Characterisation of carbapenemase-producing Acinetobacter baumannii isolates from danish patients 2014-2021: detection of a new international clone - IC11.

OBJECTIVES: This study aimed to characterise carbapenemase-producing Acinetobacter baumannii (A. baumannii) isolates from Danish patients using whole genome sequencing (WGS). It also compared typing and epidemiological data for further investigation of the spread and origin of the carbapenemase-producing A. baumannii isolates. METHODS: From 1 January 2014 to 30 September 2021, 141 carbapenemase-producing A. baumannii isolates, received at the national reference laboratory at Statens Serum Institut, were investigated using WGS. Multilocus sequence typing (MLST) and cgMLST data, obtained by SeqSphere+ software, were linked to data related to source of isolation, patient age and sex, hospital admission and travel history. RESULTS: Most of the carbapenemase-producing A. baumannii isolates were from males (n = 100, 71%). Most patients (n = 88, 63%) had travelled outside Scandinavia before admission to a Danish hospital. The most prevalent carbapenemase gene was blaOXA-23 (n = 124). Isolates belonging to the dominating international clone IC2 accounted for 78% of all isolates. A new international ST164/OXA-91 clone, proposed to be named IC11, was recognised and described. The cgMLST analysis revealed 17 clusters, reflecting both sporadic travel to similar geographical areas and confirmed outbreaks in Danish hospitals. CONCLUSIONS: The occurrence of carbapenemase-producing A. baumannii in Denmark was still low; however, isolates belonging to major international clones with a high potential to spread within hospitals, mainly IC2, dominated. OXA-23 was by far the most prevalent carbapenemase detected. Sporadic and travel-related introductions to Danish hospitals, also intra-hospital transmission, could be confirmed, emphasising the need for continuing vigilance.

Contaminated dicloxacillin capsules as the source of an NDM-5/OXA-48-producing Enterobacter hormaechei ST79 outbreak, Denmark and Iceland, 2022 and 2023.

From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory.

COVID-19 contact tracing in the hospitals located in the North Denmark region: A retrospective review.

Background: The Department of Infection Control, at our University Hospital conducted contact tracing of COVID-19 positive patients and staff members at all hospitals in the North Denmark Region. Aim: To describe the contact tracing performed during the COVID-19 pandemic in the Region and its outcomes. Methods: Data from each contact tracing were collected prospectively during 14 May 2020-26 May 2021. Data included information about the index case (patient or hospital staff member), presentation (asymptomatic vs symptomatic), probable source of transmission (community-acquired or hospital-acquired), number of close contacts and if any of these were SARS-CoV-2 PCR-test positive. Findings: 362 contact tracing were performed. A total of 573 COVID-19 positive cases were identified among 171 (30%) patients and 402 (70%) staff members. 192 (34%) of all cases were tested due to symptoms of COVID-19, whereas two-third were tested for other reasons including outbreak and systematic screening tests. A total of 1575 close contacts were identified, including 225 (14%) patients and 1350 (86%) staff members. 100 (6%) close contacts, including 24 patients and 76 staff members, were infected with SARS-CoV-2, of which 33 (43%) staff members was positive at day 0 i.e. the same day as being identified as close contacts. Discussion: We found a three to one of close contacts to each index case, but only 6% became SARS-CoV-2 positive, with a surprisingly high number of those identified at day 0. Our data confirm that regular testing of patients and staff will identify asymptomatic carriers and thereby prevent new cases.

Antibiotic stewardship based on education: minor impact on knowledge, perception and attitude.

Purpose: The purpose of this study was to implement an education-based antibiotic stewardship programme at two regional hospitals in Denmark, and thereby reduce consumption of antibiotics in general and cephalosporins and fluoroquinolones in particular. We aimed to improve physicians' knowledge, prescribing practices and perceptions and attitudes towards antibiotics, and to achieve changes in behaviour. Methods: The antibiotic stewardship programme comprised education, guidelines, audits and feedback and ward rounds by a clinical microbiologist. The ward rounds were implemented only at one hospital. The effects of the programme were evaluated using a questionnaire, audits of prescriptions (initial choice of antibiotics, indication for antibiotic treatment, re-assessment of treatment) and data on antibiotic consumption. Results: The survey revealed an improvement in junior doctors' knowledge, perception and attitude and self-reported prescribing practice. In the audit results, a larger proportion of prescribed antibiotics was in accordance with guidelines, particularly when we evaluated re-assessment of antibiotic treatment at the hospital where ward rounds had been implemented. The increase was equivalent to risk ratio (RR) 1.13 (95% confidence interval (CI): 0.95-1.35) during the intervention and RR 1.22 (95% CI 1.01-1.48) post-intervention, compared to the pre-intervention period. Penicillins as well as total antibiotic consumption increased during the study period. Conclusion: An education-based antibiotic stewardship programme can change the attitude of junior doctors and improve prescribing practices. We observed an improvement in the re-assessments of the antibiotic treatments at the hospital where a clinical microbiologist was present at ward rounds, but our persuasive methods were insufficient to reduce antibiotic consumption.

The applicability of guidelines on antimicrobials provided by the Danish Health Authority.

INTRODUCTION: Our objective was to investigate the know-ledge of "Guidelines on prescribing antibiotics for physicians and others in Denmark" published by the Danish Health Au-thority in 2012, and the usefulness of these guidelines for Danish hospital doctors. METHODS: We collected information on the perceived aware-ness and usefulness of the guidelines using a ques-tionnaire distributed at two regional hospitals. Furthermore, to evaluate the use of the guidelines, we collected data about 310 patients treated with antimicrobials at the two hospitals. RESULTS: Less than 50% of the respondents knew that the guidelines existed and among those who did know of the existence of the guidelines, less than 50% found that the guidelines were applicable to their daily clinical work. Regarding sampling of microbiological tests, we found that in 188 of the 310 cases both urine and blood had been sampled. A discontinuation date was registered in the medication module at the time of the prescription in 27% of the prescriptions. In terms of reassessment, we evaluated 282 cases and found that a reassessment had taken place in 62% of these cases. CONCLUSIONS: The results of the survey indicate that knowledge and use of the guidelines are sparse, and our data from the audits underline these data. FUNDING: The Fund for the Prevention of Hospital-acquired Infections, the Danish Ministry of Health (R. no. 1406148) and the Fund for Improving Skills in Infection Control, Central Denmark Region, funded the study. TRIAL REGISTRATION: not relevant. The Danish Data Protection Agency (R. no.: 1-16-02-84-15) and the Central Denmark Region approved the project.

Research Electronic Data Capture (REDCap®) used as an audit tool with a built-in database.

The aim of this study was to develop an audit tool with a built-in database using Research Electronic Data Capture (REDCap®) as part of an antimicrobial stewardship program at a regional hospital in the Central Denmark Region, and to analyse the need, if any, to involve more than one expert in the evaluation of cases of antimicrobial treatment, and the level of agreement among the experts. Patients treated with systemic antimicrobials in the period from 1 September 2015 to 31 August 2016 were included, in total 722 cases. Data were collected retrospectively and entered manually. The audit was based on seven flow charts regarding: (1) initiation of antimicrobial treatment (2) infection (3) prescription and administration of antimicrobials (4) discontinuation of antimicrobials (5) reassessment within 48 h after the first prescription of antimicrobials (6) microbiological sampling in the period between suspicion of infection and the first administration of antimicrobials (7) microbiological results. The audit was based on automatic calculations drawing on the entered data and on expert assessments. Initially, two experts completed the audit, and in the cases in which they disagreed, a third expert was consulted. In 31.9% of the cases, the two experts agreed on all elements of the audit. In 66.2%, the two experts reached agreement by discussing the cases. Finally, 1.9% of the cases were completed in cooperation with a third expert. The experts assessed 3406 flow charts of which they agreed on 75.8%. We succeeded in creating an audit tool with a built-in database that facilitates independent expert evaluation using REDCap. We found a large inter-observer difference that needs to be considered when constructing a project based on expert judgements. Our two experts agreed on most of the flow charts after discussion, whereas the third expert's intervention did not have any influence on the overall assessment.

Characterisation of a multidrug-resistant Bacteroides fragilis isolate recovered from blood of a patient in Denmark using whole-genome sequencing.

Here we describe a patient undergoing extensive abdominal surgery and hyperthermic intraperitoneal chemotherapy due to primary adenocarcinoma in the sigmoid colon with peritoneal carcinomatosis. During hospitalisation the patient suffered from bacteraemia with a multidrug-resistant Bacteroides fragilis isolate. Whole-genome sequencing of the isolate resulted in identification of nimE, cfiA and ermF genes corresponding to metronidazole, carbapenem and clindamycin resistance.

Expression of type III interferon (IFN) in the vaginal mucosa is mediated primarily by dendritic cells and displays stronger dependence on NF-kappaB than type I IFNs.

Interferons (IFNs) are induced as an initial response to viral infection after recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Here, we report that different PAMPs induce type I and III IFN expression at different ratios after mucosal administration in the vaginas of mice and that Toll-like receptor 9 (TLR9) stimulation evokes a particularly strong IFN-lambda response, which is essential for optimal antiviral protection. Depletion of CD11c(+) cells in vivo revealed that dendritic cells (DCs) in the vaginal epithelium are a key source of type I and III IFNs during herpes simplex virus infection and after specific stimulation of TLR9. A comparison of the signaling pathways activated by TLR9 and cytoplasmic PRRs, which induced lower levels of IFN-lambda, revealed that high-level induction of IFN-lambda correlated with strong activation of NF-kappaB p65. Inhibition of the NF-kappaB and interferon regulatory factor 3 (IRF-3) pathways with the NEMO-binding domain peptide and small interfering RNA (siRNA), respectively, revealed that transcription of the type III IFN genes was more dependent on the NF-kappaB pathway than that of the type I IFN genes, which relied more on the IRF system. Thus, the type I and III IFN genes are not induced through entirely identical pathways, which indicates differential expression of these two types of IFNs under certain conditions.

Type III IFNs: new layers of complexity in innate antiviral immunity.

Cytokines are small secreted molecules, which mediate cross-talk between cells involved in the immune response. Interferons (IFN)s, constitute a class of cytokines with antiviral activities, and the type I IFNs have been ascribed particularly important roles in the innate antiviral response. Type III IFNs (also known as IFN-lambda or interleukin 28/29) represent a class of novel cytokines with biological activities similar to the type I IFNs, but seem to have a more specialized role in antiviral defense by exerting host-protection primarily at epithelial surfaces. In this review, we describe the current knowledge on the role of type III IFNs in antiviral defense.

An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity.

Type III IFNs (IFN-lambda/IL-28/29) are cytokines with type I IFN-like antiviral activities, which remain poorly characterized. We herein show that most cell types expressed both types I and III IFNs after TLR stimulation or virus infection, whereas the ability of cells to respond to IFN-lambda was restricted to a narrow subset of cells, including plasmacytoid dendritic cells and epithelial cells. To examine the role of type III IFN in antiviral defense, we generated IL-28Ralpha-deficient mice. These mice were indistinguishable from wild-type mice with respect to clearance of a panel of different viruses, whereas mice lacking the type I IFN receptor (IFNAR(-/-)) were significantly impaired. However, the strong antiviral activity evoked by treatment of mice with TLR3 or TLR9 agonists was significantly reduced in both IL-28RA(-/-) and IFNAR(-/-) mice. The type I IFN receptor system has been shown to mediate positive feedback on IFN-alphabeta expression, and we found that the type I IFN receptor system also mediates positive feedback on IFN-lambda expression, whereas IL-28Ralpha signaling does not provide feedback on either type I or type III IFN expression in vivo. Finally, using bone-marrow chimeric mice we showed that TLR-activated antiviral defense requires expression of IL-28Ralpha only on nonhemopoietic cells. In this compartment, epithelial cells responded to IFN-lambda and directly restricted virus replication. Our data suggest type III IFN to target a specific subset of cells and to contribute to the antiviral response evoked by TLRs.

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems.

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-alpha/beta) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-alpha/beta production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.

Type III interferon (IFN) induces a type I IFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases.

Type III interferon (IFN) is a novel member of the interferon family. Type III IFN utilizes a receptor complex different from that of type I IFN, but both types of IFN induce STAT1, STAT2, and STAT3 activation. Here we describe a detailed comparison of signal transduction initiated by type I and type III IFN. Gene expression array analysis showed that IFN types I and III induced a similar subset of genes. In particular, no genes were induced uniquely by type III IFN. Next, we used chromatin immunoprecipitation (ChIP) analysis to investigate the promoter activation by types I and III IFN. The ChIP assays demonstrated that stimulation of cells with both type I and type III IFN resulted in the recruitment of ISGF3 transcription factor components to the promoter region of responsive genes and in an increase of polymerase II loading and histone acetylation. Whereas IFN type I signaling was observed for a broad spectrum of cell lines, type III IFN signaling was more restricted. The lack of IFN type III signaling was correlated with a low expression of the IL28Ra component of the IFN type III receptor, and IL28Ra overexpression was sufficient to restore IFN type III signaling. We also tested the activation of mitogen-activated protein (MAP) kinases by type III IFN and found that type III IFN relies strongly upon both p38 and JNK MAP kinases for gene induction.

IFN-lambda: novel antiviral cytokines.

The first line of defense against viral infections is mediated by interferons (IFN)s, which are produced rapidly by the infected host. Type I IFNs (IFN-alpha/beta) are known to combat viruses both directly by inhibiting viral replication in the cells and indirectly by stimulating the innate and adaptive immune responses. Recently, a novel class of cytokines was discovered and named IFN-lambda (alternatively type III IFN or interleukin-28/29 [IL- 28/29]), based on IFN-like antiviral activity and induction of typical IFN-inducible genes. Here, we review the literature on IFN-lambda and discuss the current knowledge of the functions and mechanisms of action of IFN-lambda.

Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo.

Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-alpha, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.

Age-dependent role for CCR5 in antiviral host defense against herpes simplex virus type 2.

Elimination of viral infections is dependent on rapid recruitment and activation of leukocytes with antiviral activities to infected areas. Chemokines constitute a class of cytokines that have regulatory effects on leukocyte migration and activity. In this study we have studied the role of CC chemokine receptor 1 (CCR1) and CCR5 in host defense during a generalized herpes simplex virus type 2 (HSV-2) infection. Whereas both 4- and 8-week-old CCR1(-/-) mice resembled wild-type mice (C57BL/6) with respect to defense against the infection, significantly higher virus titers were seen in the livers and brains of 4-week-old CCR5(-/-) mice. At the age of 8 weeks, CCR5(-/-) were indistinguishable from wild-type mice and cleared the infection from liver and spleen. Although 4-week-old CCR5(-/-) mice were able to recruit natural killer (NK) cells to the site of infection, these cells had reduced cytotoxic activity compared to NK cells from wild-type mice. This was not due to lower production of alpha/beta interferon or interleukin-12, two well-described activators of cytotoxic activity in NK cells. We also noted that the spleens of young CCR5(-/-) mice did not increase in size during infection as did the spleens of wild-type and CCR1(-/-) mice. This observation was accompanied by impaired proliferation of CCR5(-/-) splenocytes (SCs) ex vivo. Moreover, migration of CD8(+) T cells to the liver in response to infection was impaired in CCR5(-/-) mice, and adoptive transfer of SCs from CCR5(-/-) mice infected for 6 days into newly infected wild-type mice did not improve antiviral activity in the liver, in contrast to what was seen in mice receiving immune SCs from wild-type mice. Altogether, this study shows that CCR5 plays an age-dependent role in host defense against HSV-2 by supporting both the innate and adaptive immune response.