Porphyromonas gingivalis W83 Membrane Components Induce Distinct Profiles of Metabolic Genes in Oral Squamous Carcinoma Cells.

Periodontitis, a chronic inflammatory disease is caused by a bacterial biofilm, affecting all periodontal tissues and structures. This chronic disease seems to be associated with cancer since, in general, inflammation intensifies the risk for carcinoma development and progression. Interactions between periodontal pathogens and the host immune response induce the onset of periodontitis and are responsible for its progression, among them Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic rod, capable of expressing a variety of virulence factors that is considered a keystone pathogen in periodontal biofilms. The aim of this study was to investigate the genome-wide impact of P. gingivalis W83 membranes on RNA expression of oral squamous carcinoma cells by transcriptome analysis. Human squamous cell carcinoma cells (SCC-25) were infected for 4 and 24 h with extracts from P. gingivalis W83 membrane, harvested, and RNA was extracted. RNA sequencing was performed, and differential gene expression and enrichment were analyzed using GO, KEGG, and REACTOME. The results of transcriptome analysis were validated using quantitative real-time PCR with selected genes. Differential gene expression analysis resulted in the upregulation of 15 genes and downregulation of 1 gene after 4 h. After 24 h, 61 genes were upregulated and 278 downregulated. GO, KEGG, and REACTONE enrichment analysis revealed a strong metabolic transcriptomic response signature, demonstrating altered gene expressions after 4 h and 24 h that mainly belong to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response, signaling, and metabolism revealed upregulated expression of CCL20, CXCL8, NFkBIA, TNFAIP3, TRAF5, CYP1A1, and NOD2. This work sheds light on the RNA transcriptome of human oral squamous carcinoma cells following stimulation with P. gingivalis membranes and identifies a strong metabolic gene expression response to this periodontal pathogen. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate the basic mechanisms of periodontitis and the development of cancer.

Digestive enzyme expression in the large intestine of children with short bowel syndrome in a late stage of adaptation.

BACKGROUND AND AIMS: Intestinal adaptation in short bowel syndrome (SBS) includes morphologic processes and functional mechanisms. This study investigated whether digestive enzyme expression in the duodenum and colon is upregulated in SBS patients. METHOD: Sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH), and neutral Aminopeptidase N (ApN) were analyzed in duodenal and colonic biopsies from nine SBS patients in a late stage of adaptation as well as healthy and disease controls by immunoelectron microscopy (IEM), Western blots, and enzyme activities. Furthermore, proliferation rates and intestinal microbiota were analyzed in the mucosal specimen. RESULTS: We found significantly increased amounts of SI, LPH, and ApN in colonocytes in most SBS patients with large variation and strongest effect for SI and ApN. Digestive enzyme expression was only partially elevated in duodenal enterocytes due to a low proliferation level measured by Ki-67 staining. Microbiome analysis revealed high amounts of Lactobacillus resp. low amounts of Proteobacteria in SBS patients with preservation of colon and ileocecal valve. Colonic expression was associated with a better clinical course in single cases. CONCLUSION: In SBS patients disaccharidases and peptidases can be upregulated in the colon. Stimulation of this colonic intestinalization process by drugs, nutrients, and pre- or probiotics might offer better therapeutic approaches.

High-resolution taxonomic examination of the oral microbiome after oil pulling with standardized sunflower seed oil and healthy participants: a pilot study.

OBJECTIVES: We aimed at the high-resolution examination of the oral microbiome depending on oil pulling, compared it with saline pulling, and analyzed whether the method is capable of reducing the overall microbial burden of the oral cavity. MATERIALS AND METHODS: The study was a cohort study with three healthy subjects. Oil pulling samples, saline pulling samples, and saliva samples were microscoped and cultured under microaerophilic and anaerobic conditions; colony-forming units were counted; and cultivated bacteria were identified employing MALDI-TOF MS. The oral microbiomes (saliva) and the microbiota incorporated in oil and saline pulling samples were determined in toto by using 16S rDNA next-generation sequencing (NGS) and bioinformatics. RESULTS: Microscopy revealed that oral epithelial cells are ensheathed with distinct oil droplets during oil pulling. Oil pulling induced a higher production of saliva and the oil/saliva emulsion contained more bacteria than saline pulling samples. Oil pulling resulted in a significant and transient reduction of the overall microbial burden in comparison to saliva examined prior to and after pulling. Both oil and saline pulling samples mirrored the individual oral microbiomes in saliva. CONCLUSIONS: Within the limitations of this pilot study, it might be concluded that oil pulling is able to reduce the overall microbial burden of the oral cavity transiently and the microbiota in oil pulling samples are representative to the oral microbiome. CLINICAL RELEVANCE: Within the limitations of this pilot study, it might be concluded that oil pulling can be considered as an enlargement of standard oral hygiene techniques since it has the characteristic of an oral massage, enwrapping epithelial cells carrying bacteria in oil vesicles and reaching almost all unique habitats in oral cavity.

Depletion of Lipocalin 2 (LCN2) in Mice Leads to Dysbiosis and Persistent Colonization with Segmented Filamentous Bacteria.

Lipocalin 2 (LCN2) mediates key roles in innate immune responses. It has affinity for many lipophilic ligands and binds various siderophores, thereby limiting bacterial growth by iron sequestration. Furthermore, LCN2 protects against obesity and metabolic syndrome by interfering with the composition of gut microbiota. Consequently, complete or hepatocyte-specific ablation of the Lcn2 gene is associated with higher susceptibility to bacterial infections. In the present study, we comparatively profiled microbiota in fecal samples of wild type and Lcn2 null mice and show, in contrast to previous reports, that the quantity of DNA in feces of Lcn2 null mice is significantly lower than that in wild type mice (p < 0.001). By using the hypervariable V4 region of the 16S rDNA gene and Next-Generation Sequencing methods, we found a statistically significant change in 16 taxonomic units in Lcn2-/- mice, including eight gender-specific deviations. In particular, members of Clostridium, Escherichia, Helicobacter, Lactococcus, Prevotellaceae_UCG-001 and Staphylococcus appeared to expand in the intestinal tract of knockout mice. Interestingly, the proportion of Escherichia (200-fold) and Staphylococcus (10-fold) as well as the abundance of intestinal bacteria encoding the LCN2-sensitive siderphore enterobactin (entA) was significantly increased in male Lcn2 null mice (743-fold, p < 0.001). This was accompanied by significant higher immune cell infiltration in the ileum as demonstrated by increased immunoreactivity against the pan-leukocyte protein CD45, the lymphocyte transcription factor MUM-1/IRF4, and the macrophage antigen CD68/Macrosialin. In addition, we found a higher expression of mucosal mast cell proteases indicating a higher number of those innate immune cells. Finally, the ileum of Lcn2 null mice displayed a high abundance of segmented filamentous bacteria, which are intimately associated with the mucosal cell layer, provoking epithelial antimicrobial responses and affecting T-helper cell polarization.

High prevalence of urogenital infection/inflammation in patients with azoospermia does not impede surgical sperm retrieval.

Considering infection/inflammation to be an important risk factor in male infertility, the aim of this study was to make a comprehensive evaluation of the prevalence of urogenital tract infection/inflammation and its potential impact on sperm retrieval in azoospermic patients. In this prospective study, 71 patients with azoospermia were subjected to an extensive andrological workup including comprehensive microbiological diagnostics (2-glass test, semen, testicular swab and testicular tissue analysis) and testicular biopsy/testicular sperm extraction (TESE). Medical history suggested urogenital tract infection/inflammation in 7% of patients, 11% harboured STIs, 14% showed significant bacteriospermia, 15% had seminal inflammation, 17% fulfilled the MAGI definition, and 27% had relevant pathogens. At the testicular level, 1 patient had a swab positive for bacteria, no viruses were detected, tissue specimens never indicated pathogens, whereas histopathology revealed focal immune cell infiltrates in 23% of samples. Testicular sperm retrieval rate was 100% in obstructive and 46% in nonobstructive azoospermia. None of the infection/inflammation-related variables was associated with the success of sperm retrieval or inflammatory lesions in the testis. The high prevalence of urogenital infection/inflammation among azoospermic men underpins their role as significant aetiologic factors in male infertility. However, this observation does not refer to the chances of sperm retrieval at the time of surgery/TESE.

Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections.

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of ß-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.

Glass ionomer cement inhibits secondary caries in an in vitro biofilm model.

OBJECTIVES: The objective of this study was to investigate the effect of different glass ionomer cements on secondary caries inhibition in a fully automated in vitro biofilm model. MATERIALS AND METHODS: One hundred and twenty-four extracted third molars received class V cavities and were filled with one conventional (Ketac Molar/KM), and two resin-modified glass ionomer cements (Photac Fil/PF, Ketac N100/KN, 3M Espe). A bonded resin composite (Single Bond Plus/Filtek Supreme XTE) served as control. After 14 days water storage at 37 °C, specimens were thermocycled (10,000 × 5/55 °C). Over a period of 10 days, specimens were subjected to cariogenic challenge for 3/4/6 h/day. Demineralization was caused by Streptococcus mutans (DSM 20523) alternatingly being rinsed over specimens using artificial saliva. After biological loading, teeth were cut longitudinally and demineralization depths were evaluated at the margins and at a distance of 0.5 mm from the margins using fluorescence microscopy. Marginal quality was investigated under a SEM at ×200 magnification. RESULTS: Four-hour demineralization depths were for enamel margins (EM), enamel (E), dentin margin (DM), and dentin (D) (µm ± SD): KM: EM 12 ± 8, E 33 ± 7, DM 56 ± 11, D 79 ± 6; PF: EM 19 ± 13, E 34 ± 13, DM 53 ± 10, D 77 ± 12; and KN: EM 26 ± 5, E 38 ± 6, DM 57 ± 11, D 71 ± 7. For all glass ionomer cements (GICs), demineralization depth at the margins was less compared to 0.5 mm distance, with demineralization depth having been correlated to duration of cariogenic challenge (ANOVA [mod. LSD, p < 0.05]). Compared to the bonded resin composite, all GICs exhibited caries inhibition at restoration margins in enamel and dentin. CONCLUSIONS: Fluoride-releasing GIC materials exhibit a secondary caries inhibiting effect in vitro. CLINICAL RELEVANCE: Glass ionomer cements have a higher secondary caries inhibiting effect than resin composites.

Porphyromonas gingivalis activates NFκB and MAPK pathways in human oral epithelial cells.

BACKGROUND: The bacterial biofilm at the gingival margin induces a host immune reaction. In this local inflammation epithelial cells defend the host against bacterial challenge. Porphyromonas gingivalis (P. gingivalis), a keystone pathogen, infects epithelial cells. The aim of this study was to investigate the activation of signaling cascades in primary epithelial cells and oral cancer cell lines by a profiler PCR array. RESULTS: After infection with P. gingivalis membranes the RNA of 16 to 33 of 84 key genes involved in the antibacterial immune response was up-regulated, amongst them were IKBKB (NF-κB signaling pathway), IRF5 (TLR signaling) and JUN, MAP2K4, MAPK14 and MAPK8 (MAPK pathway) in SCC-25 cells and IKBKB, IRF5, JUN, MAP2K4, MAPK14 and MAPK8 in PHGK. Statistically significant up-regulation of IKBKB (4.7 ×), MAP2K4 (4.6 ×), MAPK14 (4.2 ×) and IRF5 (9.8 ×) (p < 0.01) was demonstrated in SCC-25 cells and IKBKB (3.1 ×), MAP2K4 (4.0 ×) MAPK 14 (3.0 ×) (p < 0.05), IRF5 (3.0 ×) and JUN (7.7 ×) (p < 0.01) were up-regulated in PHGK. CONCLUSIONS: P. gingivalis membrane up-regulates the expression of genes involved in downstream TLR, NFκB and MAPK signaling pathways involved in the pro-inflammatory immune response in primary and malignant oral epithelial cells.

TLR9 mediates S. aureus killing inside osteoblasts via induction of oxidative stress.

BACKGROUND: Staphylococcus aureus is the principle causative pathogen of osteomyelitis and implant-associated bone infections. It is able to invade and to proliferate inside osteoblasts thus avoiding antibiotic therapy and the host immune system. Therefore, development of alternative approaches to stimulate host innate immune responses could be beneficial in prophylaxis against S. aureus infection. TLR9 is the intracellular receptor which recognizes unmethylated bacterial CpG-DNA and activates immune cells. Synthetic CpG-motifs containing oligodeoxynucleotide (CpG-ODNs) mimics the stimulatory effect of bacterial DNA. RESULTS: Osteoblast-like SAOS-2 cells were pretreated with CpG-ODN type-A 2216, type-B 2006, or negative CpG-ODN 2243 (negative control) 4 h before infection with S. aureus isolate EDCC 5055 (=DSM 28763). Intracellular bacteria were streaked on BHI plates 4 h and 20 h after infection. ODN2216 as well as ODN2006 but not ODN2243 were able to significantly inhibit the intracellular bacterial growth because about 31 % as well as 43 % of intracellular S. aureus could survive the pretreatment of SAOS-2 cells with ODN2216 or ODN2006 respectively 4 h and 20 h post-infection. RT-PCR analysis of cDNAs from SAOS-2 cells showed that pretreatment with ODN2216 or ODN2006 stimulated the expression of TLR9. Pretreatment of SAOS-2 cells with ODN2216 or ODN2006 but not ODN2243 managed to induce reactive oxygen species (ROS) production inside osteoblasts as measured by flow cytometry analysis. Moreover, treating SAOS-2 cells with the antioxidant Diphenyleneiodonium (DPI) obviously reduced S. aureus killing ability of TLR9 agonists mediated by oxidative stress. CONCLUSIONS: In this work we demonstrated for the first time that CPG-ODNs have inhibitory effects on S. aureus survival inside SAOS-2 osteoblast-like cell line. This effect was attributed to stimulation of TLR9 and subsequent induction of oxidative stress. Pretreatment of infected SAOS-2 cells with ROS inhibitors resulted in the abolishment of the CPG-ODNs killing effects.

Molecular epidemiology and characterization of an outbreak causing Klebsiella pneumoniae clone carrying chromosomally located bla(CTX-M-15) at a German University-Hospital.

BACKGROUND: Multi-drug resistant Klebsiella pneumoniae strains are a common cause of health care associated infections worldwide. Clonal spread of Klebsiella pneumoniae isolates carrying plasmid mediated CTX-M-15 have been commonly reported. Limited data is available regarding dissemination of chromosomally encoded CTX-M-15 in Klebsiella pneumoniae worldwide. RESULTS: We examined 23 non-repetitive ESBL-producing Klebsiella pneumoniae strains isolated from clinical specimens over a period of 4 months in a German University Hospital. All isolates were characterized to determine their genetic relatedness using Pulsed-Field Gel Electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). PFGE revealed three clusters (B1, B2, and B3) with a sub-cluster (A3) comprising of 10 isolates with an identical PFGE pattern. All strains of the cluster B3 with similar PFGE patterns were typed as ST101, indicating an outbreak situation. The ESBL allele bla CTX-M-15 was identified in 16 (69.6 %) of all isolates, including all of the outbreak strains. Within the A3 sub-cluster, the CTX-M-15 allele could not be transferred by conjugation. DNA hybridization studies suggested a chromosomal location of bla CTX-M-15. Whole genome sequencing located CTX-M-15 within a complete ISEcp-1 transposition unit inserted into an ORF encoding for a putative membrane protein. PCR-based analysis of the flanking regions demonstrated that insertion into this region is unique and present in all outbreak isolates. CONCLUSION: This is the first characterization of a chromosomal insertion of bla CTX-M-15 in Klebsiella pneumonia ST101, a finding suggesting that in Enterobacteriaceae, chromosomal locations may also act as reservoirs for the spread of bla CTX-M-15 encoding transposition units.

Effect of microparticulate silver addition in dental adhesives on secondary caries in vitro.

OBJECTIVES: The aim of the present study was evaluate the effect of microparticulate silver additions in adhesives on secondary caries formation using an artificial mouth model. MATERIALS AND METHODS: One hundred eight intact human third molars received standardized Class-V resin composite restorations (Filtek Supreme XTE bonded with Syntac, Scotchbond 1 XT, Futurabond M). Adhesives were charged with different amounts of microparticulate silver (0 %/0.1 %/0.5 %). After storage for 4 weeks at 37 °C, teeth were subjected to 10,000 thermocycles (+5 °C and +55 °C), and impressions were taken. Streptococcus mutans 10449 was used in a nutrition medium for secondary caries simulation in a fully automated artificial mouth. After completion of thermocycling and biological load cycling, impressions were taken and replicas were investigated under a light microscope for gap widths at enamel and dentin margins. Evaluation of fluorescence was carried out using a special FITC filter. The demineralization depths at the cavity margin were evaluated using Xpert for Windows working at a pixel distance of 5 µm. RESULTS: After thermocycling, no difference in gap widths and demineralization depths was found (p > 0.05). After incubation, gap widths and demineralization depths were significantly reduced with higher amounts of silver loading in most of the adhesives (p < 0.05). The 0.5 % silver addition resulted in a slight decrease of secondary caries at resin-dentin margins (p < 0.05). CONCLUSIONS: Addition of microparticulate silver in commercially available dental adhesives has the potential of reducing secondary caries. CLINICAL RELEVANCE: The chosen setup was able to produce secondary caries with a distinct in vivo appearance. Microparticulate silver additions in dental adhesives may have an impact on inhibition of secondary caries.

Detection of bloodstream infections and prediction of bronchopulmonary dysplasia in preterm neonates with an electronic nose.

We show that smellprints of volatile organic components measured with an electronic nose (Cyranose 320; Smiths Detection Group Ltd, Watford, United Kingdom) differ between tracheal aspirates from preterm neonates with or without laboratory-confirmed bloodstream infections and with or without subsequent development of bronchopulmonary dysplasia. Tracheal aspirate smellprints could be useful noninvasive diagnostic markers for preterm neonates.

Multiresistant extended-spectrum ß-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany.

BACKGROUND: Multiresistant Gram-negative bacteria producing extended-spectrum ß-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of ß-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses. RESULTS: In this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac('6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates. CONCLUSIONS: Isolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.

Heat-related mortality in Frankfurt am Main, Germany, from 2000 to 2023.

Background: The major heatwave in Europe in August 2003 resulted in 70,000 excess deaths. In Frankfurt am Main, a city with 767,000 inhabitants in the south-west of Germany, around 200 more people died in August 2003 than expected. Soon afterwards, the city introduced adaptation measures to prevent heat-related health problems and subsequently established further mitigation measures to limit climate change. Frankfurt is rated as being one of the cities in Germany to have implemented the best climate adaptation and mitigation measures. This study addressed the following questions: is there already a downward trend in mortality from heat and can this be attributed to the measures taken? Materials and methods: The age-standardized mortality rate (ASR) was calculated for the months of June to August and for calendar weeks 23 to 34 of the individual years on the basis of population data and deaths of the inhabitants of Frankfurt am Main for the years 2000 to 2023. This was related to the meteorological data from the Frankfurt measuring station of the German National Meteorological Service. For four different heat exposure indicators (heat days, days in heat weeks, days in heatwaves and days with heat warnings), the incidence rate (death cases per 1 million person days) (IR) was calculated for days with and without exposure, and the incidence rate difference and the incidence rate ratio (IRR) were estimated to compare days with vs days without exposure. Results: Over the years, the mean daily temperatures tended to increase, and the standardized mortality rate decreased. An increase in ASR was observed during heatwaves up to 2015, but no longer in the later ones. In the summer of 2003, the incidence rate was 16.0 (95% confidence interval (CI) 12.2-19.9) per 1 million person days greater on heat days than on days not classified as heat days, and the corresponding incidence rate ratio was 1.64 (95% CI 1.48-1.82). Although the weather data for the summers of 2018 and 2022 were comparable with the record-breaking heat summer of 2003, the incidence rate differences (2018: 3.8, 95% CI 0.9-6.7; 2022: 2.3, 95% CI -0.3-4.9) and the IRR (2018: 1.20, 95% CI 1.05-1.37; 2022: 1.12, 95% CI 0.99-1.26) were considerably lower. Similar results were also obtained when comparing mortality in heat weeks and heatwaves as well as on days with heat warnings. Discussion: In summary, our study in Frankfurt am Main not only showed a decrease in heat-related mortality in the population as a whole over the years, but also a decrease in excess mortality during various heat periods (day, week, wave, warning), especially in comparison with the years with very high heat stress and drought (2003, 2018 and 2022). However, whether this development represents success of the intensive prevention measures that have been implemented in the city for years or merely describes a general trend cannot be answered with certainty by the present study. To answer this question, a comparative study should be carried out in various municipalities in the Rhine-Main region with different levels of intensity in dealing with the heat problem.

Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania.

BACKGROUND: Klebsiella pneumoniae strains expressing ESBLs are a predominant cause of hospital acquired infections. Here we describe the molecular epidemiology of these isolates in a tertiary hospital in Tanzania, as potential pathogens for neonatal infections. METHODS: Between April 2009 and March 2010 all Klebsiella pneumoniae isolates with phenotypic expression Extended Spectrum Beta Lactamase (ESBL) were collected and characterized. Identification was done using in house biochemical tests in case of ambiguous results confirmation was done using API 20E. Susceptibility testing was determined using the disc diffusion method followed by specific PCR and sequencing to determine ESBL genes. Phylogenetic analysis, Pulse field gel electrophoresis (PFGE) and Multi-Locus sequence typing (MLST) to PFGE clusters representative isolates were performed to determine clones of the isolates. Conjugation and hybridization were performed to determine the location of blaCTX-M-15 gene. RESULTS: A total of 92 non-repetitive ESBL producing K. pneumoniae representing 50.3% of Klebsiella pneumoniae isolates were characterized. These isolates were from blood 61 (66%), wound swab 13 (14%), urine 12 (13%) and pus 6 (7%) were analyzed. Most blood culture strains originated from neonatal unit 39/61(64%) and 22 (36%) of the blood culture isolates were from neonatal ICU. All isolates were resistant to gentamicin and 54% were resistant to ciprofloxacin. Using a similarity index of 80%, the isolates were assigned to thirteen clusters based on PFGE patterns and contained sub-clusters with identical strains indicating clonal outbreaks. Cluster X5, X7 and X8, and X9 were grouped into ST48, ST14 and ST348 respectively. Based on gyrA PCR- RFLP phylogenetic analysis all isolates were grouped as KpI. The predominant ESBL allele detected was blaCTX-M-15 which was found in 76% of isolates, followed by blaTEM-104 (19%), blaSHV-11 (3.2%) and blaTEM-176 (2%). The blaCTX-M-15 gene was located in multiple conjugative IncF plasmids ranging from 25 kb-485 kb in size. CONCLUSION: The high prevalence of blaCTX-M-15 observed among ESBL producing K. pneumoniae in Tanzania, is possibly due to the spread of a common IncFII 145 kb plasmid and of certain clones such as ST14 and ST48. Furthermore the 485 kb plasmid detected is the largest plasmid reported to carry blaCTX-M-15 todate.

Rescue service deployment data as an indicator of heat morbidity in Frankfurt / Main, Germany (2014-2022) - Trend association with various heat exposure indicators and considerations for outreach.

Many publications dealt with the monitoring of heat-related mortality. Fewer analyses referred to indicators of heat-related morbidity. The aim of this work was to describe the heat-related morbidity using rescue service data from the city of Frankfurt/Main, Germany for the time period 2014-2022, with regard to the questions: 1) How do rescue service deployments develop over the years? Is there a trend identifiable towards a decrease in deployments over the years, e.g. as an effect of either (physiological) adaptation of the population or of the measures for prevention of heat-related morbidity? 2) Which heat parameters (days with a heat warning, heat days, heat weeks, heat waves) are most strongly associated with heat-related morbidity in terms of rescue service deployments and might therefore be additionally used as an easily communicable and understandable heat-warning indicator? Rescue service data were provided by the interdisciplinary medical supply compass system "IVENA" and adjusted for population development including age development. The effect of various indicators for heat exposure, such as days with a heat warning from the German meteorological service based on the scientific concept of "perceived heat", heat days, heat wave days and heat week days on different endpoints for heat morbidity (deployments in total as well as for heat associated diagnoses) was calculated using both difference-based (difference ± 95% CI) and ratio-based (ratio ± 95% CI) effect estimators. Rescue services deployments in summer months increased overall from 2014 to 2022 in all age groups over the years (2698 to 3517/100.000 population). However, there was a significant decrease in 2020, which could be explained by the special situation of the COVID-19 pandemic, probably caused by the absence of tourists and commuters from the city. In addition, no data are available on the actual implementation of the measures by the population. Therefore, an effect of the measures taken to prevent heat-associated morbidity in Frankfurt am Main could not be directly demonstrated, and our first question cannot be answered on the basis of these data. Almost all heat definitions used for exposure (day with a heat warning, heat day, heat wave day, heat week day) showed significant effects on heat-associated diagnoses in every year. When analysing the effect on all deployments, the effect was in part strongly dependent on individual years: Heat wave days and heat week days even showed negative effects in some years. The definition heat day led to a significant increase in rescue service deployments in all single years between 2014 and 2022 (ratio 2014-2022 1.09 (95CI 1.07-1.11); with a range of 1.05 (95CI 1.01-1.09) in 2020 and 1.14 (95CI 1.08-1.21) in 2014), this was not the case for days with a heat warning (ratio 2014-2022 1.04 (95CI1.02-1.05); with a range of 1.01 (95CI 0.97-1.05) in 2017 and 1.16 (95CI 1.10-1.23). Thus being not inferior to the heat warning day, the "heat day" defined as ≥32 °C maximum temperature, easily obtainable from the weather forecast, can be recommended for the activities of the public health authorities (warning, surveillance etc.) regarding heat health action planning.

Development of morbidity and mortality of SARS-CoV-2 in nursing homes for the elderly in Frankfurt am Main, Germany, 2020-2022: What protective measures are still required?

Introduction: Nursing-home residents are among the highest risk group in the SARS-CoV-2 pandemic. At the onset of the SARS-CoV-2 pandemic, the majority of all deaths from or with SARS-CoV-2 occurred in long-term care facilities (LTCFs), so that maximum protective measures were mandated for these facilities. This study analyzed the impact of the new virus variants and the vaccination campaign on disease severity and mortality among nursing home residents and staff through 2022 as a basis for determining which protective measures remain necessary and appropriate. Methods: In five homes in Frankfurt am Main, Germany, with a total capacity for 705 residents, all cases occurring in the facility among residents and staff were recorded and documented (date of birth and diagnosis, hospitalization and death, vaccination status) and were descriptively analyzed with SPSS. Results: By 31st August 2022, 496 residents tested positive for SARS-CoV-2, 93 in 2020, 136 in 2021, and 267 in 2022; 14 residents presented with a second SARS-CoV-2 infection in 2022, having previously experienced an infection in 2020 or 2021. The percentage of hospitalizations decreased from 24.7% (2020) and 17.6% (2021) to 7.5% (2022), and the percentage of deaths decreased from 20.4% and 19.1% to 1.5%. In 2021, 61.8% of those infected were vaccinated (at least 2x); in 2022, 86.2% of residents had been vaccinated twice, 84% of whom had already had a booster vaccination. Hospitalization and death rates were significantly higher among the unvaccinated than the vaccinated throughout all years (unvaccinated 21.5% and 18.0%; vaccinated 9.8% and 5.5%; KW test p=0.000). However, this difference was no longer significant under the prevalence of the Omicron variant in 2022 (unvaccinated 8.3% and 0%; p=0.561; vaccinated 7.4% and 1.7%; p=0.604). From 2020 to 2022, 400 employees were documented as infected, with 25 having second infections in 2022. Only one employee showed a second infection in 2021 following the first in 2020. Three employees were hospitalized; no deaths occurred. Discussion and conclusion: Severe COVID-19 courses occurred with the Wuhan Wild type in 2020, with a high death rate among nursing-home residents. In contrast, during the waves in 2022 with the relatively mildly pathogenic Omicron variant, many infections but few severe courses and deaths were observed among the now mostly vaccinated and boostered nursing-home residents. Given the high immunity of the population and the low pathogenicity of the circulating virus - even in nursing-home residents - protective measures in nursing homes that restrict people's right to self-determination and quality of life no longer seem justified. Instead, the general hygiene rules and the recommendations of the KRINKO (German Commission for Hospital Hygiene and Infection Prevention) on infection prevention should be followed, and the recommendations of the STIKO (German Standing Commission on Vaccination) on vaccination not only against SARS-CoV-2 but also against influenza and pneumococci should be observed.

Daptomycin-Impregnated PMMA Cement against Vancomycin-Resistant Germs: Dosage, Handling, Elution, Mechanical Stability, and Effectiveness.

BACKGROUND: The number of periprosthetic joint infections caused by vancomycin-resistant pathogens is increasing. Currently, no PMMA cement is commercially available to cover VRE. Daptomycin shows promising results in treating infection, offering a good safety profile and a reduced risk of developing resistance. The purpose of this in vitro study was to investigate the mechanical stability, handling properties, elution behavior, and antimicrobial effectiveness of PMMA cement loaded with three different daptomycin concentrations in comparison to commercially available antibiotic-loaded bone cement (ALBC). METHODS: Mechanical properties and handling characteristics (ISO 5833, DIN 53435), HPLC elution, antimicrobial effectiveness with proliferation assay (DIN 17025), and inhibition zone testing were investigated. RESULTS: All tested daptomycin concentrations met the ISO and DIN standards for mechanical strength. Loading of 40 g of PMMA cement with 0.5 g of daptomycin did not show any antimicrobial effectiveness, in contrast to 1.0 g and 1.5 g. PMMA cement with 1.5 g of daptomycin was the best in terms of elution and effectiveness, and it showed good ISO mechanical strength; ISO doughing was sticky for a little longer and setting was faster compared to the vancomycin-containing reference cement. CONCLUSION: PMMA cement containing 0.5 g of gentamicin and 1.5 g of daptomycin could be a good alternative to the already established COPAL® (Wehrheim, Germany) G+V for the treatment of PJIs caused by VRE.

Complete sequences of plasmids from the hemolytic-uremic syndrome-associated Escherichia coli strain HUSEC41.

The complete and annotated sequences of four plasmids from a historical enteroaggregative Shiga toxin-producing Escherichia coli (HUSEC) serotype O104:H4 strain, HUSEC41/01-09591, isolated in 2001 in Germany are reported.

Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. RESULTS: The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. CONCLUSION: Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.