Historic methicillin-resistant Staphylococcus aureus: expanding current knowledge using molecular epidemiological characterization of a Swiss legacy collection.

BACKGROUND: Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes. METHODS: We accessed 451 historic (1965-1987) MRSA isolates stored in the Culture Collection of Switzerland, mostly collected from the Zurich region. We determined phenotypic antimicrobial resistance (AMR) and performed whole genome sequencing (WGS) using Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international assemblies from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes. RESULTS: Among the 451 historic Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-III. Early MRSA were predominantly from clonal complex (CC)8. From 1980 to the end of the twentieth century, we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally. CONCLUSIONS: The combined analysis of 1761 historic and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss-German and international MRSA across time.

Unbiased screen for pathogens in human paraffin-embedded tissue samples by whole genome sequencing and metagenomics.

Identification of bacterial pathogens in formalin fixed, paraffin embedded (FFPE) tissue samples is limited to targeted and resource-intensive methods such as sequential PCR analyses. To enable unbiased screening for pathogens in FFPE tissue samples, we established a whole genome sequencing (WGS) method that combines shotgun sequencing and metagenomics for taxonomic identification of bacterial pathogens after subtraction of human genomic reads. To validate the assay, we analyzed more than 100 samples of known composition as well as FFPE lung autopsy tissues with and without histological signs of infections. Metagenomics analysis confirmed the pathogenic species that were previously identified by species-specific PCR in 62% of samples, showing that metagenomics is less sensitive than species-specific PCR. On the other hand, metagenomics analysis identified pathogens in samples, which had been tested negative for multiple common microorganisms and showed histological signs of infection. This highlights the ability of this assay to screen for unknown pathogens and detect multi-microbial infections which is not possible by histomorphology and species-specific PCR alone.

Direct antimicrobial resistance prediction from clinical MALDI-TOF mass spectra using machine learning.

Early use of effective antimicrobial treatments is critical for the outcome of infections and the prevention of treatment resistance. Antimicrobial resistance testing enables the selection of optimal antibiotic treatments, but current culture-based techniques can take up to 72 hours to generate results. We have developed a novel machine learning approach to predict antimicrobial resistance directly from matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra profiles of clinical isolates. We trained calibrated classifiers on a newly created publicly available database of mass spectra profiles from the clinically most relevant isolates with linked antimicrobial susceptibility phenotypes. This dataset combines more than 300,000 mass spectra with more than 750,000 antimicrobial resistance phenotypes from four medical institutions. Validation on a panel of clinically important pathogens, including Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, resulting in areas under the receiver operating characteristic curve of 0.80, 0.74 and 0.74, respectively, demonstrated the potential of using machine learning to substantially accelerate antimicrobial resistance determination and change of clinical management. Furthermore, a retrospective clinical case study of 63 patients found that implementing this approach would have changed the clinical treatment in nine cases, which would have been beneficial in eight cases (89%). MALDI-TOF mass spectra-based machine learning may thus be an important new tool for treatment optimization and antibiotic stewardship.

Cognitive correlates of α4ß2 nicotinic acetylcholine receptors in mild Alzheimer's dementia.

In early Alzheimer's dementia, there is a need for PET biomarkers of disease progression with close associations to cognitive dysfunction that may aid to predict further cognitive decline and neurodegeneration. Amyloid biomarkers are not suitable for that purpose. The α4ß2 nicotinic acetylcholine receptors (α4ß2-nAChRs) are widely abundant in the human brain. As neuromodulators they play an important role in cognitive functions such as attention, learning and memory. Post-mortem studies reported lower expression of α4ß2-nAChRs in more advanced Alzheimer's dementia. However, there is ongoing controversy whether α4ß2-nAChRs are reduced in early Alzheimer's dementia. Therefore, using the recently developed α4ß2-nAChR-specific radioligand (-)-18F-flubatine and PET, we aimed to quantify the α4ß2-nAChR availability and its relationship to specific cognitive dysfunction in mild Alzheimer's dementia. Fourteen non-smoking patients with mild Alzheimer's dementia, drug-naïve for cholinesterase therapy, were compared with 15 non-smoking healthy controls matched for age, sex and education by applying (-)-18F-flubatine PET together with a neuropsychological test battery. The one-tissue compartment model and Logan plot method with arterial input function were used for kinetic analysis to obtain the total distribution volume (VT) as the primary, and the specific binding part of the distribution volume (VS) as the secondary quantitative outcome measure of α4ß2-nAChR availability. VS was determined by using a pseudo-reference region. Correlations between VT within relevant brain regions and Z-scores of five cognitive functions (episodic memory, executive function/working memory, attention, language, visuospatial function) were calculated. VT (and VS) were applied for between-group comparisons. Volume of interest and statistical parametric mapping analyses were carried out. Analyses revealed that in patients with mild Alzheimer's dementia compared to healthy controls, there was significantly lower VT, especially within the hippocampus, fronto-temporal cortices, and basal forebrain, which was similar to comparisons of VS. VT decline in Alzheimer's dementia was associated with distinct domains of impaired cognitive functioning, especially episodic memory and executive function/working memory. Using (-)-18F-flubatine PET in patients with mild Alzheimer's dementia, we show for the first time a cholinergic α4ß2-nAChR deficiency mainly present within the basal forebrain-cortical and septohippocampal cholinergic projections and a relationship between lower α4ß2-nAChR availability and impairment of distinct cognitive domains, notably episodic memory and executive function/working memory. This shows the potential of (-)-18F-flubatine as PET biomarker of cholinergic α4ß2-nAChR dysfunction and specific cognitive decline. Thus, if validated by longitudinal PET studies, (-)-18F-flubatine might become a PET biomarker of progression of neurodegeneration in Alzheimer's dementia.

Influence of Sonication on Bacterial Regrowth from Antibiotic Loaded PMMA Scaffolds - An In-vitro Study.

Periprosthetic joint infection (PJI) is one of the most challenging complications after joint replacement. However, when treated correctly, chances of recovery are good. The most important step in correct diagnosis and management of PJI is the detection of the causative germ. In the last years, the use of sonication in the diagnostic process has become more important. However, this diagnostic methodology has been controversially discussed when used in combination with antibiotic loaded bone cement (PMMA), which is frequently used in joint replacement surgeries. The aim of this study was thus to analyse in vitro bacterial growth in sonication fluid cultures obtained from antibiotic loaded PMMA which were contaminated with various bacterial biofilms. Sonication fluid obtained from antibiotic loaded PMMA (Copal G+V and Copal G+C) and plain Palacos R (control) contaminated either with S. aureus, E. faecalis, S. sanguinis or P.acnes, were analysed for bacterial re-growth in a standardised in-vitro setting. In vitro bacterial growth was not interfered by released antibiotics from sonication of antibiotic loaded PMMA for S. aureus, E. faecalis and S. sanguinis. However, for P. acnes bacterial counts were affected by the released antibiotics as well as by the time delay between sonication and analysis. The in-vitro data suggest sonication to be an easy and sensitive diagnostic modality to detect easy-to-detect bacteria, however, results are alarming for the difficult-to-detect bacteria P. acnes, indicating that further attention and research is necessary to improve the detection of difficult-to-detect bacteria.

Material and biofilm load of K wires in toe surgery: titanium versus stainless steel.

BACKGROUND: Recurrence rates for toe deformity correction are high and primarily are attributable to scar contractures. These contractures may result from subclinical infection. QUESTIONS/PURPOSES: We hypothesized that (1) recurrence of toe deformities and residual pain are related to low-grade infections from biofilm formation on percutaneous K wires, (2) biofilm formation is lower on titanium (Ti) K wires compared with stainless steel (SS) K wires, and (3) clinical outcome is superior with the use of Ti K wires compared with SS K wires. METHODS: In this prospective nonrandomized, comparative study, we investigated 135 lesser toe deformities (61 patients; 49 women; mean ± SD age, 60 ± 15 years) temporarily fixed with K wires between August 2010 and March 2011 (81 SS, 54 Ti). K wires were removed after 6 weeks. The presence of biofilm-related infections was analyzed by sonication. RESULTS: High bacterial loads (> 500 colony-forming units [CFU]/mL) were detected on all six toes requiring revision before 6 months. Increased bacterial load was associated with pain and swelling but not recurrence of the deformity. More SS K wires had greater than 100 CFU/mL bacteria than Ti K wires. For K wires with a bacterial count greater than 100 CFU/mL, toes with Ti K wires had a lower recurrence rate, less pain, and less swelling than toes with SS K wires. CONCLUSIONS: Ti K wires showed superior clinical outcomes to SS K wires. This appears to be attributable to reduced infection rates. Although additional study is needed, we currently recommend the use of Ti K wires for the transfixation of toe deformities. LEVEL OF EVIDENCE: Level II, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Intratracheal perfluorocarbons diminish LPS-induced increase in systemic TNF-alpha.

Perfluorocarbons (PFC) reduce the production of various inflammatory cytokines, including TNF-alpha. The anti-inflammatory effect is not entirely understood. If anti-inflammatory properties are caused by a mechanical barrier, PFC in the alveoli should have no effect on the inflammatory response to intravenous LPS administration. To test that hypothesis, rats (n=31) were administered LPS intravenously and were either spontaneously breathing (Spont), conventionally ventilated (CMV), or receiving partial liquid ventilation (PLV). Serum concentration of TNF-alpha was measured. The pulmonary expressions of TNF-alpha and TNF-alpha receptor 1 protein and of TNF-alpha and ICAM-1 mRNA were determined. LPS caused a significant (P<0.001) increase in serum TNF-alpha. Serum TNF-alpha concentration was similar in LPS/Spont (525+/-180 pg/ml) and LPS/CMV (504+/-154 pg/ml) but was significantly (P<0.001) lower in animals of the LPS/PLV group (274+/-101 pg/ml). Immunohistochemical data on TNF-alpha protein expression showed a LPS-induced increase in TNF-alpha and TNF-alpha receptor 1 expression that was diminished by partial liquid ventilation. PCR measurements revealed a lower expression of TNF-alpha and ICAM-1 mRNA in LPS/PLV than in LPS/CMV or LPS/Spont animals. Semiquantitative histological evaluation revealed only minor alveolar inflammation with no significant differences between the groups. Low serum TNF-alpha concentration in PFC-treated animals is most likely explained by a decreased production of TNF-alpha in the lung.

Propionibacterium spp. in prosthetic joint infections: a diagnostic challenge.

INTRODUCTION: Propionibacterium species are common inhabitants of the skin and usually non-pathogenic for humans. However, Propionibacterium spp. can occasionally cause infections, but are estimated to play a minor role in prosthetic joint infections (PJI). The relative frequency of these anaerobes and their potential to cause surgical site infection may be clinically underestimated. An unknown proportion of these infections might be missed, since little is known about their clinical presentation, and since growth of Propionibacterium spp. in diagnostic samples is often interpreted as contamination. Thus, a hypothesis is being tested, stating that Propionibacterium spp. is not as rare as often reported, and it can cause severe soft-tissue damages in PJI. MATERIALS AND METHODS: In this retrospective analysis, we reviewed all PJI that had been treated in our institution from 2000 to 2005, and assessed the relative frequency of those caused by Propionibacterium spp. In the identified cases, features that led to the diagnosis (clinical, laboratory, radiological, microbiological and histopathological characteristics) were analysed. RESULTS: Of 139 cases of prosthetic joint infections, 8(6%) were caused by Propionibacterium spp. Seven patients complained of pain as the main symptom, and four had damaged soft-tissue. Analysis of the diagnostic procedures showed a median of 39% positive samples out of all cultured biopsies (median 9.5 biopsies per case), with a median time-to-positivity of 8 days. Results of histopathological examinations of the periprosthetic tissue correlated well with the clinical courses. CONCLUSIONS: Our data suggest that Propionibacterium associated prosthetic joint infections occur at a relative frequency that is comparable to many other pathogens. Clinical signs are generally subtle, but the spectrum includes also significant soft-tissue damages. In this study, a median of 9.5 biopsies per case, an incubation time of 14 days, and the aid of histopathological examinations proved to be helpful in establishing the diagnosis.

Increased resistin blood levels are not associated with insulin resistance in patients with renal disease.

BACKGROUND: Resistin is a newly discovered insulin inhibitor secreted by adipocytes. We explored the potential role of resistin in the pathophysiological process of insulin resistance encountered in patients with renal disease. METHODS: Resistin blood concentrations, insulin sensitivity index (by intravenous glucose tolerance test), and glomerular filtration rate (GFR; by inulin clearance) were assessed in 30 male patients with immunoglobulin A glomerulonephritis in different stages of renal disease. RESULTS: Patients with increased resistin blood concentrations had more advanced renal failure and were significantly older. Plasma resistin levels correlated significantly with GFR (r = -0.82; P < 0.0001), plasma homocysteine concentration (r = 0.68; P < 0.001), and age (r = 0.42; P = 0.05), but not with fasted plasma insulin (r = -0.34; P = 0.12), glucose (r = 0.25; P = 0.19), and leptin (r = -0.24; P = 0.21) concentrations; body mass index (r = -0.06; P = 0.75), waist-hip ratio (r = 0.09; P = 0.63), or insulin sensitivity (r = -0.05; P = 0.79). In multiple regression analysis, GFR was the only independent predictor of plasma resistin concentrations in renal patients (r = -0.812; P < 0.0001). CONCLUSION: Resistin blood concentrations increase with progressive impairment of renal function. Thus, the kidney seems to be an important site of resistin elimination. However, the greater than 5-fold increase in resistin blood levels apparently is not associated with deterioration in insulin sensitivity in patients with renal disease.