Friedrich-Loeffler-Institut für Medizinische Mikrobiologie
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Summary
Roundup® is the brand name for herbicide solutions containing glyphosate, which specifically inhibits the 5‐enolpyruvyl‐shikimate‐3‐phosphate (EPSP) synthase of the shikimate pathway. The inhibition of the EPSP synthase causes plant death because EPSP is required for biosynthesis of aromatic amino acids. Glyphosate also inhibits the growth of archaea, bacteria, Apicomplexa, algae and fungi possessing an EPSP synthase. Here, we have characterized two glyphosate‐resistant bacteria from a Roundup solution. Taxonomic classification revealed that the isolates 1CH1 and 2CH1 are Burkholderia anthina and Burkholderia cenocepacia strains respectively. Both isolates cannot utilize glyphosate as a source of phosphorus and synthesize glyphosate‐sensitive EPSP synthase variants. Burkholderia. anthina 1CH1 and B. cenocepacia 2CH1 tolerate high levels of glyphosate because the herbicide is not taken up by the bacteria. Previously, it has been observed that the exposure of soil bacteria to herbicides like glyphosate promotes the development of antibiotic resistances. Antibiotic sensitivity testing revealed that the only the B. cenocepacia 2CH1 isolate showed increased resistance to a variety of antibiotics. Thus, the adaptation of B. anthina 1CH1 and B. cenocepacia 2CH1 to glyphosate did not generally increase the antibiotic resistance of both bacteria. However, our study confirms the genomic adaptability of bacteria belonging to the genus Burkholderia.
Upon antigen recognition by the T cell receptor (TCR), a complex signaling network orchestrated by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) regulates the transmission of the extracellular signal to the nucleus. The role of the PTPs Src-homology 2 (SH2) domain-containing phosphatase 1 (SHP1, Ptpn6) and Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2, Ptpn11) have been studied in various cell types including T cells. Whereas SHP1 acts as an essential negative regulator of the proximal steps in T cell signalling, the role of SHP2 in T cell activation is still a matter of debate. Here, we analyzed the role of the constitutively active SHP2-D61Y-mutant in T cell activation using knock-in mice expressing the mutant form Ptpn11D61Y
in T cells. We observed reduced numbers of CD8+ and increased numbers of CD4+ T cells in the bone marrow and spleen of young and aged SHP2-D61Y-mutant mice as well as in Influenza A Virus (IAV)-infected mice compared to controls. In addition, we found elevated frequencies of effector memory CD8+ T cells and an upregulation of the programmed cell death protein 1 (PD-1)-receptor on both CD4+ and CD8+ T cells. Functional analysis of SHP2-D61Y-mutated T cells revealed an induction of late apoptosis/necrosis, a reduced proliferation and altered signaling upon TCR stimulation. However, the ability of D61Y-mutant mice to clear viral infection was not affected. In conclusion, our data indicate an important regulatory role of SHP2 in T cell function, where the effect is determined by the kinetics of SHP2 phosphatase activity and differs in the presence of the permanently active and the temporally regulated phosphatase. Due to interaction of SHP2 with the PD-1-receptor targeting the protein-tyrosine phosphatase might be a valuable tool to enhance T cell activities in immunotherapy.
Melioidosis is a seasonal infectious disease in tropical and subtropical areas caused by the soil bacterium Burkholderia pseudomallei. In many parts of the world, including South West India, most cases of human infections are reported during times of heavy rainfall, but the underlying causes of this phenomenon are not fully understood. India is among the countries with the highest predicted melioidosis burden globally, but there is very little information on the environmental distribution of B. pseudomallei and its determining factors. The present study aimed (i) to investigate the prevalence of B. pseudomallei in soil in South West India, (ii) determine geochemical factors associated with B. pseudomallei presence and (iii) look for potential seasonal patterns of B. pseudomallei soil abundance. Environmental samplings were performed in two regions during the monsoon and post-monsoon season and summer from July 2016 to November 2018. We applied direct quantitative real time PCR (qPCR) together with culture protocols to overcome the insufficient sensitivity of solely culture-based B. pseudomallei detection from soil. A total of 1,704 soil samples from 20 different agricultural sites were screened for the presence of B. pseudomallei. Direct qPCR detected B. pseudomallei in all 20 sites and in 30.2% (517/1,704) of all soil samples, whereas only two samples from two sites were culture-positive. B. pseudomallei DNA-positive samples were negatively associated with the concentration of iron, manganese and nitrogen in a binomial logistic regression model. The highest number of B. pseudomallei-positive samples (42.6%, p < 0.0001) and the highest B. pseudomallei loads in positive samples [median 4.45 × 103 genome equivalents (GE)/g, p < 0.0001] were observed during the monsoon season and eventually declined to 18.9% and a median of 1.47 × 103 GE/g in summer. In conclusion, our study from South West India shows a wide environmental distribution of B. pseudomallei, but also considerable differences in the abundance between sites and within single sites. Our results support the hypothesis that nutrient-depleted habitats promote the presence of B. pseudomallei. Most importantly, the highest B. pseudomallei abundance in soil is seen during the rainy season, when melioidosis cases occur.
Antibiotic resistance is increasing worldwide making it necessary to search for alternative antimicrobials. Sodium bituminosulfonate is a long-known substance, whose antimicrobial inhibitory activity has recently been re-evaluated. However, to the best of our knowledge, the bactericidal mode of action of this substance has not been systematically characterized. The aim of this study was to investigate the in vitro bactericidal activity of sodium bituminosulfonate by determining the minimal bactericidal concentrations (MBC), as well as the rapidity of bactericidal effect by time-kill curves. Clinical isolates of methicillin-susceptible (MSSA, n = 20) and methicillin-resistant (mecA/mecC-MRSA, n = 20) Staphylococcus aureus were used to determine MBC by a broth microdilution method. Sodium bituminosulfonate (Ichthyol® light) was tested in double-dilution concentration steps ranging from 0.03 g/L to 256 g/L. For time-kill analysis, two reference and two clinical S. aureus strains were tested with different concentrations of sodium bituminosulfonate (1× minimal inhibitory concentration (MIC), 2× MIC, 4× MIC, 16× MIC and 256× MIC). For MSSA isolates, MBC50, MBC90 and the MBC range were 0.5 g/L, 1.0 g/L and 0.125–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 4, 4 and 1–8, respectively. Among MRSA isolates, MBC50, MBC90 and the MBC range amounted to 0.5 g/L, 1.0 g/L and 0.06–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 2, 4 and 1–8, respectively. Time-kill kinetics revealed a bactericidal effect after 30 min for sodium bituminosulfonate concentrations of 16× MIC and 256× MIC. The bactericidal activity against MSSA and MRSA was demonstrated for sodium bituminosulfonate. The killing was very rapid with the initial population reduced by 99.9% after only short incubation with concentrations of 16× MIC and higher.
T cell activation plays a central role in supporting and shaping the immune response. The induction of a functional adaptive immune response requires the control of signaling processes downstream of the T cell receptor (TCR). In this regard, protein phosphorylation and dephosphorylation have been extensively studied. In the past decades, further checkpoints of activation have been identified. These are E3 ligases catalyzing the transfer of ubiquitin or ubiquitin-like proteins to protein substrates, as well as specific peptidases to counteract this reaction, such as deubiquitinating enzymes (DUBs). These posttranslational modifications can critically influence protein interactions by targeting proteins for degradation by proteasomes or mediating the complex formation required for active TCR signaling. Thus, the basic aspects of T cell development and differentiation are controlled by defining, e.g., the threshold of activation in positive and negative selection in the thymus. Furthermore, an emerging role of ubiquitination in peripheral T cell tolerance has been described. Changes in the function and abundance of certain E3 ligases or DUBs involved in T cell homeostasis are associated with the development of autoimmune diseases. This review summarizes the current knowledge of E3 enzymes and their target proteins regulating T cell signaling processes and discusses new approaches for therapeutic intervention.
Hand hygiene is a cornerstone of infection prevention. However, few data are available for school children on their knowledge of infectious diseases and their prevention. The aim of the study was to develop and apply a standardized questionnaire for children when visiting primary schools to survey their knowledge about infectious diseases, pathogen transmission and prevention measures. Enrolling thirteen German primary schools, 493 questionnaires for grade three primary school children were included for further analyses, comprising 257 (52.1%) girls and 236 (47.9%) boys with an age range of 8–11 years. Out of 489 children, 91.2% participants indicated that they knew about human-to-human transmissible diseases. Of these, 445 children responded in detail, most frequently mentioning respiratory and gastrointestinal diseases, followed by childhood diseases. Addressing putative hygiene awareness-influencing factors, it was worrisome that more than 40.0% of the children avoided visiting the sanitary facilities at school. Most of the children (82.9%) noted that they did not like to use the sanitary facilities at school because of their uncleanliness and the poor hygienic behavior of their classmates. In conclusion, basic infection awareness exists already in primary school age children. Ideas about the origin and prevention of infections are retrievable, however, this knowledge is not always accurate and adequately contextualized. Since the condition of sanitary facilities has a strong influence on usage behavior, the child’s perspective should be given more consideration in the design and maintenance of sanitary facilities.
Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent’s stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.
Background: For years, coagulase-negative staphylococci (CoNS) were not considered a cause of bloodstream infections (BSIs) and were often regarded as contamination. However, the association of CoNS with nosocomial infections is increasingly recognized. The identification of more than 40 different CoNS species has been driven by the introduction of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Yet, treatment guidelines consider CoNS as a whole group, despite increasing antibiotic resistance (ABR) in CoNS. This retrospective study provides an in-depth data analysis of CoNS isolates found in human blood culture isolates between 2013 and 2019 in the entire region of the Northern Netherlands. Methods: In total, 10,796 patients were included that were hospitalized in one of the 15 hospitals in the region, leading to 14,992 CoNS isolates for (ABR) data analysis. CoNS accounted for 27.6% of all available 71,632 blood culture isolates. EUCAST Expert rules were applied to correct for errors in antibiotic test results. Results: A total of 27 different CoNS species were found. Major differences were observed in occurrence and ABR profiles. The top five species covered 97.1% of all included isolates: S. epidermidis, S. hominis, S. capitis, S. haemolyticus, and S. warneri. Regarding ABR, methicillin resistance was most frequently detected in S. haemolyticus (72%), S. cohnii (65%), and S. epidermidis (62%). S. epidermidis and S. haemolyticus showed 50–80% resistance to teicoplanin and macrolides while resistance to these agents remained lower than 10% in most other CoNS species. Conclusion: These differences are often neglected in national guideline development, prompting a focus on ‘ABR-safe’ agents such as glycopeptides. In conclusion, this multi-year, full-region approach to extensively assess the trends in both the occurrence and phenotypic resistance of CoNS species could be used for evaluating treatment policies and understanding more about these important but still too often neglected pathogens.
Population-based studies of Staphylococcus aureus contribute to understanding the epidemiology of S. aureus infection. We enrolled surgical inpatients admitted to an African tertiary-care hospital in order to prospectively analyze the nosocomial impact of S. aureus. Data collection included an active sampling of the anterior nares and infectious foci within 48 h after admission and subsequently when clinically indicated. All S. aureus isolates were spa and agr genotyped. Possession of Panton-Valentine leukocidin (PVL) and other toxin genes was determined. We analyzed antibiotic susceptibility profiles by VITEK 2 systems and verified methicillin-resistant S. aureus (MRSA) by mecA/C PCR. Among 325 patients, 15.4% carried methicillin-susceptible S. aureus (MSSA) at admission, while 3.7% carried MRSA. The incidence densities of nosocomial infections due to MSSA and MRSA were 35.4 and 6.2 infections per 10,000 patient-days, respectively. Among all 47 nosocomial infections, skin and soft-tissue (40.4%) and bones or joints’ (25.5%) infections predominated. Six (12.7%) infection-related S. aureus isolates harbored PVL genes including two (4.2%) MRSA: overall, seventeen (36.2%) isolates carried pyrogenic toxin superantigens or other toxin genes. This study illustrates the considerable nosocomial impact of S. aureus in a Nigerian University hospital. Furthermore, they indicate a need for effective approaches to curtail nosocomial acquisition of multidrug-resistant S. aureus.
Antimicrobial resistance (AMR) is a serious global health threat with extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales as the most critical ones. Studies on AMR in wild birds imply a possible dissemination function and indicate their potential role as sentinel animals. This study aimed to gain a deeper insight into the AMR burden of wild waterfowl by sampling semi-wild mallard ducks used as sentinels and to identify if AMR bacteria could be recommended to be added to the pathogens of public health risks to be screened for. In total, 376 cloacal and pooled fecal samples were collected from the sentinel plant over a period of two years. Samples were screened for ESBL-carrying E. coli and isolates found further analyzed using antimicrobial susceptibility testing and whole-genome sequencing. Over the sampling period, 4.26% (16/376) of the samples were positive for ESBL-producing E. coli. BlaCTX-M-1 and blaCTX-M-32 were the most abundant CTX-M types. Although none of the top global sequence types (ST) could be detected, poultry-derived ST115 and non-poultry-related STs were found and could be followed over time. The current study revealed low cases of ESBL-producing E. coli in semi-wild mallard ducks, which proves the suitability of sentinel surveillance for AMR detection in water-associated wildlife.