InfectoGnostics Research Campus Jena

This pilot study investigated the presence of methicillin-resistant Staphylococcus aureus (MRSA) and third-generation cephalosporin-resistant (3GC-R) Enterobacterales in conventionally kept domestic pigs and their environment across four districts in Rwanda. A total of 114 swabs (nasal, rectal, manure, dust) from 29 farms were collected and processed to isolate resistant bacteria. Thirty-two 3GC-R Enterobacterales were detected. Escherichia coli predominantly harboring blaCTX-M group 1 β-lactamase genes, alongside Klebsiella pneumoniae isolates, all displaying extended-spectrum β-lactamase (ESBL) phenotypes. Four MRSA isolates, all belonging to clonal complex 398 and SCCmec type IV, the typical livestock MRSA, were recovered from nasal and environmental samples. Multidrug resistance was frequently observed. The co-occurrence of β-lactamase genes, non-β-lactam resistance genes, and virulence factors such as fimH and loci associated with extraintestinal pathogenic and enteropathogenic E. coli. The detection of both MRSA and 3GC-R Enterobacterales in the present study indicates pigs and their farm environments as reservoirs of WHO priority pathogens in Rwanda, highlighting a potential public health risk in the context of extensive human-animal-environment interaction. These findings emphasize the urgent need for integrated One Health surveillance and comprehensive AMR control strategies addressing both animal and environmental reservoirs to support Rwanda's National Action Plan on Antimicrobial Resistance.

Background/Objectives: Vancomycin-resistant enterococci (VRE) are one of the leading causes of antibiotic-resistant infections in the hospital setting worldwide, and this has become a major issue, because most patients infected with this strain are difficult to treat. Multiplex real-time polymerase chain reaction (RT PCR) is an advantageous technique that can amplify multiple targets in a single reaction, and can be used to quickly detect specific targets in VRE within two hours, starting from suspected colonies of bacterial cultures, without sample preparation. Methods: In this study, we selected the glycopeptide/vancomycin resistance genes that are most common in clinical settings, vanA and vanB, in combination with the species markers ddl_faecium and ddl_faecalis for the most common VRE species-Enterococcus faecium and Enterococcus faecalis. Results: DNA from forty clinical VRE strains was prepared using a fast and economic heat lysis method, and a multiplex real-time PCR assay was optimized and carried out subsequently. The results were in concordance with the results from recombinase polymerase amplification (RPA) of the same VRE samples. Conclusions: Multiplex RT PCR and RPA for VRE detection proffers a second method for the confirmation of vancomycin resistance, and it can be developed as a fast screening assay for patients before admission into high-risk settings.

Given the rapid cross-country spread of SARS-CoV-2 and the resulting difficulty in tracking lineage spread, we investigated the potential of combining mobile service data and fine-granular metadata (such as postal codes and genomic data) to advance integrated genomic surveillance of the pandemic in the federal state of Thuringia, Germany. We sequenced over 6500 SARS-CoV-2 Alpha genomes (B.1.1.7) across 7 months within Thuringia while collecting patients’ isolation dates and postal codes. Our dataset is complemented by over 66,000 publicly available German Alpha genomes and mobile service data for Thuringia. We identified the existence and spread of nine persistent mutation variants within the Alpha lineage, seven of which formed separate phylogenetic clusters with different spreading patterns in Thuringia. The remaining two are subclusters. Mobile service data can indicate these clusters’ spread and highlight a potential sampling bias, especially of low-prevalence variants. Thereby, mobile service data can be used either retrospectively to assess surveillance coverage and efficiency from already collected data or to actively guide part of a surveillance sampling process to districts where these variants are expected to emerge. The latter concept was successfully implemented as a proof-of-concept for a mobility-guided sampling strategy in response to the surveillance of Omicron sublineage BQ.1.1. The combination of mobile service data and SARS-CoV-2 surveillance by genome sequencing is a valuable tool for more targeted and responsive surveillance.

ObjectivesGerman general practitioners (GPs) either order laboratory tests from external laboratories (initiated laboratory services (ILS)) or perform point-of-care tests (POCTs) within the GP practice. This study investigated the current use and time trends of laboratory testing in German GP practices with a particular focus on POC testing.DesignRetrospective consecutive cross-sectional study by analysis of complete outpatient healthcare claims data (2017–2022).SettingPrimary care in Thuringia, a federal state in central Germany.ParticipantsData from 2061 GPs.Primary and secondary outcome measuresNumber of performed laboratory analyses per year, mean number of laboratory analyses per patient and year.ResultsIn 2022, 1601 GPs performed 16.1 million laboratory analyses, with 14.9 million (92.5%) being ILS and 1.2 million (7.5%) being POCTs conducted within GP practices. The mean number of laboratory tests per patient was 15.5 in 2022. The number of laboratory tests performed per patient decreased by 18.8% from 2017 to 2022. An especially pronounced reduction was observed in the use of POCTs (−36.0%). During the whole study period, the most common initiated laboratory analyses included creatinine, blood cell count, glucose, thyroid-stimulating hormone and HbA1/HbA1c. The most frequently employed POCTs were glucose, urine test strip, prothrombin time/international normalised ratio, HbA1/HbA1c, erythrocyte sedimentation rate and microalbuminuria. POC testing instead of ILS was more likely performed in older patient groups (≥40 years) compared with young adult patients (18–39 years) (adjusted OR (aOR) 40–59 years: 1.19 (95% CI 1.14 to 1.24); aOR 60–79 years: 1.48 (95% CI 1.40 to 1.57); aOR >79 years: 1.43 (95% CI 1.34 to 1.54)).ConclusionsGerman GPs regularly employ laboratory testing, primarily utilising external laboratory service, while POC testing plays a relatively minor role. The notable decline in the use of POCT testing may be attributed to several factors, including regulations from healthcare authorities, reimbursement guidelines and increasing test costs.

The development and improvement of catalytic processes require a detailed understanding of catalyst dynamics, reaction mechanisms, and structure–activity relationships inside catalytic reactors, from the laboratory to production scale. This paper presents the methodology of iso-potential operando DRIFTS for measuring the profiles of surface adsorbates inside catalytic reactors. Iso-potential operando spectroscopy (IPOS) in general and iso-potential operando DRIFTS in particular separate the functionality “catalytic reactor” and “spectroscopic cell” from each other. The catalytic reactor is equipped with a mechanism of spatial sampling and spatial temperature measurement. A small fraction of the reaction mixture is sampled locally in the reactor and transferred continuously into a spectroscopic cell containing a very small amount of the same catalyst as in the reactor. The temperature is set to the same value as is locally measured in the reactor. In this way, the catalyst in the spectroscopic cell is exposed to the same chemical potential as that locally in the catalytic reactor. It is hypothesized that it takes on the same structure, the same surface adsorbates, and shows the same reactivity. IPO DRIFTS is applied to CO2 methanation on Ni/γ-Al2O3 catalysts. Two surface adsorbate species, adsorbed carbonyl (*COads) and adsorbed formate (*HCOOads), are detected. The band intensity of *HCOOads decreases along the catalyst bed with the CO2 concentration in the gas phase, identifying surface formate as a kinetically relevant intermediate. This finding is in line with an associative mechanism where CO2 adsorbs on γ-Al2O3 forming carbonate or bicarbonate, being rapidly hydrogenated to formate. Formate reduction is the rate-determining step, with all subsequent hydrogenation steps to CH4 being fast. The band intensity of *COads does not change, irrespective of position in the catalyst bed. This invariance of *COads can be interpreted in two ways. *COads could be a spectator species that is present at the catalyst surface but not involved in any kinetically relevant reaction channel. Alternatively, *COads could be formed by rapid dissociative adsorption of CO2 at the surface of the Ni nanoparticles with a high adsorption equilibrium constant, leading to an almost constant *COads coverage within the investigated CO2 conversion range. If the rate-determining step in the reaction sequence to CH4 would then occur after the formation of *COads, e.g., *COads → *Cads + *Oads or *COads + *Hads → *HCOads, an almost constant *COads signal would result as well.

Brucellosis is a bacterial zoonotic disease that requires major attention for both health and financial facilities in many parts of the world including the Mediterranean and the Middle East. The existing gold standard diagnosis relies on the culturing technique, which is costly and time-consuming with a duration of up to 45 days. The Brucella protease biosensor represents a new detection approach that will lead to low-cost point-of-care devices for sensitive Brucella detection. In addition, the described diagnostic device is portable and simple to operate by a nurse or non-skilled clinician making it appropriate for the low-resource setting. In this study, we rely on the total extracellular protease proteolytic activity on specific peptide sequences identified using the FRET assay by high-throughput screening from the library of peptide (96 short peptides such as dipeptides and tripeptides) substrates for Brucella melitensis (B. melitensis). The B. melitensis-specific protease substrate was utilized in the development of the paper-based colorimetric assay. Two specific and highly active dipeptide substrates were identified (FITC-Ahx-K-r-K-Ahx-DABCYL and FITC-Ahx-R-r-K-Ahx-DABCYL). The peptide-magnetic bead nanoprobe sensors developed based on these substrates were able to detect the Brucella with LOD as low as 1.5 × 102 and 1.5 × 103 CFU/mL using K-r dipeptide and R-r dipeptide substrates, respectively, as the recognition element. The samples were tested using this sensor in few minutes. Cross-reactivity studies confirmed that the other proteases extracted from closely related pathogens have no significant effect on the sensor. To the best of our knowledge, this assay is the first assay that can be used with low-cost, rapid, direct, and visual detection of B. melitensis.

To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda. A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected. These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock.

Abstract Our study investigated the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a three-year-longKlebsiella pneumoniaeoutbreak with Illumina short read sequencing data as the point of reference.We detected considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besidesK. pneumoniae. Based on this data, we explored PCR-based sequencing and a masking strategy, which both successfully addressed these inaccuracies and ensured accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA is freely available on GitHub under the GNUv3 license:github.com/replikation/MPOA) to identify and mask problematic genome positions in a reference-free manner.Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investing outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either PCR-based sequencing or using our provided bioinformatic workflow. We would advise that read mapping-based quality control of genomes should be provided when publishing results.

Bovine mastitis is a common disease of dairy cattle causing major economic losses due to reduced yield and poor quality of milk worldwide. The current investigation aimed to gain insight into the genetic diversity, antimicrobial resistance profiles and virulence associated factors of Staphylococcus (S.) aureus isolated from clinical bovine mastitis in dairy farms in Thuringia, Germany. Forty Staphylococcus aureus isolates collected from clinical bovine mastitis cases from 17 Thuringian dairy farms were phenotyped and genetically characterized using whole genome sequencing. Out of 40 S. aureus, 30 (75%) were confirmed as methicillin resistant isolates. The isolates showed elevated antimicrobial resistance against penicillin, tetracycline and oxacillin, i.e., 77.5, 77.5, and 75%, respectively. Lower resistance rates were found against moxifloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, i.e., 35, 35, 30, and 22.5%, respectively. While resistance against clindamycin and erythromycin was rarely found (5 and 2.5%, respectively). All isolates were susceptible to linezolid, teicoplanin, vancomycin, tigecycline, fosfomycin, fusidic acid and rifampicin. These isolates were further allocated into five different sequence types: ST398 (n = 31), ST1074 (n = 4), ST504 (n = 3), ST582 (CC15) (n = 1) and ST479 (n = 1). These isolates were also assigned to seven clusters with up to 100 SNP which has facilitated geographical mapping and epidemiological distribution in Thuringia. Strains belonging to ST398 were classified into clusters 1, 2, 3, 4 and 7. The isolates of ST504 were of cluster 5, those of ST1074 were belonging to cluster 6. Resistance genes blaZ, blaI and blaR associated with penicillin resistance were found in 32 (80%) strains, all except one were belonging to ST398. Methicillin resistance associated mecA was identified in 30 (96.8%) isolates of ST398. All tetracycline and erythromycin resistant isolates were of ST398, and all harbored both tetM and ermA. About 90.3% of tetracycline resistant isolates assigned to ST398 were also carrying tetK gene. The point mutations parC_S80F, gyrA_S84L and parC_S80Y in gyrA and parC associated with quinolone resistance were found in all phenotypically resistant isolates to ciprofloxacin and moxifloxacin (n = 14). Sixty-eight virulence genes were identified among isolates. Both lukD/E and lukM/F-PV-P83 were identified in 22.5% of isolates, all were non-ST398. In this study, ST398 had the highest potential to cause disease and had a massive prevalence in bovine mastitis cases. Five different sequence types and seven clusters were identified in the federal state of Thuringia. The circulation of some clusters in the same region over several years shows the persistence of cluster-associated infection despite the intensive veterinary care. On the other hand, some regions had different clusters at the same year or in different consecutive years. Different sequence types and associated different clusters of S. aureus were geographically widely distributed among dairy farms in Thuringia. The findings of this study show that various clusters have the potential to spread over a large geographical scale. The detection of LA-MRSA on dairy farms, which is known for cabapility to widely spread among different groups of animals, humans and their environment urges for the implementation of national wide strategic programs. The identification of CA-MRSA among the isolates such as ST398 poses a significant risk for the transmission of such strains between animals and humans on dairy farms.

The spatial profile measurement technique was applied for the first time to a liquid phase reaction to study the selective oxidation of propylene to propylene oxide with hydrogen peroxide on titanium silicalite-1 catalyst, also known as the HPPO process. A spatial profiling reactor based on minimal invasive capillary sampling measurements was used to obtain the experimental data. The reactor is able to measure spatially resolved concentration and temperature profiles at industrially relevant conditions, that is, 40 °C and 20–35 bar pressure, with propylene in the liquid phase. The reaction was carried out at different contact times of LHSV 3.0, 6.0, and 12.0 h–1, respectively, achieving high conversion and selectivity. The experimental data show that the concentration profiles of the main epoxidation reaction follows a pseudo-zeroth-order behavior. In contrast, the side product concentration profiles exhibit a nonlinear trend. The primary side products of the reaction 1-methoxy-2-propanol, 2-methoxy-1-propanol, and propylene glycol were quantified. Furthermore, hydroxacetone and methoxyacetone from the consecutive reaction of 1-methoxy-2-propanol, 2-methoxy-1-propanol, and propylene glycol with hydrogen peroxide were detected as secondary side products. Formic acid, methylformate, dipropylene glycol methyl ether isomers, and dipropylene glycol isomers were detected in trace amounts. Quantitative data for the formation of hydroxyacetone is provided for the first time. The experimental concentration profiles were simulated using an axial 1D-pseudo-homogeneous dispersion model with two kinetic models reported in the literature. Additionally, a kinetic model based on the Eley–Rideal mechanism is proposed. Upon linearization, the derived model shows a correlation of R2 = 0.99 and R2 = 0.98 with the linearized form of the experimental differential rate. Statistical analysis of the models in this work shows that the derived Eley–Rideal mechanism has the highest correlation with the concentration gradients measured within the catalyst bed.