. In this paper, we describe the implementation of tillage-related practices in the global ecosystem model LPJmL. The extended model is evaluated against reported differences between tillage and no-till management on several soil properties. To this end, simulation results are compared with published meta-analyses on tillage effects. In general, the model is able to reproduce observed tillage effects on global, as well as regional, patterns of carbon and water fluxes. However, modelled N fluxes deviate from the literature values and need further study. The addition of the tillage module to LPJmL5 opens up opportunities to assess the impact of agricultural soil management practices under different scenarios with implications for agricultural productivity, carbon sequestration, greenhouse gas emissions, and other environmental indicators

Antigen-Specific vs. Neutralizing Antibodies Against Conditioned Media of Patients With Clostridioides difficile Infection: A Prospective Exploratory Study

The immunological response against Clostridioides difficile (C. difficile) is crucial for an improved understanding of disease mechanisms and the development of novel therapeutic strategies. From April 2014 to February 2015, adult patients with C. difficile infection (CDI) were recruited, and the clinical course and treatment response were carefully monitored. On day 1, 3, and 6 after diagnosis, patient plasma samples were screened for anti-GDH (glutamate dehydrogenase), anti-TcdA, anti-TcdB, and anti-CWP84 (cell-wall protein 84) antibodies by ELISA. Additionally, neutralization assays of toxins from conditioned media of clinical isolates (RT010, RT014, and RT027) were performed. Most patients with CDI (n=46) had antibodies against GDH (85%) and CWP84 (61%), but only few had antibodies against TcdA (11%) and TcdB (28%). We found patients with neutralizing antibodies against C. difficile toxins (conditioned media) produced by RT027 (26%). A subgroup of these samples could neutralize both toxins from RT027 and RT014 [11%, (5/46)]; however, no single sample neutralized only RT014. Overall, neutralizing antibody titers were low (≤1:16). In a one week follow-up of acute infection, we never observed an early booster effect with seroconversion or antibody increases, irrespective of disease severity. No correlation was found between the presence of antigen-specific (ELISA) or neutralizing antibodies and the clinical course of disease. Anti-TcdB but not anti-TcdA antibodies correlated with the occurrence of neutralizing antibodies. In conclusion, natural antibody titers against C. difficile toxins were absent or low and were not associated with disease severity. The correlation between the anti-TcdB with toxin neutralization confirms the importance of TcdB for virulence of CDI. Alternative sensitization strategies, e.g., through vaccine development, are required to overcome the regular low-titer antibody production following natural intestinal C. difficile exposure

Active Cytomegalovirus Infection in Patients with Septic Shock

Cytomegalovirus reactivation occurred in one third of patients and was associated with prolonged ventilation and stay in an intensive care unit

Discrimination between hypervirulent and non-hypervirulent ribotypes of Clostridioides difficile by MALDI-TOF mass spectrometry and machine learning

Hypervirulent ribotypes (HVRTs) of Clostridioides difcile such as ribotype (RT) 027 are epidemiologically important. This study evaluated whether MALDI-TOF can distinguish between strains of HVRTs and non-HVRTs commonly found in Europe. Obtained spectra of clinical C. difcile isolates (training set, 157 isolates) covering epidemiologically relevant HVRTs and non-HVRTs found in Europe were used as an input for diferent machine learning (ML) models. Another 83 isolates were used as a validation set. Direct comparison of MALDI-TOF spectra obtained from HVRTs and non-HVRTs did not allow to discriminate between these two groups, while using these spectra with certain ML models could diferentiate HVRTs from non-HVRTs with an accuracy >95% and allowed for a sub-clustering of three HVRT subgroups (RT027/ RT176, RT023, RT045/078/126/127). MALDI-TOF combined with ML represents a reliable tool for rapid identifcation of major European HVRTs

Molecular epidemiology and antimicrobial resistance of Clostridioides difficile in Germany, 2014-2019

Clostridioides difficile is a Gram positive spore-forming rod and mainly responsible for nosocomial diarrhea in developed nations. Molecular and antimicrobial surveillance is important for monitoring the strain composition including genotypes of high epidemiological importance such as ribotype 027 (RT027) and corresponding resistance patterns. 1535 isolates obtained from samples sent between 2014 and 2019 to the German National Reference Center (NRC) for diagnostic reasons (NRC strain set), and 1143 isolates from a Tertiary Care University Center in Saarland, Germany (non-NRC strain set), were evaluated using antibiotic susceptibility testing and ribotyping. In the NRC strain set, RT027 overtook RT001, the main RT found in the preceding studies, and dominated with 36.2%, followed by RT001 (13.3%), and RT014 (8.5%). Of note, since 2016 a constant decrease of RT027 could be noticed. In the non-NRC strain set a large strain diversity was present with RT014 (18%) and RT001 (8.9%) being most prevalent. In NRC samples, resistance towards metronidazole, vancomycin, moxifloxacin, clarithromycin and rifampicin was 2.7%, 0%, 57.1%, 53.2% and 19.2%, respectively. Metronidazole resistance was almost exclusively found in RT027 isolates. Rifampicin resistance was also observed predominantly in isolates of RT027, constituting an almost four-fold increase, when compared to preceeding studies in this region. In conclusion these data demonstrate that RT027 is a driver for rifampicin and metronidazole resistance, underlining the importance of continuous surveillance efforts

Molecular and culture-based diagnosis of Clostridium difficile isolates from Côte d'Ivoire after prolonged storage at disrupted cold chain conditions

Background Although Clostridium difficile is a major cause of diarrhoea, its epidemiology in tropical settings is poorly understood. Strain characterisation requires work-up in specialised laboratories, often after prolonged storage without properly maintained cold chain. Methods We screened 298 human faecal samples from Côte d'Ivoire using a rapid test for C. difficile glutamate dehydrogenase (GDH). GDH-positive samples were aerobically stored at disrupted cold chain conditions (mean duration: 11 days) before transfer to a reference laboratory for anaerobic culture, susceptibility testing, PCR assays and ribotyping. Results Sixteen samples (5.4%) had a positive GDH screening test. C. difficile infection was confirmed in six specimens by culture and PCR, while no nucleic acids of C. difficile were detected in the culture-negative samples. Further analysis of stool samples harbouring toxigenic C. difficile strains confirmed that both GDH and toxins remained detectable for at least 28 days, regardless of storage conditions (aerobic storage at 4°C or 20°C). Conclusions Storage conditions only minimally affect recovery of C. difficile and its toxins in stool culture. A rapid GDH screening test and subsequent transfer of GDH-positive stool samples to reference laboratories for in-depth characterisation may improve our understanding of the epidemiology of C. difficile in the tropic

Molecular characterization, toxin detection and resistance testing of human clinical Clostridium difficile isolates from Lebanon

Clostridium (Clostridioides) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen’s occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon. From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing. Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene (tcdA) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively. The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area