YSMR : A video tracking and analysis program for bacterial motility

Background: Motility in bacteria forms the basis for taxis and is in some pathogenic bacteria important for virulence. Video tracking of motile bacteria allows the monitoring of bacterial swimming behaviour and taxis on the level of individual cells, which is a prerequisite to study the underlying molecular mechanisms. Results: The open-source python program YSMR (Your Software for Motility Recognition) was designed to simultaneously track a large number of bacterial cells on standard computers from video files in various formats. In order to cope with the high number of tracked objects, we use a simple detection and tracking approach based on grey-value and position, followed by stringent selection against suspicious data points. The generated data can be used for statistical analyses either directly with YSMR or with external programs. Conclusion: In contrast to existing video tracking software, which either requires expensive computer hardware or only tracks a limited number of bacteria for a few seconds, YSMR is an open-source program which allows the 2-D tracking of several hundred objects over at least 5 minutes on standard computer hardware. The code is freely available at https://github.com/schwanbeck/YSMR © 2020 The Author(s)

Chimaeribacter arupi a new member of the Yersineacea family has the characteristics of a human pathogen

Chimaeribacter arupi (heterotypic synonym: “Nissabacter archeti”) is a facultative anaerobic, newly described Gram-negative rod and belongs to the Yersineacea family. Here, we report the case of a 19-month-old female infant patient who presented to the emergency unit with somnolence and fever. C. arupi was isolated from a positive blood culture, taken via an implanted Broviac catheter, proving a bloodstream infection by the pathogen. The objective of this study was to utilize whole genome sequencing to assess the genes encoding potential virulence associated factors, which may play a role in host tropism, tissue invasion and the subsequent stages in the pathogenesis of a bloodstream infection with C. arupi. The genome of the isolate was completely sequenced employing Illumina MiSeq and Nanopore MinION sequencing and the presumptive virulence associated factors and antimicrobial resistance genes were investigated in more detail. Additionally, we performed metabolic profiling and susceptibility testing by microdilution. The presence of predicted TcfC-like α-Pili suggests that C. arupi is highly adapted to humans as a host. It utilizes flagellar and type IV pili-mediated motility, as well as a number of γ1-pili and a σ-pilus, which may be used to facilitate biofilm formation and adherence to host epithelia. Additionally, long polar fimbriae may aid in tissue invasion. The bacterium possesses antioxidant factors, which may enable temporary survival in phagolysosomes, and a capsule that potentially provides protection from phagocytosis. It may acquire iron ions from erythrocytes through the type 6 secretion system and hemolysins. Furthermore, the isolate exhibits beta-lactamase-mediated penicillin and aminopenicillin resistance. Based on the analysis of the whole genome, we conclude that C. arupi possesses virulence factors associated with tissue invasion and may thus be a potential opportunistic pathogen of bloodstream infections

Artificially designed pathogens – a diagnostic option for future military deployments

Diagnostic microbial isolates of bio-safety levels 3 and 4 are difficult to handle in medical field camps under military deployment settings. International transport of such isolates is challenging due to restrictions by the International Air Transport Association. An alternative option might be inactivation and sequencing of the pathogen at the deployment site with subsequent sequence-based revitalization in well-equipped laboratories in the home country for further scientific assessment. A literature review was written based on a PubMed search. First described for poliovirus in 2002, de novo synthesis of pathogens based on their sequence information has become a well-established procedure in science. Successful syntheses have been demonstrated for both viruses and prokaryotes. However, the technology is not yet available for routine diagnostic purposes. Due to the potential utility of diagnostic sequencing and sequence-based de novo synthesis of pathogens, it seems worthwhile to establish the technology for diagnostic purposes over the intermediate term. This is particularly true for resource-restricted deployment settings, where safe handling of harmful pathogens cannot always be guaranteed. peerReviewed Document type: Articl

Intracellular Persisting Staphylococcus aureus Is the Major Pathogen in Recurrent Tonsillitis

BACKGROUND: The two major indications for tonsillectomy are recurrent tonsillitis (RT) and peritonsillar abscess (PTA). Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. METHODS: In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS), fluorescent in situ hybridization (FISH) and immunohistochemistry. FINDINGS: S. aureus was the predominant species (57.7%) in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2%) in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay) showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. CONCLUSIONS: Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1)

Rapid Discrimination of Salmonella enterica Serovar Typhi from Other Serovars by MALDI-TOF Mass Spectrometry

Systemic infections caused by Salmonella enterica are an ongoing public health problem especially in Sub-Saharan Africa. Essentially typhoid fever is associated with high mortality particularly because of the increasing prevalence of multidrug-resistant strains. Thus, a rapid blood-culture based bacterial species diagnosis including an immediate sub-differentiation of the various serovars is mandatory. At present, MALDI-TOF based intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi. In this study, we investigated the appropriateness of ICMS to identify pathogenic bacteria derived from Sub-Saharan Africa and tested the potential of this technology to discriminate S. enterica subsp. enterica serovar Typhi (S. Typhi) from other serovars. Among blood culture isolates obtained from a study population suffering from febrile illness in Ghana, no major misidentifications were observed for the species identification process, but serovars of Salmonella enterica could not be distinguished using the commercially available Biotyper database. However, a detailed analysis of the mass spectra revealed several serovar-specific biomarker ions, allowing the discrimination of S. Typhi from others. In conclusion, ICMS is able to identify isolates from a sub-Saharan context and may facilitate the rapid discrimination of the clinically and epidemiologically important serovar S. Typhi and other non-S. Typhi serovars in future implementations

Novel Murine Infection Models Provide Deep Insights into the “Ménage à Trois” of Campylobacter jejuni, Microbiota and Host Innate Immunity

BACKGROUND: Although Campylobacter jejuni-infections have a high prevalence worldwide and represent a significant socioeconomic burden, it is still not well understood how C. jejuni causes intestinal inflammation. Detailed investigation of C. jejuni-mediated intestinal immunopathology is hampered by the lack of appropriate vertebrate models. In particular, mice display colonization resistance against this pathogen. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations we developed a novel C. jejuni-infection model using gnotobiotic mice in which the intestinal flora was eradicated by antibiotic treatment. These animals could then be permanently associated with a complete human (hfa) or murine (mfa) microbiota. After peroral infection C. jejuni colonized the gastrointestinal tract of gnotobiotic and hfa mice for six weeks, whereas mfa mice cleared the pathogen within two days. Strikingly, stable C. jejuni colonization was accompanied by a pro-inflammatory immune response indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils and apoptotic cells, as well as increased concentrations of TNF-α, IL-6, and MCP-1 in the colon mucosa of hfa mice. Analysis of MyD88(-/-), TRIF(-/-), TLR4(-/-), and TLR9(-/-) mice revealed that TLR4- and TLR9-signaling was essential for immunopathology following C. jejuni-infection. Interestingly, C. jejuni-mutant strains deficient in formic acid metabolism and perception induced less intestinal immunopathology compared to the parental strain infection. In summary, the murine gut flora is essential for colonization resistance against C. jejuni and can be overcome by reconstitution of gnotobiotic mice with human flora. Detection of C. jejuni-LPS and -CpG-DNA by host TLR4 and TLR9, respectively, plays a key role in immunopathology. Finally, the host immune response is tightly coupled to bacterial formic acid metabolism and invasion fitness. CONCLUSION/SIGNIFICANCE: We conclude that gnotobiotic and "humanized" mice represent excellent novel C. jejuni-infection and -inflammation models and provide deep insights into the immunological and molecular interplays between C. jejuni, microbiota and innate immunity in human campylobacteriosis

Impact of Case Definitions on Efficacy Estimation in Clinical Trials—A Proof-of-Principle Based on Historical Examples

Efficacy estimations in clinical trials are based on case definitions. Commonly, they are a more or less complex set of conditions that have to be fulfilled in order to define a clinical case. In the simplest variant, such a case is identical with a single positive diagnostic test result. Frequently, however, case definitions are more complex. Further, their conditions often ignore the inherent logical structure of symptoms and disease: A symptom or a set of symptoms may be necessary but not sufficient for the unambiguous identification of a case. After describing the structure of case definitions and its impact on efficacy estimations, we exemplify this impact using data from two clinical trials dealing with the effectiveness of the vaginal application of tenofovir gel for the prevention of HIV infections and with the therapeutic effects of fecal transplantation on recurrent Clostridium difficile infections. We demonstrate that the diagnostic performance of case definitions affects efficacy estimations for interventions in clinical trials. The potential risk of bias and uncertainty is high, irrespective of the complexity of the case definition. Accordingly, case definitions in clinical trials should focus on specificity in order to avoid the risk of bias

Risk Assessment for Molds in the Vicinity of a Child Requiring Peritoneal Dialysis Living in a Rural Northern German Area

As well as severe immunosuppression, other predisposing factors may facilitate invasive mycosis caused by molds. Chronic kidney disease and the resulting peritoneal dialysis have been reported as factors putting patients at risk of fungal infections from environmental sources. We describe an environmental investigation undertaken to guide exposure prevention for a peritoneal dialysis patient with transient colonization of her nostrils by Lichtheimia corymbifera in a rural area of northern Germany. Systematic screening for airborne and surface-deposited molds enabled targeted recommendations to be made, although Lichtheimia corymbifera itself was not grown from the collected environmental samples. This communication is intended to illustrate how such an investigation can be performed on the basis of the environmental distribution of the molds and how preventive recommendations can be derived from the results

Infectious Causes of Cholesteatoma and Treatment of Infected Ossicles prior to Reimplantation by Hydrostatic High-Pressure Inactivation

Chronic inflammation, which is caused by recurrent infections, is one of the factors contributing to the pathogenesis of cholesteatoma. If reimplantation of autologous ossicles after a surgical intervention is intended, inactivation of planktonic bacteria and biofilms is desirable. High hydrostatic pressure treatment is a procedure, which has been used to inactivate cholesteatoma cells on ossicles. Here we discuss the potential inactivating effect of high hydrostatic pressure on microbial pathogens including biofilms. Recent experimental data suggest an incomplete inactivation at a pressure level, which is tolerable for the bone substance of ossicles and results at least in a considerable reduction of pathogen load. Further studies are necessary to access how far this quantitative reduction of pathogens is sufficient to prevent ongoing chronic infections, for example, due to forming of biofilms