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Since 2012, have we in Denmark observed an increase of invasive pneumococcal infections (IPD) due to Streptococcus pneumoniae serotype 24F. We here present epidemiological data on 24F IPD cases, and characterization of 48 24F clinical isolates based on clonal relationship, antimicrobial resistance (AMR) determinants and virulence factors. IPD surveillance data from (1999–2016) were used to calculate the incidence and age-distribution of serotype 24F IPD and the effect of pneumococcal conjugated vaccines (PCV). Characterization of forty-eight 24F isolates (14.7% of all 24F isolates from the period) was based on whole-genome sequencing analysis (WGS). The IPD cases of serotype 24F showed a significant increase (p < 0.05) for all age groups after the PCV-13 introduction in 2010. The majority of tested 24F isolates consisted of two MLST types, i.e. the ST72 and the ST162. Serotype 24F IPD increased in Denmark after the PCV-13 introduction in parallel with an increase of the ST162 clone. The genotypic penicillin binding protein (PBP) profile agreed with the phenotypical penicillin susceptibility. The virulence genes lytA , ply , piaA , piaB , piaC , rspB and the cpsA / wzg were detected in all 24F isolates, while the pspA and zmpC genes were absent.

Denmark has monitored invasive pneumococcal diseases (IPD) for decades, observing shifts in serotype prevalence, partly due to pneumococcal conjugate vaccines in children. The COVID-19 pandemic and the Danish government’s 2020 vaccination program with the 23-valent pneumococcal polysaccharide vaccine (PPV23) for older adults further influenced IPD epidemiology. This study explores the dynamics of Global Pneumococcal Sequence Clusters (GPSCs) in Denmark from 2019 to 2023 using whole-genome sequencing (WGS) on IPD isolates from all age groups received at Statens Serum Institut (SSI). Serotyping, multilocus sequence typing (MLST), GPSC identification, and phylogenetic analysis to assess clonal relationships were conducted. Of the 1,999 sequenced isolates, representing 93.3% of reported cases, 79 different GPSCs were identified, with GPSC3, GPSC12, and GPSC19 being dominant. GPSC3/ST53 (serotype 8) declined significantly from 24.6% in 2019 to 14.4% in 2023 (p < 0.05), whereas GPSC12/ST180 (serotype 3) increased significantly from 8.2% to 15.1% (p < 0.05). The PPV23 vaccine and pandemic restrictions decreased IPD incidence, particularly for vaccine-covered serotypes, yet serotype 3 remained problematic, indicating challenges in achieving broad serotype coverage. Although pneumococcal vaccination and pandemic-related public health measures influenced the distribution of serotypes and sequence types (STs), only two dominant GPSCs showed clear changes over time. This reflects that a single GPSC can encompass multiple serotype–ST combinations, including both vaccine-covered and non-vaccine variants. As a result, while GPSCs provide a useful high-level overview of pneumococcal lineages, they may lack the resolution needed to detect finer-scale shifts in serotype–ST composition, especially those critical for evaluating vaccine impact and identifying emerging clones.

As a follow-up to a previous study, we investigated vaccine effectiveness (VE) of 23-valent pneumococcal polysaccharide vaccine (PPSV23) against invasive pneumococcal disease (IPD) among 1,254,498 persons >65 years of age as part of a vaccination program in Denmark during April 2020-January 2023. We assessed VE by using a Cox regression model and adjusted for age, sex, and underlying conditions. Using nationwide data, we estimated a VE of PPSV23 against all-type IPD of 32% and against PPSV23-serotype IPD of 41%. Because this follow-up study had more statistical power than the original study, we also estimated VE against IPD caused by PPSV23-serotypes excluding serotype 3; serotype 3; serotype 8; serotype 22F; PPSV23 non-PCV15 serotypes; PPSV23 non-PCV20 serotypes; and IPD over time. Our findings suggest PPSV23 vaccination can protect persons >65 years of age against IPD caused by all serotypes or serotype groupings, except serotype 3.

Worldwide the increase in multi-resistant bacteria due to misuse of traditional antibiotics is a growing threat for our health. Finding alternatives to traditional antibiotics is thus timely. Probiotic bacteria have numerous beneficial effects and could offer safer alternatives to traditional antibiotics. Here, we use the nematode Caenorhabditis elegans ( C. elegans ) to screen a library of different lactobacilli to identify potential probiotic bacteria and characterize their mechanisms of action. We show that pretreatment with the Lactobacillus spp. Lb21 increases lifespan of C. elegans and results in resistance towards pathogenic methicillin-resistant Staphylococcus aureus (MRSA) . Using genetic analysis, we find that Lb21-mediated MRSA resistance is dependent on the DBL-1 ligand of the TGF-β signaling pathway in C. elegans . This response is evolutionarily conserved as we find that Lb21 also induces the TGF-β pathway in porcine epithelial cells. We further characterize the host responses in an unbiased proteome analysis and identify 474 proteins regulated in worms fed Lb21 compared to control food. These include fatty acid CoA synthetase ACS-22, aspartic protease ASP-6 and vitellogenin VIT-2 which are important for Lb21-mediated MRSA resistance. Thus, Lb21 exerts its probiotic effect on C. elegans in a multifactorial manner. In summary, our study establishes a mechanistic basis for the antimicrobial potential of lactobacilli.

Non-vaccine-serotypes (non-VT) pose a challenge to reducing invasive pneumococcal disease (IPD). Since 2023, serotype 38 IPD has increased in Denmark promoting investigation of this serotype’s characteristics. We included all non-VT IPD cases from 2014 to 2024 to calculate annual incidences per 100,000 individuals with 95% confidence intervals (CI). Clinical characteristics and outcomes of serotype 38 IPD were compared with other non-VT IPD in 2022 to 2024. Incidence of serotype 38 IPD increased mainly in children < 2 years, from 0.87 (95% CI 0.02–4.84) to 5.99 (95% CI 2.41–12.34) cases per 100,000 population, whereas the incidence for other non-VT remained stable. SNP analysis, conducted for serotype 38 isolates, revealed that the rise was driven by the ST393 clone with isolates not linked to a region or outbreak. Baseline characteristics and outcomes were similar between the 42 cases of serotype 38 IPD and the 412 other non-VT IPD cases, except for age distribution ( p  < 0.001) with serotype 38 IPD more frequent in children aged < 2 years (21.4% vs. 3.4%). In conclusion, serotype 38 IPD, driven by the ST393 clone, was the dominant serotype causing non-VT IPD in children < 2 years the last two years, however disease severity was similar to other non-VT IPD.

This study assesses a novel bile solubility test and MALDI-TOF for the differentiation of Streptococcus pneumoniae from other mitis group streptococci, including differentiation of S. pneumoniae from Streptococcus pseudopneumoniae . Eighty-four species verified mitis group isolates were subjected to our bile solubility test (which measures and calculates the differences of absorbance in the test tube containing 10% sodium deoxycholate versus a blank control tube, after incubation for 10 minutes at 36 °C using a spectrophotometer) and MALDI-TOF MS (both the standard result output and by visual spectra evaluation). Applying a calculated optimal cut-off absorbance-value of 2.1, differentiated S. pneumoniae from all but one other mitis group streptococci (one S. mitis isolate generated an OD-value above 2.1). MALDI-TOF score value identification identified correctly 46 S. pneumoniae and 4 S. pseudopneumoniae but misidentified 16 other mitis group strains. Visual spectra evaluation correctly identified all S. pneumoniae and S. pseudopneumoniae strains but misidentified 13 other mitis group strains. The bile solubility test based on spectrophotometric reading described in this study can differentiate S. pneumoniae from other Streptococcus species. Combining the bile solubility test and the MALDI-TOF spectra results provide a correct identification of all S. pneumoniae and S. pseudopneumoniae isolates.

Background Streptococcus pneumoniae serotype 8 incidence has increased in Denmark after the introduction of pneumococcal conjugated vaccines (PCV). The mechanism behind the serotype 8 replacement is not well understood. In this study, we aimed to present epidemiological data on invasive pneumococcal disease (IPD) and molecular characterization of 96 serotype 8 clinical isolates. Methods IPD data from 1999 to 2019 were used to calculate the incidence and age distribution. Whole-genome sequencing (WGS) analysis was performed on 96 isolates (6.8% of the total serotype 8 IPD isolates in the period) to characterize the isolates with respect to pneumococcal lineage traits, a range of genes with potential species discrimination, presence of colonization and virulence factors, and molecular resistance pattern. Results The serotype 8 IPD incidence increased significantly ( P  < 0.05) for the age groups above 15 years after the introduction of PCV13, primarily affecting the elderly (65+). All isolates were phenotypically susceptible to penicillin, erythromycin and clindamycin. Molecular characterization revealed seven different MLST profiles with ST53 as the most prevalent lineage (87.5%) among the analyzed serotype 8 isolates. The genes covering the cell-surface proteins: lytA , rspB , pspA , psaA & Xisco and the pneumococcal toxin pneumolysin =  ply were present in all isolates, while genes for the membrane transporter proteins: piaA/piaB/piaC ; the capsular genes: cpsA (wzg) & psrP ; the metallo-binding proteins zmpB & zmpC ; and the neuroamidase proteins: nanA/nanB were variably present. Surprisingly, the putative transcriptional regulator gene SP2020 was not present in all isolates (98%). Susceptibility to penicillin, erythromycin and clindamycin was molecularly confirmed. Conclusion The observed serotype 8 replacement was not significantly reflected with a change in the MLST profile or changes in antibiotic resistance- or virulence determinants.

The number of invasive Streptococcus agalactiae (GBS) non-typeable (NT) isolates in Denmark received since 1999 has in general accounted for 10% of all invasive GBS isolates. We present data on 55 clinical NT isolates based on clinical manifestations, clonal relationship, antimicrobial resistance (AMR) determinants, and virulence factors. The GBS isolates included in this study were phenotypic-based NT obtained from 2015 to 2017, as well as 10 reference isolates. Whole genome sequencing (WGS) was performed on all isolates and the data were analyzed for the presence of both species specific genes, capsular genes (genotype), and other relevant genes. We furthermore compared different procedures for detection of serotype specific capsular genes. Overall we were able to genotype 54 of the 55 isolates. After retesting the isolates a phenotype was detected for 20 (36%) isolates, of which the initial phenotyping problem for 13 isolates was found to be due to a problem with serotype Ia specific antiserum. Thirty-five isolates remained phenotypic non-typeable with a majority of genotype V isolates which do not express a capsular gene. From all the Danish invasive GBS isolates from 2015 to 2017, the 35 NT isolates were all detected in the age group above 21 years with bacteremia. The 35 NT isolates belonged to six different well-known human pathogenic clonal complexes. The CDC recommended sequences for capsule genotyping were the most optimal for serotype prediction, because of the sequence simplicity and clear cutoff values. However we recommend to also use other capsular sequences for the NT isolates, if they cannot be genotyped by the CDC method.

Entamoeba gingivalis has been associated with periodontal diseases. Baseline data from the background population, which could help delimit the role of the parasite in health and disease, remain limited. To describe epidemiological features, genetic diversity, and associations with oral microbiome signatures of E. gingivalis colonisation in Tanzanians with non-oral/non-dental diseases. DNAs from 92 oral washings from 52 participants were subject to metabarcoding of ribosomal genes. DNA sequences were identified to genus level and submitted to oral microbiota diversity analyses. Sixteen (31%) of the 52 study participants were E. gingivalis-positive, with no difference in positivity rate according to gender or age. Only one subtype (ST1) was found. Individuals testing positive for E. gingivalis had higher oral microbiota alpha diversity than those testing negative (P = 0.03). Eight of the top-ten most common bacterial genera were shared between the two groups (Alloprevotella, Fusobacterium, Gemella, Haemophilus, Neisseria, Porphyromonas, Prevotella, Streptococcus, and Veillonella). Meanwhile, E. gingivalis carriers and non-carriers were more likely to have Aggregatibacter and Rothia, respectively, among the top-ten most common genera. About one third of the cohort carried E. gingivalis ST1, and carriers had higher oral microbiome diversity and were more predominantly colonized by Aggregatibacter.

The frequencies of non-susceptibility against common antibiotics among pneumococci vary greatly across the globe. When compared to other European countries antibiotic resistance against penicillin and macrolides has been uncommon in Sweden in recent years. Multidrug resistance (MDR) is, however, of high importance since relevant treatment options are scarce. The purpose of this study was to characterize the molecular epidemiology, presence of resistance genes and selected virulence genes of extensively drug-resistant (XDR) ( =15) and MDR ( =10) detected in clinical respiratory tract samples isolated from patients in a southern Swedish county 2016-2018. With the aim of relating them to global MDR pneumococci. Whole genome sequencing (WGS) was performed to determine molecular epidemiology, resistance genes and presence of selected virulence factors. Antimicrobial susceptibility profiles were determined using broth microdilution testing. Further analyses were performed on isolates from the study and from the European nucleotide archive belonging to global pneumococcal sequence cluster (GPSC) 1 ( 86), GPSC9 ( 55) and GPSC10 ( 57). Bacteria were analyzed regarding selected virulence determinants (pilus islet 1, pilus islet 2 and Zinc metalloproteinase C) and resistance genes. Nineteen of 25 isolates were related to dominant global MDR lineages. Seventeen belonged to GPSC1, GPSC9 or GPSC10 with MDR non-PCV serotypes in GPSC9 (serotype 15A and 15C) as well as GPSC10 (serotype 7B, 15B and serogroup 24). Pilus islet-1 and pilus islet-2 were present in most sequence types belonging to GPSC1 and in two isolates within GPSC9 but were not detected in isolates belonging to GPSC10. Zinc metalloproteinase C was well conserved within all analyzed isolates belonging to GPSC9 but were not found in isolates from GPSC1 or GPSC10. Although MDR is relatively uncommon in Sweden compared to other countries, virulent non-PCV serotypes that are MDR may become an increasing problem, particularly from clusters GPSC9 and GPSC10. Since the incidence of certain serotypes (3, 15A, and 19A) found among our MDR Swedish study isolates are persistent or increasing in invasive pneumococcal disease further surveillance is warranted.