Proof-of-Concept Standardized Approach Using a Single-Disk Method Analogous to Antibiotic Disk Diffusion Assays for Routine Phage Susceptibility Testing in Diagnostic Laboratories.

Application of bacteriophages is increasingly being implemented in clinical therapies. Prior susceptibility testing should be regarded as mandatory, but standards are lacking. The objective of this research was to develop a highly standardized methodology to facilitate phage susceptibility testing (PST) in clinical microbiology routine laboratories. Therefore, EUCAST methods established for single disk-based antibiotic susceptibility testing (AST) were adapted. In a first step, basic parameters were evaluated using well-studied Escherichia phage T4-Escherichia coli combinations. In addition, test results were compared to those from conventional spot test and efficiency of plating (EOP) approaches. In a second step, the applicability of the methodology and the most promising test parameters were demonstrated for five other frequently isolated clinical bacterial species and their corresponding phages. At present, the method predominantly leads to qualitative rather than quantitative results. This disk-based approach provides a standardized, easy-to-handle, reproducible and reliable PST protocol by relying on well-established routine procedures in diagnostic laboratories. IMPORTANCE Application of bacteriophages in clinical therapies is attractive due to increasing rates of isolation of multidrug-resistant bacteria worldwide. As the phage effect is highly specific, prior susceptibility testing of target bacteria is mandatory. Of note, established standards are lacking. In this research, we adapted the single-disk method for antibiotic susceptibility testing to phage susceptibility testing (PST) in order to provide a standardized, easy-to-handle, reproducible, and reliable PST protocol for application in diagnostic routine laboratories.

Next-generation sequencing for hypothesis-free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples - a proof-of-principle assessment.

BACKGROUND: The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question. RESULTS: For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis. CONCLUSIONS: The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin-fixed, paraffin-embedded tissue requires further standardization.

Detection of Tropheryma whipplei in stool samples by one commercial and two in-house real-time PCR assays.

OBJECTIVE: Tropheryma whipplei, the causative agent of Whipple's disease, can also be identified in stool samples of humans without systemic disease. It is much more frequently detected in human stool samples in tropical environments than in industrialized countries. PCR-screening has been applied for point prevalence studies and environmental assessments in tropical settings, but results depend on the applied assay. We compared one commercial qPCR kit with two well-described in-house assays for detection of T. whipplei from stool. METHODS: Residual materials from nucleic acid extractions of stool samples from two groups with presumably different prevalences and increased likelihood of being colonized or infected by T. whipplei were tested. One group comprised 300 samples from study participants from western Africa (group 1); the second group was of 300 returnees from tropical deployments (group 2). Each sample was assessed with all three qPCR assays. Cycle threshold (Ct ) values were descriptively compared. RESULTS: Based solely on mathematical modeling, the three PCR assays showed considerably different detection rates of T. whipplei DNA in stool samples (kappa 0.67 (95% confidence interval [0.60, 0.73])). Considering the calculated test characteristics, prevalence of 28.3% for group 1 and 5.0% for group 2 was estimated. Discordant test results were associated with later Ct values. The study did not validate the assays for the detection of T. whipplei in Whipple's disease and for diagnostic purposes since clinical specificity and sensitivity were not investigated. CONCLUSIONS: In spite of the observed diagnostic uncertainty, PCR-based screening approaches can be used for epidemiological purposes and environmental samples to define the source and reservoir in resource-limited tropical settings if prevalence is calculated using diagnostic accuracy-adjusted methods.

Comparison of the etiological relevance of Staphylococcus haemolyticus and Staphylococcus hominis.

The study was performed to assess potential differences in the etiological relevance of two coagulase-negative staphylococci (CoNS), Staphylococcus haemolyticus and Staphylococcus hominis, in an observational single-center study. Over a 5-year interval, patients in whom there was detected S. haemolyticus or S. hominis of presumed etiological relevance were assessed for the primary endpoint death during hospital stay and the secondary endpoint transfer to an intensive care unit (ICU) after the detection of S. haemolyticus or S. hominis. Patients with S. haemolyticus or S. hominis died in 11.3% (50 out of 444) and 9.5% (60 out of 631) of cases, respectively, and were transferred to ICU after S. haemolyticus and S. hominis detection in 8.7% (19 out of 219) and 11.7% (44 out of 377) of cases, respectively. There was no significance for species-related influence on the primary outcome parameter (P > 0.1), while ICU transfers were more likely for patients with S. hominis detections (P = 0.016). Delayed diagnosis of both CoNS species was associated with an increased probability of death (P = 0.009). The study revealed comparable morbidity caused by S. haemolyticus and S. hominis identified in a clinically relevant context.

Arthralgia and blood culture-negative endocarditis in middle Age Men suggest tropheryma whipplei infection: report of two cases and review of the literature.

BACKGROUND: Whipple's disease is a rare, often multisystemic chronic infectious disease caused by the rod-shaped bacterium Tropheryma whipplei. Very rarely the heart is involved in the process of the disease, leading to culture-negative infective endocarditis. Up to 20 % of all infective endocarditis are blood culture-negative and therefore a diagnostic challenge. We present two unusual cases of culture-negative infective endocarditis encountered in two different patients with prior history of arthralgia. A history of rheumatic arthritis or even a transient arthralgia should put Tropheryma whipplei on the top of differentials in patients of this age group presenting with culture-negative infective endocarditis, especially in cases of therapy resistance to antirheumatic agents. CASE PRESENTATION: The first patient was a 55 year-old Caucasian male with culture-negative Whipple-related adhesive pericarditis and endocarditis of the aortic valve. Importantly, the patient reported a 15-year history of therapy resistant sero-negative migratory polyarthritis. Aortic valve endocarditis developed during treatment with tocilizumab. The second patient was a 65-year-old male patient with no prior history of the classic Whipple's disease who presented with a culture-negative aortic valve endocarditis. His past medical history revealed episodes of transient arthralgia, which he was not treated for however, due to the self-limiting nature of the symptoms. Both patients underwent aortic valve replacement surgery. During surgery, pericardectomy was necessary in the first patient due to adhesive pericarditis. Post surgery both patients were started on long-term treatment with trimetoprim-sulfamethoxazol. At 1-year follow-up of both patients, echocardiographic and clinical assessment revealed no signs of persistent infection. Both men reported negative history of arthralgia during the one year period post surgery. CONCLUSION: Tropheryma whipplei culture negative-infective endocarditis is an emerging clinical entity, predominantly found in middle-aged and older men with a history of arthralgia. These data highlight the need for ruling out Whipple's disease in patients with a history of arthralgia prior to initiation of biological agents in treatment of rheumatoid arthritis. There is also a need to assess for Tropheryma whipplei in all patients with culture- negative infective endocarditis.

Preanalytical, Analytical and Postanalytical Analyses on Corynebacterium spp. and Actinomycetaceae in Urine Samples of Patients with Suspected Urinary Tract Infection-A Hypothesis-Forming Observational Study.

A hypothesis-forming exploratory cross-sectional assessment was conducted to assess the occurrence and relevance of Gram-positive rod-shaped bacteria like Corynebacterium spp. and Actinomycetaceae in human urine samples. In total, 1170 urine samples from 1031 inpatients with suspected urinary tract infection were assessed for culture-based growth of Gram-positive rod-shaped bacteria applying API Coryne assays, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and in-house 16S rRNA gene sequencing. Overall, 502 different bacterial colonies from 346 urine samples taken from 324 inpatients were observed. The three quantitatively most abundant genera or genus clusters were Corynebacterium (254 isolates, 62%), Actinomyces/Winkia (79 isolates, 19%), and Actinotignum/Actinobaculum (29 isolates, 7%). Compared to sequencing, the diagnostic accuracy of all assessed competitor assays from the diagnostic routine was <80% for differentiation on the genus level and <30% for differentiation on the species level. Prolongated incubation for 4 days compared to 2 days resulted in additional detection of 15% of the totally recorded Gram-positive rod-shaped bacteria. An approximately 5-fold increased detection rate in mid-stream urine compared to urine acquired applying alternative sampling strategies was observed. In conclusion, in the rare event of the suspected clinical relevance of such findings, confirmatory testing with invasively sampled urine should be considered due to the high contamination rate observed in mid-stream urine. Confirmatory testing by DNA-sequencing methods should be considered if an exact identification of genus or species is regarded as relevant for the individual choice of the therapeutic strategy.

Heterologous expression of sahH reveals that biofilm formation is autoinducer-2-independent in Streptococcus sanguinis but is associated with an intact activated methionine cycle.

Numerous studies have claimed deleterious effects of LuxS mutation on many bacterial phenotypes, including bacterial biofilm formation. Genetic complementation mostly restored the observed mutant phenotypes to WT levels, leading to the postulation that quorum sensing via a family of molecules generically termed autoinducer-2 (AI-2) is essential for many phenotypes. Because LuxS mutation has dual effects, this hypothesis needs to be investigated into the details for each bacterial species. In this study we used S. sanguinis SK36 as a model biofilm bacterium and employed physiological characterization and transcriptome approaches on WT and luxS-deficient strains, in combination with chemical, luxS, and sahH complementation experiments. SahH enables a direct conversion of SAH to homocysteine and thereby restores the activated methionine cycle in a luxS-negative background without formation of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione. With this strategy we were able to dissect the individual contribution of LuxS and AI-2 activity in detail. Our data revealed that S. sanguinis biofilm formation is independent from AI-2 substance pools and is rather supported by an intact activated methyl cycle. Of 216 differentially transcribed genes in the luxS mutant, 209 were restored by complementation with a gene encoding the S-adenosylhomocysteine hydrolase. Only nine genes, mainly involved in natural competence, were directly affected by the AI-2 quorum-sensing substance pool. Cumulatively, this suggested that biofilm formation in S. sanguinis is not under control of AI-2. Our study suggests that previously evaluated LuxS mutants in other species need to be revisited to resolve the precise contribution of AI-2 substance pools and the methionine pathways.

Protective mechanisms of respiratory tract Streptococci against Streptococcus pyogenes biofilm formation and epithelial cell infection.

Streptococcus pyogenes (group A streptococci [GAS]) encounter many streptococcal species of the physiological microbial biome when entering the upper respiratory tract of humans, leading to the question how GAS interact with these bacteria in order to establish themselves at this anatomic site and initiate infection. Here we show that S. oralis and S. salivarius in direct contact assays inhibit growth of GAS in a strain-specific manner and that S. salivarius, most likely via bacteriocin secretion, also exerts this effect in transwell experiments. Utilizing scanning electron microscopy documentation, we identified the tested strains as potent biofilm producers except for GAS M49. In mixed-species biofilms, S. salivarius dominated the GAS strains, while S. oralis acted as initial colonizer, building the bottom layer in mixed biofilms and thereby allowing even GAS M49 to form substantial biofilms on top. With the exception of S. oralis, artificial saliva reduced single-species biofilms and allowed GAS to dominate in mixed biofilms, although the overall two-layer structure was unchanged. When covered by S. oralis and S. salivarius biofilms, epithelial cells were protected from GAS adherence, internalization, and cytotoxic effects. Apparently, these species can have probiotic effects. The use of Affymetrix array technology to assess HEp-2 cell transcription levels revealed modest changes after exposure to S. oralis and S. salivarius biofilms which could explain some of the protective effects against GAS attack. In summary, our study revealed a protection effect of respiratory tract bacteria against an important airway pathogen and allowed a first in vitro insight into local environmental processes after GAS enter the respiratory tract.

Serological survey of HIV and syphilis in pregnant women in Madagascar.

OBJECTIVES: Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow-up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. METHODS: Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow-up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. RESULTS: Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. CONCLUSIONS: Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.

In vivo examination of the local inflammatory response after implantation of Ti6Al4V samples with a combined low-temperature plasma treatment using pulsed magnetron sputtering of copper and plasma-polymerized ethylenediamine.

Copper (Cu) could serve as antibacterial coating for Ti6Al4V implants. An additional cell-adhesive layer might compensate Cu cytotoxicity. This study aimed at in vitro and in vivo evaluation of low-temperature plasma treatment of Ti6Al4V plates with Ti/Cu magnetron sputtering (Ti6Al4V-Ti/Cu), plasma-polymerized ethylenediamine (Ti6Al4V-PPEDA), or both (Ti6Al4V-Ti/Cu-PPEDA). Ti6Al4V-Ti/Cu and Ti6Al4V-Ti/Cu-PPEDA had comparable in vitro Cu release and antibacterial effectiveness. Following intramuscular implantation of Ti6Al4V-Ti/Cu, Ti6Al4V-PPEDA, Ti6Al4V-Ti/Cu-PPEDA and Ti6Al4V controls for 7, 14 and 56 days with 8 rats/day, peri-implant tissue was immunohistochemically examined for different inflammatory cells. Ti6Al4V-PPEDA had more mast cells and NK cells than Ti6Al4V, and more tissue macrophages, T lymphocytes, mast cells and NK cells than Ti6Al4V-Ti/Cu-PPEDA. Ti6Al4V-Ti/Cu had more mast cells than Ti6Al4V and Ti6Al4V-Ti/Cu-PPEDA. Results indicate that PPEDA-mediated cell adhesion counteracted Cu cytotoxicity. Ti6Al4V-Ti/Cu-PPEDA differed from Ti6Al4V only for mast cells on day 56. Altogether, implants with both plasma treatments had antibacterial properties and did not increase inflammatory reactions.

A model of implant-associated infection in the tibial metaphysis of rats.

OBJECTIVE: Implant-associated infections remain serious complications in orthopaedic and trauma surgery. A main scientific focus has thus been drawn to the development of anti-infective implant coatings. Animal models of implant-associated infections are considered helpful in the in vivo testing of new anti-infective implant coatings. The aim of the present study was to evaluate a novel animal model for generation of implant-associated infections in the tibial metaphysis of rats. MATERIALS AND METHODS: A custom-made conical implant made of Ti6Al4V was inserted bilaterally at the medial proximal tibia of 26 female Sprague-Dawley rats. Staphylococcus aureus in amounts spanning four orders of magnitude and each suspended in 15 µ l phosphate buffered saline (PBS) was inoculated into the inner cavity of the implant after the implantation into the defined position. Controls were treated accordingly with PBS alone. Animals were then followed for six weeks until sacrifice. Implant-associated infection was evaluated by microbiological investigation using swabs and determination of viable bacteria in the bone around the implant and the biofilm on the implants after sonification. RESULTS: Irrespective of the initial inoculum, all animals in the various groups harbored viable bacteria in the intraoperative swabs as well as the sonication fluid of the implant and the bone samples. No correlation could be established between initially inoculated CFU and population sizes on implant surfaces at sacrifice. However, a significantly higher viable count was observed from peri-implant bone samples for animals inoculated with 10(6) CFU. Macroscopic signs of animal infection (pus and abscess formation) were only observed for implants inoculated with at least 10(5) CFU S. aureus. DISCUSSION/CONCLUSION: The results demonstrate the feasibility of this novel animal model to induce an implant-associated infection in the metaphysis of rats, even with comparatively low bacterial inocula. The specific design of the implant allows an application of bacteria in reproducible numbers at well-defined contact sites to the animal bone.

Epidemiology of vancomycin-resistant enterococci in the United Arab Emirates: a retrospective analysis of 12 years of national AMR surveillance data.

Introduction: Enterococci are usually low pathogenic, but can cause invasive disease under certain circumstances, including urinary tract infections, bacteremia, endocarditis, and meningitis, and are associated with peritonitis and intra-abdominal abscesses. Increasing resistance of enterococci to glycopeptides and fluoroquinolones, and high-level resistance to aminoglycosides is a concern. National antimicrobial resistance (AMR) surveillance data for enterococci from the Middle East and North Africa (MENA) and the Gulf region is scarce. Methods: A retrospective 12-year analysis of N = 37,909 non-duplicate diagnostic Enterococcus spp. isolates from the United Arab Emirates (UAE) was conducted. Data was generated by routine patient care during 2010-2021, collected by trained personnel and reported by participating surveillance sites to the UAE National AMR Surveillance program. Data analysis was conducted with WHONET. Results: Enterococcus faecalis was the most commonly reported species (81.5%), followed by Enterococcus faecium (8.5%), and other enterococci species (4.8%). Phenotypically vancomycin-resistant enterococci (VRE) were found in 1.8% of Enterococcus spp. isolates. Prevalence of VRE (%VRE) was highest for E. faecium (8.1%), followed by E. faecalis (0.9%). A significant level of resistance to glycopeptides (%VRE) for these two species has been observed in the majority of observed years [E. faecalis (0-2.2%), 2010: 0%, 2021: 0.6%] and E. faecium (0-14.2%, 2010: 0%, 2021: 5.8%). Resistance to fluoroquinolones was between 17 and 29% (E. faecalis) and was higher for E. faecium (between 42 and 83%). VRE were associated with higher patient mortality (RR: 2.97), admission to intensive care units (RR: 2.25), and increased length of stay (six excess inpatient days per VRE case), as compared to vancomycin-susceptible Enterococcus spp. Discussion: Published data on Enterococcus infections, in particular VRE-infections, in the UAE and MENA region is scarce. Our data demonstrates that VRE-enterococci are relatively rare in the UAE, however showing an increasing resistance trend for several clinically important antibiotic classes, causing a concern for the treatment of serious infections caused by enterococci. This study also demonstrates that VRE were associated with higher mortality, increased intensive care unit admission rates, and longer hospitalization, thus poorer clinical outcome and higher associated costs in the UAE. We recommend the expansion of current surveillance techniques (e.g., local VRE screening), stricter infection prevention and control strategies, and better stewardship interventions. Further studies on the molecular epidemiology of enterococci are needed.

Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes.

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes.

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.

Serum profile of pro- and anti-inflammatory cytokines in rats following implantation of low-temperature plasma-modified titanium plates.

Surface modification of Titanium (Ti) by low-temperature plasma influences cell-material interactions. Therefore, this study aimed at examining serum cytokine levels and associations after intramuscular implantation (n = 8 rats/group) of Ti-plates with Plasma Polymerized Allyl Amine (Ti-PPAAm), Plasma Polymerized Acrylic Acid (Ti-PPAAc), and without such layers (Ti-Controls). Pro-inflammatory (IL-2, IFNγ, IL-6) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines were measured weekly for 56 days. Ti-PPAAm caused increased IL-2 (d7-14, d35), increased IFNγ (d35) and decreased IL-10 (d35, d49-56). Ti-PPAAc induced divergent anti-inflammatory cytokine changes with increased IL-4 (d28-56) and decreased IL-10 (d42-56). Ti-Controls elicited increased IL-2 (d42) and IFNγ (d35-42, d56). IL-6 was not detected and IL-13 only in three samples, thus they do not influence the response against these Ti implants. Correlation analysis revealed surface-dependent associations between cytokines indicating the involvement of different inflammatory cell populations. Concluding, different plasma modifications induce specific serum cytokine profiles and associations indicating distinct inflammatory responses.

Culture-negative infective endocarditis of the aortic valve due to Aerococcus urinae: a rare aetiology.

Bacteria of the species Aerococcus urinae are Gram-positive, catalase-negative cocci that are arranged in pairs, tetrads, or clusters resembling enterococci or staphylococci. They are rare causative agents of infective endocarditis. Repetitive urinary tract infections based upon underlying genitourinary tract abnormalities could involve these bacteria. Due to their similarity to other Gram-positive cocci misinterpretation may occur along the line of microbiologic differentiation, which could potentially lead to a fatal outcome. We herein report on the clinical course of a 68 year-old male patient who in the setting of an embolic stroke was initially diagnosed with a culture-negative acute infective endocarditis of the aortic valve.

Imaging and Clinical Parameters for Distinction between Infected and Non-Infected Fluid Collections in CT: Prospective Study Using Extended Microbiological Approach.

The aim of this investigation was to evaluate predictive CT imaging features and clinical parameters to distinguish infected from sterile fluid collections. Detection of infectious agents by advanced microbiological analysis was used as the reference standard. From April 2018 to October 2019, all patients undergoing CT-guided drainages were prospectively enrolled, if drainage material volume was at least 5 mL. Univariate analysis revealed attenuation (p = 0.001), entrapped gas (p < 0.001), fat stranding (p < 0.001), wall thickness (p < 0.001) and enhancement (p < 0.001) as imaging biomarkers and procalcitonin (p = 0.003) as clinical predictive parameters for infected fluid collections. On multivariate analysis, attenuation > 10 HU (p = 0.038), presence of entrapped gas (p = 0.027) and wall enhancement (p = 0.028) were independent parameters for distinguishing between infected and non-infected fluids. Gas entrapment had high specificity (93%) but low sensitivity (48%), while wall enhancement had high sensitivity (91%) but low specificity (50%). CT attenuation > 10 HU showed intermediate sensitivity (74%) and specificity (70%). Evaluation of the published proposed scoring systems did not improve diagnostic accuracy over independent predictors in our study. In conclusion, this prospective study confirmed that CT attenuation > 10 HU, entrapped gas and wall enhancement are the key imaging features to distinguish infected from sterile fluid collections on CT.

Genomic organization, structure, regulation and pathogenic role of pilus constituents in major pathogenic Streptococci and Enterococci.

Oligocomponent pilus structures, recently discovered in many important Gram-positive pathogens, represent a new class of virulence factors with adhesive and matrix protein-binding activity. Some of these proteins have emerged as very promising lead components of protein-based vaccines against Streptococci. These extended surface structures play key roles in host cell and tissue adherence, paracellular translocation, and biofilm formation of major Gram-positive pathogens such as Streptococcus pyogenes, S. agalactiae, S. pneumoniae as well as in opportunistic and nosocomial pathogens like Enterococci. Here, we discuss the similarities and differences of: (1) the genomic organization of the various regions encoding pilus proteins, (2) the number, type, and assembly of the proteins constituting the pili, (3) their expression and regulation mechanisms, (4) their role in bacterial virulence, and (5) their potential as vaccine candidate antigens.