Super-resolution fluorescence microscopy for investigating bacterial cell biology.

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.

Live imaging of Yersinia translocon formation and immune recognition in host cells.

Yersinia enterocolitica employs a type three secretion system (T3SS) to translocate immunosuppressive effector proteins into host cells. To this end, the T3SS assembles a translocon/pore complex composed of the translocator proteins YopB and YopD in host cell membranes serving as an entry port for the effectors. The translocon is formed in a Yersinia-containing pre-phagosomal compartment that is connected to the extracellular space. As the phagosome matures, the translocon and the membrane damage it causes are recognized by the cell-autonomous immune system. We infected cells in the presence of fluorophore-labeled ALFA-tag-binding nanobodies with a Y. enterocolitica strain expressing YopD labeled with an ALFA-tag. Thereby we could record the integration of YopD into translocons and its intracellular fate in living host cells. YopD was integrated into translocons around 2 min after uptake of the bacteria into a phosphatidylinositol-4,5-bisphosphate enriched pre-phagosomal compartment and remained there for 27 min on average. Damaging of the phagosomal membrane as visualized with recruitment of GFP-tagged galectin-3 occurred in the mean around 14 min after translocon formation. Shortly after recruitment of galectin-3, guanylate-binding protein 1 (GBP-1) was recruited to phagosomes, which was accompanied by a decrease in the signal intensity of translocons, suggesting their degradation or disassembly. In sum, we were able for the first time to film the spatiotemporal dynamics of Yersinia T3SS translocon formation and degradation and its sensing by components of the cell-autonomous immune system.

Population of invasive group A streptococci isolates from a German tertiary care center is dominated by the hypertoxigenic virulent M1UK genotype.

PURPOSE: Hypertoxigenic Streptococcus pyogenes emm1 lineage M1UK has recently been associated with upsurges of invasive infections and scarlet fever in several countries, but whole-genome sequencing surveillance data of lineages circulating in Germany is lacking. In this study, we investigated recent iGAS isolates from our laboratory at a German tertiary care center for the presence of the M1UK lineage. METHODS: Whole-genome sequencing was employed to characterize a collection of 47 consecutive non-copy isolates recovered from blood cultures (21) and tissue samples (26) in our laboratory between October 2022 and April 2023. RESULTS: M protein gene (emm) typing distinguished 14 different emm types, with emm1 (17) being the dominant type. Single-nucleotide polymorphism (SNP) analysis confirmed the presence of all 27 SNPs characteristic for the M1UK lineage in 14 of 17 emm1 isolates. CONCLUSION: This study has shown for the first time that M1UK is present in Germany and might constitute a driving force in the observed surge of GAS infections. This observation mirrors developments in the UK and other countries and underscores the importance of WGS surveillance to understand the epidemiology of GAS.

Visualization of translocons in Yersinia type III protein secretion machines during host cell infection.

Type III secretion systems (T3SSs) are essential virulence factors of numerous bacterial pathogens. Upon host cell contact the T3SS machinery-also named injectisome-assembles a pore complex/translocon within host cell membranes that serves as an entry gate for the bacterial effectors. Whether and how translocons are physically connected to injectisome needles, whether their phenotype is related to the level of effector translocation and which target cell factors trigger their formation have remained unclear. We employed the superresolution fluorescence microscopy techniques Stimulated Emission Depletion (STED) and Structured Illumination Microscopy (SIM) as well as immunogold electron microscopy to visualize Y. enterocolitica translocons during infection of different target cell types. Thereby we were able to resolve translocon and needle complex proteins within the same injectisomes and demonstrate that these fully assembled injectisomes are generated in a prevacuole, a PI(4,5)P2 enriched host cell compartment inaccessible to large extracellular proteins like antibodies. Furthermore, the operable translocons were produced by the yersiniae to a much larger degree in macrophages (up to 25% of bacteria) than in HeLa cells (2% of bacteria). However, when the Rho GTPase Rac1 was activated in the HeLa cells, uptake of the yersiniae into the prevacuole, translocon formation and effector translocation were strongly enhanced reaching the same levels as in macrophages. Our findings indicate that operable T3SS translocons can be visualized as part of fully assembled injectisomes with superresolution fluorescence microscopy techniques. By using this technology, we provide novel information about the spatiotemporal organization of T3SS translocons and their regulation by host cell factors.

Potential Role for Urine Polymerase Chain Reaction in the Diagnosis of Whipple's Disease.

BACKGROUND: Whipple's disease (WD) is a rare infection with Tropheryma whipplei that is fatal if untreated. Diagnosis is challenging and currently based on invasive sampling. In a case of WD diagnosed from a kidney biopsy, we observed morphologically-intact bacteria within the glomerular capsular space and tubular lumens. This raised the questions of whether renal filtration of bacteria is common in WD and whether polymerase chain reaction (PCR) testing of urine might serve as a diagnostic test for WD. METHODS: We prospectively investigated urine samples of 12 newly-diagnosed and 31 treated WD patients by PCR. As controls, we investigated samples from 110 healthy volunteers and patients with excluded WD or acute gastroenteritis. RESULTS: Out of 12 urine samples from independent, therapy-naive WD patients, 9 were positive for T. whipplei PCR. In 3 patients, fluorescence in situ hybridization visualized T. whipplei in urine. All control samples were negative, including those of 11 healthy carriers with T. whipplei-positive stool samples. In our study, the detection of T. whipplei in the urine of untreated patients correlated in all cases with WD. CONCLUSIONS: T. whipplei is detectable by PCR in the urine of the majority of therapy-naive WD patients. With a low prevalence but far-reaching consequences upon diagnosis, invasive sampling for WD is mandatory and must be based on a strong suspicion. Urine testing could prevent patients from being undiagnosed for years. Urine may serve as a novel, easy-to-obtain specimen for guiding the initial diagnosis of WD, in particular in patients with extra-intestinal WD.

Acting on Actin: Rac and Rho Played by Yersinia.

Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.

Antibiotic resistance and clonal diversity of invasive Staphylococcus aureus in the rural Ashanti Region, Ghana.

BACKGROUND: Staphylococcus aureus is among the most common pathogens isolated from blood cultures in Ghana; yet the epidemiology of blood infections in rural settings is poorly described. This study aims to investigate antimicrobial susceptibility and clonal diversity of S. aureus causing bloodstream infections in two hospitals in the Ashanti Region, Ghana. METHODS: Blood cultures were performed for all febrile patients (≥37.5 °C) on hospital admission. Antibiotic susceptibility testing for S. aureus isolates was carried out by the VITEK 2 system. Multiplex polymerase chain reaction (PCR) was used to detect S. aureus-specific nuc gene, Panton-Valentine leukocidin (PVL), and methicillin-resistant S. aureus (MRSA)-specific mecA and mecC genes. The population structure of S. aureus was assessed by spa typing. RESULTS: In total, 9,834 blood samples were cultured, out of which 0.6% (n = 56) were positive for S. aureus. Multidrug resistance (MDR) was detected in 35.7% (n = 20) of the S. aureus strains, of which one was a MRSA. The highest rate of antibiotic resistance was seen for commonly available antibiotics, including penicillin (n = 55; 98.2%), tetracycline (n = 32; 57.1%) and trimethoprim/sulfamethoxazole (n = 26; 46.4%). Of all S. aureus strains, 75.0% (n = 42) carried the PVL-encoding genes. We found 25 different spa types with t355 (n = 11; 19.6%), t314 (n = 8; 14.3%), t084 (n = 8; 14.3%) and t311 (n = 5; 8.9%) being predominant. CONCLUSION: The study exhibited an alarmingly large level of antibiotic resistance to locally available antibiotics. The frequency of genetically diverse and PVL-positive methicillin-sensitive S. aureus (MSSA) was high and could represent a reservoir for the emergence of virulent PVL-positive MRSA clones.

Emergence of daptomycin non-susceptibility in colonizing vancomycin-resistant Enterococcus faecium isolates during daptomycin therapy.

Infections due to vancomycin-resistant enterococci (VRE) are of significant importance in high-risk populations, and daptomycin is a bactericidal antibiotic to treat multidrug-resistant VRE in these patients. The emergence of daptomycin non-susceptibility invasive VRE during daptomycin therapy is a major clinical issue. Here the hypothesis was tested that systemic daptomycin therapy also induces the emergence of daptomycin non-susceptible (DNS-) isolates in colonizing VRE populations. 11 vancomycin-resistant Enterococcus faecium strain pairs recovered from rectal swabs were available for analysis. All initial isolates exhibited daptomycin MICs within the wild type MIC distribution of E. faecium (MIC≤4 mg/L). In follow-up isolates from five patients a 4-16-fold daptomycin MIC increase was detected. All patients carrying DNS-VRE received daptomycin (14-28 days) at 4 mg/kg body weight, while two patients in whom no DNS-VRE emerged were only treated with daptomycin for 1 and 4 days, respectively. Comparative whole genome sequencing identified DNS-VRE-specific single nucleotide polymorphisms (SNP), including mutations in cardiolipin synthase (Cls), and additional SNPs in independent genes potentially relevant for the DNS phenotype. Mutations within cls were also identified in three additional, colonizing DNS-VRE. Of these, at least one strain was transmitted within the hospital. In none of the VRE isolates tested, pre-existing or de novo mutations in the liaFSR operon were detected. This is the first report documenting the emergence of DNS-VRE in colonizing strains during daptomycin treatment, putting the patient at risk for subsequent DNS-VRE infections and priming the spread of DNS-VRE within the hospital environment.

Cytotoxic necrotizing factor-Y boosts Yersinia effector translocation by activating Rac protein.

Pathogenic Yersinia spp. translocate the effectors YopT, YopE, and YopO/YpkA into target cells to inactivate Rho family GTP-binding proteins and block immune responses. Some Yersinia spp. also secrete the Rho protein activator cytotoxic necrotizing factor-Y (CNF-Y), but it has been unclear how the bacteria may benefit from Rho protein activation. We show here that CNF-Y increases Yop translocation in Yersinia enterocolitica-infected cells up to 5-fold. CNF-Y strongly activated RhoA and also delayed in time Rac1 and Cdc42, but when individually expressed, constitutively active mutants of Rac1, but not of RhoA, increased Yop translocation. Consistently, knock-out or knockdown of Rac1 but not of RhoA, -B, or -C inhibited Yersinia effector translocation in CNF-Y-treated and control cells. Activation or knockdown of Cdc42 also affected Yop translocation but much less efficiently than Rac. The increase in Yop translocation induced by CNF-Y was essentially independent of the presence of YopE, YopT, or YopO in the infecting Yersinia strain, indicating that none of the Yops reported to inhibit translocation could reverse the CNF-Y effect. In summary, the CNF-Y activity of Yersinia strongly enhances Yop translocation through activation of Rac.

Complete genome sequence of a Staphylococcus condimenti isolated from a port catheter-associated bloodstream infection.

Here, we describe the complete genome sequence of a Staphylococcus condimenti blood culture isolate from a catheter-related bloodstream infection in a male patient.

MINFLUX imaging of a bacterial molecular machine at nanometer resolution.

The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machinein situ, i.e., the injectisome of the enteropathogenY. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule levelin situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

MALDI-TOF MS fingerprinting allows for discrimination of major methicillin-resistant Staphylococcus aureus lineages.

Early detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and initiation of adequate infection control measures are important objectives in hospital hygiene. To reach these goals, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods like pulsed-field gel electrophoresis (PFGE), spa typing, or multilocus sequence typing (MLST) have a high discriminatory power, however, these methods are time consuming and cost intensive. The aim of this study was to investigate the potential of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for discrimination of major MRSA lineages. By analysis of mass spectra from 25 representative MRSA isolates belonging to the 5 major hospital-acquired (HA) MRSA clonal complexes (CC5, CC8, CC22, CC30, CC45; deduced from spa typing), reproducible spectrum differences were observed at 13 characteristic m/z values allowing robust discrimination of the clonal complexes. When 60 independent clinical MRSA isolates were tested for the presence or absence of the 13 characteristic MALDI-TOF MS peaks, 15 different profiles (MALDI types) could be detected. Hierarchical clustering of the MALDI types showed high concordance with the clonal complexes. Our results suggest that MALDI-TOF MS has the potential to become a valuable first-line tool for inexpensive and rapid typing of MRSA in infection control.

Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting.

Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

Molecular Characterization of Staphylococcus aureus Isolated from Chronic Infected Wounds in Rural Ghana.

BACKGROUND: Globally, Staphylococcus aureus is an important bacterial pathogen causing a wide range of community and hospital acquired infections. In Ghana, resistance of S. aureus to locally available antibiotics is increasing but the molecular basis of resistance and the population structure of S. aureus in particular in chronic wounds are poorly described. However, this information is essential to understand the underlying mechanisms of resistance and spread of resistant clones. We therefore subjected 28 S. aureus isolates from chronic infected wounds in a rural area of Ghana to whole genome sequencing. RESULTS: Overall, resistance of S. aureus to locally available antibiotics was high and 29% were Methicillin resistant Staphylococcus aureus (MRSA). The most abundant sequence type was ST88 (29%, 8/28) followed by ST152 (18%, 5/28). All ST88 carried the mecA gene, which was associated with this sequence type only. Chloramphenicol resistance gene fexB was exclusively associated with the methicillin-resistant ST88 strains. Panton-Valentine leukocidin (PVL) carriage was associated with ST121 and ST152. Other detected mechanisms of resistance included dfrG, conferring resistance to trimethoprim. CONCLUSIONS: This study provides valuable information for understanding the population structure and resistance mechanisms of S. aureus isolated from chronic wound infections in rural Ghana.

A Giant Extracellular Matrix Binding Protein of Staphylococcus epidermidis Binds Surface-Immobilized Fibronectin via a Novel Mechanism.

Although it is normally an innocuous part of the human skin microbiota, Staphylococcus epidermidis has emerged as a major nosocomial pathogen, and implanted foreign materials are an essential risk factor for the development of an infection. The extraordinary efficiency of S. epidermidis to colonize artificial surfaces is particularly related to the ability to form biofilms. Biofilm formation itself critically depends on stable pathogen binding to extracellular host matrix components, e.g. fibronectin (Fn), covering inserted devices in vast amounts. Extracellular matrix binding protein (Embp) and its subdomains referred to as the F-repeat and the FG-repeat are critical for adherence of S. epidermidis to surface-immobilized Fn. Embp-Fn interactions preferentially occur with surface-bound, but not folded, globular Fn via binding to the F3 domain. High-resolution structure analysis of F- and FG-repeats revealed that both repeats are composed of two tightly connected triple α-helix bundles, exhibiting an elongated but rather rigid structural organization in solution. Both F- and FG-repeat possess Fn-binding capacity via interactions with type III subdomain FN12, involving residues within the C and F ß-sheet. FN12 essentially supports stability of the globular Fn state, and thus these findings reasonably explain why Embp-mediated interaction of S. epidermidis necessitates Fn surface immobilization. Thus, Embp employs an uncharacterized bacterial Fn-binding mechanism to promote staphylococcal adherence.IMPORTANCEStaphylococcus epidermidis is a leading pathogen in implant-associated hospital infections. The pathogenesis critically depends on bacterial binding to ECM components, specifically fibronectin (Fn). The cell surface-localized, 1-MDa extracellular matrix binding protein (Embp) is essentially characterized by 10 F- and 40 FG-repeats. These repetitive units, each characterized by two α-helical bundles, organize themselves in a rigid, elongated form. Embp binds preferentially to surface-localized but not soluble Fn, with both F- and FG-repeats being sufficient for Fn binding and resulting bacterial adherence. Binding preferentially involves Fn type III domain, specifically residues of FN12 ß-sheets C and F. Both play key role in stabilizing the globular Fn conformation, explaining the necessity of Fn surface immobilization for a subsequent interaction with Embp. In comparison to many other bacterial Fn-binding proteins using the Fn N terminus, Embp employs a previously undescribed mechanism supporting the adhesion of S. epidermidis to surface-immobilized Fn.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Rapid MALDI-TOF mass spectrometry strain typing during a large outbreak of Shiga-Toxigenic Escherichia coli.

BACKGROUND: In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification. METHODS: Specific peaks in the outbreak strain's spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak. RESULTS: Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates. CONCLUSIONS: MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.

CMY-42, a novel plasmid-mediated CMY-2 variant AmpC beta-lactamase.

We isolated a clinical Escherichia coli strain with an antimicrobial resistance phenotype characteristic for the expression of an AmpC beta-lactamase. Molecular methods revealed a novel, plasmid-localized variant of CMY-2 with a substitution of valine 231 for serine (V231S), which was designated CMY-42. Like the CMY-2-like AmpC beta-lactamase CMY-30, carrying the substitution V231G, CMY-42 displayed increased activity toward expanded spectrum cephalosporins. This finding supports the hypothesis that a bulky side chain at position 231 (Ambler's position 211) may pose a steric clash with certain cephalosporins hindering the access of the AmpC beta-lactamase; however, additional phenomena may account for the observed hydrolytic properties.