Association Between Vegetation Size and Outcome in the Partial Oral Antibiotic Endocarditis Treatment Trial.

Step-down oral antibiotic therapy is associated with a non-inferior long-term outcome compared with continued intravenous antibiotic therapy in the treatment of left-sided infective endocarditis. We aimed to analyze whether step-down oral therapy compared with continued intravenous antibiotic therapy is also associated with a non-inferior outcome in patients with large vegetations (vegetation length ≥ 10 mm) or among patients who underwent surgery before step-down oral therapy. We included patients without presence of aortic root abscess at diagnosis from the POET (Partial Oral Antibiotic Endocarditis Treatment) study. Multivariable Cox regression analyses were used to find associations between large vegetation, cardiac surgery, step-down oral therapy, and the primary end point (composite of all-cause mortality, unplanned cardiac surgery, embolic event, or relapse of positive blood cultures during follow-up). A total of 368 patients (age 68 ± 12, 77% men) were included. Patients with large vegetations (n = 124) were more likely to undergo surgery compared with patients with small vegetations (n = 244) (65% vs 20%, p <0.001). During a median 1,406 days of follow-up, 146 patients reached the primary end point. Large vegetations were not associated with the primary end point (hazard ratio 0.74, 95% confidence interval 0.47 to 1.18, p = 0.21). Step-down oral therapy was non-inferior to continued intravenous antibiotic in all subgroups when stratified by the presence of a large vegetation at baseline and early cardiac surgery. Step-down oral therapy is safe in the presence of a large vegetation at diagnosis and among patients who underwent early cardiac surgery.

Follow-Up Study of Effectiveness of 23-Valent Pneumococcal Polysaccharide Vaccine Against All-Type and Serotype-Specific Invasive Pneumococcal Disease, Denmark.

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.

Hepatic granulomas following liver transplantation: A retrospective survey, and analysis of possible microbiological etiology.

BACKGROUND: Liver granulomas have always been a diagnostic challenge for pathologists. They have been described in up to 15% of liver biopsies and can also be seen in liver allograft biopsy specimens, but there is a paucity of information regarding the prevalence and associated etiologic factors of granulomas in liver transplanted patients. The aim of this study is to shed light on the etiology of liver granulomas. METHODS: Liver biopsies from liver transplanted patients, in the period from 01.01.2011 - 01.05.2017, were examined. We registered the histo-morphological characteristics and clinicopathological data of all biopsies and performed next-generation sequencing (NGS) to detect possible pathogens (bacteria, fungi, and parasites) in the biopsies containing granulomas. RESULTS: We reviewed a total of 400 liver biopsies from 217 liver transplant patients. Of these, 131 liver biopsies (32.8%) from 98 patients (45.2%) revealed granulomas. Most were epithelioid granulomas located parenchymal and were detected in 115 (87.7%) biopsies. We also identified 10 cases (7.6%) with both lobular and portal granulomas and six biopsies (4.5%) with portal granulomas alone. In 54 biopsies (41.2%), granulomas were found in biopsies with acute cellular rejection (ACR). Fifty (51%) patients with granulomas underwent liver transplantation for autoimmune-related end-stage liver disease (AILD). The granulomas were found most frequently in the first six months after transplantation, where patients also more often were biopsied. NGS analysis did not reveal any potential infectious agent, and no significant differences were observed in the microbiological diversity (microbiome) between clinical- and granuloma characteristics concerning bacteria, fungi, and parasites. CONCLUSION: Our study confirmed that granulomas are frequently seen in liver allograft biopsy specimens, and most often localized in the parenchyma, occurring in the first post-transplant period in patients with AILD, and often seen simultaneously with episodes of ACR. Neither a specific microbiological etiological agent nor a consistent microbiome was detected in any case.

JAK2V617F drives gut microbiota differences in patients with myeloproliferative neoplasms.

BACKGROUND: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. METHODS: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. RESULTS: MPN patients had a higher observed richness (median, 245 [range, 49-659]) compared with HCs (191.5 [range, 111-300; p = .003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p < .001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. CONCLUSION: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

National Danish surveillance of invasive clinical Haemophilus influenzae isolates and their resistance profile.

Introduction: This study aimed to investigate the epidemiology, serotype distribution, phenotypical antibiogram, and molecular resistance gene characteristics of invasive Haemophilus influenzae infections in Denmark from 2014 to 2022. Additionally, the potential impact of outdoor temperature and COVID-19 restrictions on the epidemiology of H. influenzae was assessed. Materials and methods: Invasive H. influenzae isolates were received from patients with positive culture results from cerebrospinal fluid, blood, or other sterile sites. Sample data were obtained from the Danish laboratory surveillance system/MiBa database, and whole-genome sequencing (WGS) was performed on the isolates. The incidence rates and distribution of H. influenzae cases were analyzed, and antibiotic susceptibility were assessed. Results: A total of 1,007 invasive H. influenzae cases were identified, with serotyping conducted for 752 (74.7%) isolates. The median incidence per year of H. influenzae was 2.0 cases per 100,000, with the highest incidence in 2014 and the lowest in 2020. The majority of H. influenzae isolates were non-typeable H. influenzae (NTHi), while the most prominent serotypes were serotype f followed by serotype b. Bacteremia cases accounted for the majority (88.6%) of occurrences, although meningitis cases showed an increasing trend during the time period. The age group 85+ exhibited the highest incidence. The implementation of COVID-19 preventive interventions in 2020 resulted in a significant reduction in H. influenzae incidence, which returned to pre-COVID levels in 2021. A negative correlation was observed between monthly H. influenzae cases and outdoor temperature. An overall level of genetic beta-lactamase resistance of 26.3% was observed divided into 10.6% beta-lactamase-positive ampicillin-resistant (gBLPAR), 13.6% beta-lactamase-negative ampicillin-resistant (gBLNAR) and 2.1% beta-lactamase-positive amoxicillin clavulanate-resistant (gBLPACR). Other non-beta-lactam resistance traits were detected in 7.6% of isolates (primarily aminoglycoside-modifying enzymes). Conclusion: The overall incidence of H. influenzae in Denmark returned to stable levels after the COVID-19 epidemic, with NTHi strains dominating. The COVID-19 preventive interventions led to a major reduction in incidence. A significant negative correlation between the incidence of H. influenzae and temperature was observed. The study revealed an overall genetic beta-lactam resistance rate of 26.3%, and the concordance between genotypic and phenotypic beta-lactam resistance was high (98.2%).

Rifampicin reduces plasma concentration of linezolid in patients with infective endocarditis.

BACKGROUND: Linezolid in combination with rifampicin has been used in treatment of infective endocarditis especially for patients infected with staphylococci. OBJECTIVES: Because rifampicin has been reported to reduce the plasma concentration of linezolid, the present study aimed to characterize the population pharmacokinetics of linezolid for the purpose of quantifying an effect of rifampicin cotreatment. In addition, the possibility of compensation by dosage adjustments was evaluated. PATIENTS AND METHODS: Pharmacokinetic measurements were performed in 62 patients treated with linezolid for left-sided infective endocarditis in the Partial Oral Endocarditis Treatment (POET) trial. Fifteen patients were cotreated with rifampicin. A total of 437 linezolid plasma concentrations were obtained. The pharmacokinetic data were adequately described by a one-compartment model with first-order absorption and first-order elimination. RESULTS: We demonstrated a substantial increase of linezolid clearance by 150% (95% CI: 78%-251%), when combined with rifampicin. The final model was evaluated by goodness-of-fit plots showing an acceptable fit, and a visual predictive check validated the model. Model-based dosing simulations showed that rifampicin cotreatment decreased the PTA of linezolid from 94.3% to 34.9% and from 52.7% to 3.5% for MICs of 2 mg/L and 4 mg/L, respectively. CONCLUSIONS: A substantial interaction between linezolid and rifampicin was detected in patients with infective endocarditis, and the interaction was stronger than previously reported. Model-based simulations showed that increasing the linezolid dose might compensate without increasing the risk of adverse effects to the same degree.

Unveiling Rare Pathogens and Antibiotic Resistance in Tanzanian Cholera Outbreak Waters.

The emergence of antibiotic resistance is a global health concern. Therefore, understanding the mechanisms of its spread is crucial for implementing evidence-based strategies to tackle resistance in the context of the One Health approach. In developing countries where sanitation systems and access to clean and safe water are still major challenges, contamination may introduce bacteria and bacteriophages harboring antibiotic resistance genes (ARGs) into the environment. This contamination can increase the risk of exposure and community transmission of ARGs and infectious pathogens. However, there is a paucity of information on the mechanisms of bacteriophage-mediated spread of ARGs and patterns through the environment. Here, we deploy Droplet Digital PCR (ddPCR) and metagenomics approaches to analyze the abundance of ARGs and bacterial pathogens disseminated through clean and wastewater systems. We detected a relatively less-studied and rare human zoonotic pathogen, Vibrio metschnikovii, known to spread through fecal--oral contamination, similarly to V. cholerae. Several antibiotic resistance genes were identified in both bacterial and bacteriophage fractions from water sources. Using metagenomics, we detected several resistance genes related to tetracyclines and beta-lactams in all the samples. Environmental samples from outlet wastewater had a high diversity of ARGs and contained high levels of blaOXA-48. Other identified resistance profiles included tetA, tetM, and blaCTX-M9. Specifically, we demonstrated that blaCTX-M1 is enriched in the bacteriophage fraction from wastewater. In general, however, the bacterial community has a significantly higher abundance of resistance genes compared to the bacteriophage population. In conclusion, the study highlights the need to implement environmental monitoring of clean and wastewater to inform the risk of infectious disease outbreaks and the spread of antibiotic resistance in the context of One Health.

Pronounced gut microbiota signatures in patients with JAK2V617F-positive essential thrombocythemia.

Essential thrombocythemia (ET) is part of the Philadelphia chromosome-negative myeloproliferative neoplasms. It is characterized by an increased risk of thromboembolic events and also to a certain degree hypermetabolic symptoms. The gut microbiota is an important initiator of hematopoiesis and regulation of the immune system, but in patients with ET, where inflammation is a hallmark of the disease, it is vastly unexplored. In this study, we compared the gut microbiota via amplicon-based 16S rRNA gene sequencing of the V3-V4 region in 54 patients with ET according to mutation status Janus-kinase 2 (JAK2V617F)-positive vs JAK2V617F-negative patients with ET, and in 42 healthy controls (HCs). Gut microbiota richness was higher in patients with ET (median-observed richness, 283.5; range, 75-535) compared with HCs (median-observed richness, 191.5; range, 111-300; P < 0.001). Patients with ET had a different overall bacterial composition (beta diversity) than HCs (analysis of similarities [ANOSIM]; R = 0.063, P = 0.004). Patients with ET had a significantly lower relative abundance of taxa within the Firmicutes phylum compared with HCs (51% vs 59%, P = 0.03), and within that phylum, patients with ET also had a lower relative abundance of the genus Faecalibacterium (8% vs 15%, P < 0.001), an important immunoregulative bacterium. The microbiota signatures were more pronounced in patients harboring the JAK2V617F mutation, and highly similar to patients with polycythemia vera as previously described. These findings suggest that patients with ET may have an altered immune regulation; however, whether this dysregulation is induced in part by, or is itself inducing, an altered gut microbiota remains to be investigated. IMPORTANCE Essential thrombocythemia (ET) is a cancer characterized by thrombocyte overproduction. Inflammation has been shown to be vital in both the initiation and progression of other myeloproliferative neoplasms, and it is well known that the gut microbiota is important in the regulation of our immune system. However, the gut microbiota of patients with ET remains uninvestigated. In this study, we characterized the gut microbiota of patients with ET compared with healthy controls and thereby provide new insights into the field. We show that the gut microbiota of patients with ET differs significantly from that of healthy controls and the patients with ET have a lower relative abundance of important immunoregulative bacteria. Furthermore, we demonstrate that patients with JAK2V617F-positive ET have pronounced gut microbiota signatures compared with JAK2V617F-negative patients. Thereby confirming the importance of the underlying mutation, the immune response as well as the composition of the microbiota.

Evaluating the diagnostic accuracy and clinical utility of 16S and 18S rRNA gene targeted next-generation sequencing based on five years of clinical experience.

BACKGROUND: The use of 16S/18S rRNA targeted next-generation sequencing (tNGS) has improved microbial diagnostics, however, the use of tNGS in a routine clinical setting requires further elucidation. We retrospectively evaluated the diagnostic accuracy and clinical utility of 16S/18S tNGS, routinely used in the North Denmark Region between 2017 and 2021. METHODS: We retrieved 544 tNGS results from 491 patients hospitalised with suspected infection (e.g. meningitis, pneumonia, intraabdominal abscess, osteomyelitis and joint infection). The tNGS assays was performed using the Illumina MiSeq desktop sequencer, and BION software for annotation. The patients' diagnosis and clinical management was evaluated by medical chart review. We calculated sensitivity and specificity, and determined the diagnostic accuracy of tNGS by defining results as true positive, true negative, false positive, and false negative. RESULTS: Overall, tNGS had a sensitivity of 56% and a specificity of 97%. tNGS was more frequently true positive compared to culture (32% vs 18%), and tNGS detected a greater variety of bacteria and fungi, and was more frequently polymicrobial. However, the total diagnostic turnaround time was 16 days, and although 73% of tNGS results were true positive or true negative, only 4.4% of results led to changes in clinical management. CONCLUSIONS: As a supplement to culture, tNGS improves identification of pathogenic microorganisms in a broad range of clinical specimens. However, the long turnaround time of tNGS in our setting may have contributed to a limited clinical utility. An improved turnaround time can be the key to improved clinical utility in a future setting.

Trends in invasive bacterial diseases during the first 2 years of the COVID-19 pandemic: analyses of prospective surveillance data from 30 countries and territories in the IRIS Consortium.

BACKGROUND: The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. METHODS: For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. FINDINGS: Overall, 116 841 cases were analysed: 76 481 in 2018-19, before the pandemic, and 40 360 in 2020-21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40-0·55), H influenzae (0·51; 0·40-0·66) and N meningitidis (0·26; 0·21-0·31), while no significant changes were observed for S agalactiae (1·02; 0·75-1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145-55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. INTERPRETATION: COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. FUNDING: Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization.

Predicting ß-lactam susceptibility from the genome of Streptococcus pneumoniae and other mitis group streptococci.

Introduction: For Streptococcus pneumoniae, ß-lactam susceptibility can be predicted from the amino acid sequence of the penicillin-binding proteins PBP1a, PBP2b, and PBP2x. The combination of PBP-subtypes provides a PBP-profile, which correlates to a phenotypic minimal inhibitory concentration (MIC). The non-S. pneumoniae Mitis-group streptococci (MGS) have similar PBPs and exchange pbp-alleles with S. pneumoniae. We studied whether a simple BLAST analysis could be used to predict phenotypic susceptibility in Danish S. pneumoniae isolates and in internationally collected MGS. Method: Isolates with available WGS and phenotypic susceptibility data were included. For each isolate, the best matching PBP-profile was identified by BLAST analysis. The corresponding MICs for penicillin and ceftriaxone was retrieved. Category agreement (CA), minor-, major-, and very major discrepancy was calculated. Genotypic-phenotypic accuracy was examined with Deming regression. Results: Among 88 S. pneumoniae isolates, 55 isolates had a recognized PBP-profile, and CA was 100% for penicillin and 98.2% for ceftriaxone. In 33 S. pneumoniae isolates with a new PBP-profile, CA was 90.9% (penicillin) and 93.8% (ceftriaxone) using the nearest recognized PBP-profile. Applying the S. pneumoniae database to non-S. pneumoniae MGS revealed that none had a recognized PBP-profile. For Streptococcus pseudopneumoniae, CA was 100% for penicillin and ceftriaxone in 19 susceptible isolates. In 33 Streptococcus mitis isolates, CA was 75.8% (penicillin) and 86.2% (ceftriaxone) and in 25 Streptococcus oralis isolates CA was 8% (penicillin) and 100% (ceftriaxone). Conclusion: Using a simple BLAST analysis, genotypic susceptibility prediction was accurate in Danish S. pneumoniae isolates, particularly in isolates with recognized PBP-profiles. Susceptibility was poorly predicted in other MGS using the current database.

Attainment of Target Antibiotic Levels by Oral Treatment of Left-Sided Infective Endocarditis: A POET Substudy.

BACKGROUND: In the POET (Partial Oral Endocarditis Treatment) trial, oral step-down therapy was noninferior to full-length intravenous antibiotic administration. The aim of the present study was to perform pharmacokinetic/pharmacodynamic analyses for oral treatments of infective endocarditis to assess the probabilities of target attainment (PTAs). METHODS: Plasma concentrations of oral antibiotics were measured at day 1 and 5. Minimal inhibitory concentrations (MICs) were determined for the bacteria causing infective endocarditis (streptococci, staphylococci, or enterococci). Pharmacokinetic/pharmacodynamic targets were predefined according to literature using time above MIC or the ratio of area under the curve to MIC. Population pharmacokinetic modeling and pharmacokinetic/pharmacodynamic analyses were done for amoxicillin, dicloxacillin, linezolid, moxifloxacin, and rifampicin, and PTAs were calculated. RESULTS: A total of 236 patients participated in this POET substudy. For amoxicillin and linezolid, the PTAs were 88%-100%. For moxifloxacin and rifampicin, the PTAs were 71%-100%. Using a clinical breakpoint for staphylococci, the PTAs for dicloxacillin were 9%-17%.Seventy-four patients at day 1 and 65 patients at day 5 had available pharmacokinetic and MIC data for 2 oral antibiotics. Of those, 13 patients at day 1 and 14 patients at day 5 did only reach the target for 1 antibiotic. One patient did not reach target for any of the 2 antibiotics. CONCLUSIONS: For the individual orally administered antibiotic, the majority reached the target level. Patients with sub-target levels were compensated by the administration of 2 different antibiotics. The findings support the efficacy of oral step-down antibiotic treatment in patients with infective endocarditis.

Antibody avidity to pertussis toxin after acellular pertussis vaccination and infection.

Pertussis toxin (PT) is a unique virulence factor of Bordetella pertussis, and therefore a key component of acellular pertussis vaccines. Although immunity after infection seems to persist longer than after vaccination, the exact mechanisms are not fully known. In this study the overall binding strength (avidity) of anti-PT IgG antibodies was compared after acellular booster vaccination and infection, as a parameter to evaluate long-lasting protection.Danish and Finnish serum samples from a total of 134 serologically confirmed patients and 112 children who received acellular booster vaccines were included in this study. The concentration of anti-PT IgG was first determined by ELISA, followed by two separate ELISAs to evaluate antibody avidity: either with a dilution series of urea as a bond-breaking agent of antibody and antigen binding and a constant anti-PT IgG concentration between the samples or with a constant dilution ratio of sera and detergent. In addition to urea, the use of diethylamine and ammonium thiocyanate as disruptive agents were first compared between each other.A strong Spearman correlation (R > 0.801) was noted between avidity and concentration of anti-PT IgG antibodies if a constant serum dilution method was used, and avidity was noted to be higher in patients in comparison to vaccinees in Denmark, but not in Finland. However, no correlation between antibody concentration and avidity was found if a constant anti-PT IgG concentration was used (R = -0.157). With this method, avidity after vaccination was significantly higher in comparison to that after infection in both Danish and Finnish subjects (p < 0.01). A shorter time since the latest booster vaccination was found to affect avidity positively on the next PT-antigen exposure with either vaccination or infection.

The gut microbiota in patients with polycythemia vera is distinct from that of healthy controls and varies by treatment.

Chronic inflammation is believed to play an important role in the development and disease progression of polycythemia vera (PV). Because an association between gut microbiota, hematopoiesis, and inflammation is well established, we hypothesized that patients with PV have a gut microbiota distinct from healthy control participants (HCs). Recombinant interferon alfa 2 (IFN-α2)-treatment of patients with PV is reportedly disease modifying in terms of normalization of elevated blood cell counts in concert with a reduction in the JAK2V617F allelic burden. Therefore, we hypothesized that patients treated with IFN-α2 might have a composition of the gut microbiota toward normalization. Herein, via amplicon-based next-generation sequencing of the V3 to V4 regions of the 16S ribosomal RNA gene, we report on an abnormal gut microbiota in 102 patients with PV compared with 42 HCs. Patients with PV had a lower alpha diversity and a lower relative abundance of several taxa belonging to Firmicutes (45%) compared with HCs (59%, P <.001). Furthermore, we report the composition of the gut microbiota to differ between the treatment groups (IFN-α2, hydroxyurea, no treatment, and combination therapy with IFN-α2 and ruxolitinib) and the HCs. These observations are highly interesting considering the potential pathogenetic importance of an altered gut microbiota for development of other diseases, including chronic inflammatory diseases. Our observations call for further gut microbiota studies to decipher potential causal associations between treatment and the gut microbiota in PV and related neoplasms.

Evaluation of molecular typing for national surveillance of invasive clinical Haemophilus influenzae isolates from Denmark.

Haemophilus influenzae is a gram-negative coccobacillus known to cause respiratory and invasive infections. It can possess a polysaccharide capsule that can be categorized into six different serotypes (i.e., Hia, Hib, Hic, Hid, Hie, and Hif) and non-encapsulated strains that are defined as non-typeable. Furthermore, H. influenzae can be characterized into eight biotypes (I-VIII). Traditionally, isolates have been serotyped and biotyped using phenotypic methods; however, these methods are not always reliable. In this study, we evaluate the use of whole-genome sequencing (WGS) for national surveillance and characterization of clinical Danish H. influenzae isolates. In Denmark, all clinical invasive isolates between 2014 and 2021 have been serotyped using a traditional phenotypic latex agglutination test as well as in silico serotyped using the in silico programs "hinfluenzae_capsule_characterization" and "hicap" to compare the subsequent serotypes. Moreover, isolates were also biotyped using a phenotypic enzyme test and the genomic data for the detection of the genes encoding ornithine, tryptophan, and urease. The results showed a 99-100% concordance between the two genotypic approaches and the phenotypic serotyping, respectively. The biotyping showed a 95% concordance between genotyping and phenotyping. In conclusion, our results show that in a clinical surveillance setting, in silico serotyping and WGS-based biotyping are a robust and reliable approach for typing clinical H. influenzae isolates.

Enrichment of antibiotic resistance genes within bacteriophage populations in saliva samples from individuals undergoing oral antibiotic treatments.

Spread of antibiotic resistance is a significant challenge for our modern health care system, and even more so in developing countries with higher prevalence of both infections and resistant bacteria. Faulty usage of antibiotics has been pinpointed as a driving factor in spread of resistant bacteria through selective pressure. However, horizontal gene transfer mediated through bacteriophages may also play an important role in this spread. In a cohort of Tanzanian patients suffering from bacterial infections, we demonstrate significant differences in the oral microbial diversity between infected and non-infected individuals, as well as before and after oral antibiotics treatment. Further, the resistome carried both by bacteria and bacteriophages vary significantly, with bla CTX-M1 resistance genes being mobilized and enriched within phage populations. This may impact how we consider spread of resistance in a biological context, as well in terms of treatment regimes.