Severe Human Case of Zoonotic Infection with Swine-Origin Influenza A Virus, Denmark, 2021.

During routine surveillance at the National Influenza Center, Denmark, we detected a zoonotic swine influenza A virus in a patient who became severely ill. We describe the clinical picture and the genetic characterization of this variant virus, which is distinct from another variant found previously in Denmark.

Combination of thioridazine and dicloxacillin as a possible treatment strategy of staphylococci.

We have previously shown that the phenothiazine, thioridazine, acts in synergy with the beta-lactam antibiotic, dicloxacillin, to kill methicillin-resistant Staphylococcus aureus. In this study, we investigated whether synergy by combining these two drugs could also be observed in vancomycin intermediate susceptible S. aureus (VISA) and methicillin-resistant Staphylococcus epidermidis (MRSE). Synergy was observed in three of four tested VISA strains, suggesting that the thickening of cell wall does not interfere with the effects of thioridazine. In S. epidermidis, no synergy was observed in all tested strains, suggesting that synergy by combining thioridazine and dicloxacillin is isolated to S. aureus species.

Investigation of a possible outbreak of carbapenem-resistant Acinetobacter baumannii in Odense, Denmark using PFGE, MLST and whole-genome-based SNPs.

OBJECTIVES: The objectives were to study a possible outbreak of carbapenem-resistant Acinetobacter baumannii by comparing three different typing methods (PFGE, MLST and whole-genome SNPs) and to compare the resistance gene profiles of the isolates. METHODS: From December 2012 to October 2013, eight carbapenem-resistant A. baumannii were detected at Odense University Hospital, Odense, Denmark. These isolates were typed by PFGE, with ApaI and SmaI, respectively, and subjected to WGS. The WGS data were used for in silico extraction of MLST types using two different schemes, resistance genes and SNPs, to which 31 publicly available A. baumannii genomes were added. RESULTS: Using ApaI, the eight isolates had four different PFGE profiles, which were further differentiated using SmaI, separating one of the profiles into two distinct PFGE types. Five ST2 (Pasteur MLST) OXA-23-producing isolates, two ST1 OXA-72-producing isolates and one ST158 OXA-23-producing isolate were detected. The five ST2 isolates were subdivided into ST195, ST208 and ST218 using the Oxford MLST scheme. The phylogenetic analysis based on the core genome showed that six of the eight Danish A. baumannii isolates were located in three distinct clusters. The two remaining isolates did not cluster with other Danish or international isolates included in the study. Isolates that clustered using PFGE, Oxford MLST and phylogenetic analysis also shared similar resistance gene profiles. CONCLUSIONS: The SNP profile, Oxford MLST, PFGE and resistance gene profiles clearly indicated spread of three different A. baumannii strains.

Two cases of Ruminococcus gnavus bacteremia associated with diverticulitis.

We report two cases of bacteremia with the anaerobic bacterium Ruminococcus gnavus. In both cases, the bacteremia was associated with diverticular disease. Preliminary conventional identification suggested peptostreptococci, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis did not produce scores high enough for species identification. Finally, the bacteria were identified by 16S rRNA gene sequencing.

Need for species-specific detection for the diagnosis of amoebiasis in a non-endemic setting.

The diagnosis of amoebiasis caused by Entamoeba histolytica is traditionally based on microscopy. However, the specificity of this method may be questioned, especially in areas where infections by E. histolytica are rare. In the present study, a species-specific real-time PCR was used for the identification of the morphologically similar species E. histolytica and Entamoeba dispar. Out of 15 microscopy-positive stool samples, all were negative for E. histolytica and positive for E. dispar. In 2 cases, a suspicion of amoebic liver abscesses was confirmed by detection of E. histolytica DNA in stored sample material. Microscopy alone is clearly insufficient for the detection of E. histolytica in a setting where this parasite is rare. Microscopy-positive stool samples should be further tested by species-specific tests to distinguish E. histolytica from the non-pathogenic parasite E. dispar. On specific suspicion of amoebiasis, such as the suspicion of amoebic liver abscesses, species-specific tests can be applied even after storage.

Development and Clinical Application of a Multilocus Sequence Typing Scheme for Bacteroides fragilis Based on Whole-Genome Sequencing Data.

Bacteroides fragilis is among the most abundant and pathogenic bacterial species in the gut microbiota and is associated with diarrheal disease in children, inflammatory bowel disease, and the development of colorectal cancer. It is increasingly resistant to potent antimicrobial agents such as carbapenems and metronidazole, making it among the most resistant anaerobic bacteria. These factors combined call for increased monitoring of B. fragilis and its population structure on a worldwide scale. Here, we present a possible solution through the development of a multilocus sequence typing scheme (MLST). The scheme is based on seven core gene fragments: groL (hsp60), rpoB, recA, dnaJ, rprX, prfA, and fusA. These gene fragments possess high discriminatory power while retaining concordance with whole core genome-based phylogenetic analysis. The scheme proved capable of differentiating B. fragilis isolates at the strain level. It offers a standardized method for molecular typing and can be applied to isolates from various sampling backgrounds, such as patient isolates, environmental samples, and strains obtained from food and animal sources. In total, 567 B. fragilis genomes were sequence typed and their isolate data collected. The MLST scheme clearly divided the B. fragilis population into two divisions based on the presence of the cfiA and cepA resistance genes. However, no other specific subpopulations within the analyzed genomes were found to be associated with any specific diseases or geographical location. With this MLST scheme, we hope to provide a powerful tool for the study and monitoring of B. fragilis on an international scale. IMPORTANCE Here, we present the first MLST scheme for Bacteroides fragilis, one of the most abundant pathogenic bacteria in the human gut microbiota. The scheme enables standard classification and monitoring of B. fragilis on a worldwide scale and groups the majority of current isolate data in one place. A more unified approach to the collection and analysis of B. fragilis data could provide crucial insights into how the pathogen operates and develops as a species. Close monitoring of B. fragilis is especially relevant, as it is increasingly resistant to potent antimicrobial agents and engages in horizontal gene transfer with other bacteria. Hopefully, this approach will guide new discoveries into how B. fragilis evolves and interacts with its human host. Additionally, the scheme could potentially be applied to other species of the genus Bacteroides.

Detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid samples following pre-enrichment culture.

Molecular methods for diagnosing Lyme neuroborreliosis (LNB) have shown suboptimal diagnostic sensitivities. The objective of this study was to improve the clinical sensitivity of PCR detection of Borrelia burgdorferi sensu lato spirochetes by inoculating cerebrospinal fluid (CSF) from patients suspected of LNB directly into culture medium at the time of lumbar puncture, with this pursuing enrichment of Borrelia spirochetes before PCR analysis. Adult patients with symptoms suggestive of LNB were prospectively enrolled at two hospitals in the Region of Southern Denmark. The CSF-culture samples were incubated for at least eight weeks. During this period, culture sample aliquots were analysed for the presence of Borrelia DNA by separate PCR protocols in two independent clinical laboratories. The included patients were diagnosed with definite (n=12) or possible (n=2) LNB, and non-LNB (n=171) based on clinical and paraclinical findings. Patients in the LNB and the non-LNB group had a median duration from symptom onset to lumbar puncture of 40 days (IQR [23-90] days) and 120 days (IQR [32-365] days), respectively. Pre-enrichment growth of Borrelia spirochetes was accomplished from three patients (21 %) in the LNB group. The positive culture samples were confirmed by both the digital droplet PCR and the real-time PCR methods employed. All CSF samples were PCR negative in the non-LNB group. The results of this study do not support the use of Borrelia-specific PCR as a general routine diagnostic tool in adults. Still, they suggest it may prove of additional value in selected patients with a limited time from symptom onset to sample collection.

Vancomycin-sensitive Enterococcus faecium bacteraemia - hospital transmission and mortality in a Danish University Hospital.

Introduction. The emergence of vancomycin-resistant Enterococcus faecium (VREfm) has left the vancomycin-sensitive E. faecium (VSEfm) strains almost unnoticed.Hypothesis. Molecular characteristics, hospital transmission patterns and clinical impact of VSEfm have changed, and VSEfm is a predictor of VREfm introduction.Aim. We wanted to do a molecular characterization of VSEfm to identify hospital transmissions and links between VSEfm and VREfm, and to investigate the demographics, treatment and impact on mortality of VSEfm bacteraemia.Methodology. VSEfm and VREfm blood culture isolates from Odense University Hospital, Denmark, from 2015 to 2019 were characterized using whole-genome sequencing and core-genome multilocus sequence typing (cgMLST). Clonal shifts and diversity of the VREfm isolates were compared to the VSEfm isolates. Hospital records were used for clinical data and transmission investigation of VSEfm cases.Results. Six-hundred and thirty VSEfm isolates from 599 patients belonged to 42 sequence types (STs) and 131 complex types (CTs) in several clusters. Multiple types were involved in putative transmission, occurring over the entire period. Twenty-seven VREfm bacteraemia cases were included. No correlation between the VSEfm and VREfm clones was identified. The 30 day mortality was 40 %, but only in 6.3 % of the cases, VSEfm bacteraemia was the likely cause of death.Conclusion. The molecular types of VSEfm bacteraemia isolates are changing and diverse. No direct correlation between VSEfm and the introduction of VREfm was found, but widespread hospital transmission indicates a presence of risk factors that could facilitate transmission of other micro-organisms as well. VSEfm bacteraemia is rarely the cause of death, indicating that 30 day mortality does not reflect the cause of death.

Absence of N-Acetylglucosamine Glycosylation on Listeria monocytogenes Wall Teichoic Acids Promotes Fatty Acid Tolerance by Repulsion From the Bacterial Surface.

Free fatty acids (FFAs) have strong antimicrobial properties against pathogenic bacteria and are known as natural protective agents against bacterial infections. Growth of the foodborne pathogen Listeria monocytogenes is highly affected by the presence of antimicrobial FFAs, however, the response of L. monocytogenes toward FFAs is not fully understood. Here, we explore how L. monocytogenes gains tolerance toward FFAs and present a novel mechanism conferring bacterial protection against FFA toxicity. Strains tolerant against the antimicrobial FFA palmitoleic acid were isolated and whole genome sequenced, and mutations were found in genes involved in wall teichoic acid (WTA) glycosylations. We show that mutation or deletion of lmo1079, which is essential for N-acetylglucosamine (GlcNAc) glycosylation of WTAs, confer tolerance against several antimicrobial FFAs. The FFA tolerant strains are lacking GlcNAc on their WTAs, which result in a more hydrophilic surface. In line with this, we observed a reduced binding of FFAs to the surface of the FFA tolerant strains. Additionally, lack of GlcNAc on WTAs confers tolerance toward acid stress. Altogether, these findings support that GlcNAc modification of WTA plays an important role in the response of L. monocytogenes toward stress conditions encountered during growth as a saprophyte and pathogen, including FFA-rich environments. Most importantly, our data revealed that L. monocytogenes strains lacking GlcNAc on their WTAs are protected against FFA toxicity, because the FFAs are repulsed from the bacterial surface of GlcNAc-deficient strains.

The Global Regulator CcpA of Listeria monocytogenes Confers Sensitivity to Antimicrobial Fatty Acids.

Free fatty acids (FFAs) are known to exhibit antimicrobial and anti-virulent properties against bacterial pathogens. Specific FFAs, such as lauric acid (LA; C12:0), exert both effects against the foodborne pathogen Listeria monocytogenes: at low levels, LA acts to inhibit the activity of the virulence regulator PrfA, whereas at higher levels, LA inhibits bacterial growth. Deletion of prfA is known to promote tolerance toward antimicrobial FFAs, suggesting that the response of L. monocytogenes to anti-virulent and antimicrobial FFAs could be linked. In this study, we explored the response of L. monocytogenes toward antimicrobial FFAs holding an anti-virulence activity by isolating strains that can grow at high concentrations of LA. We found that LA-tolerant isolates carry mutations in the gene encoding the global regulator CcpA. Importantly, we discovered that mutation or deletion of ccpA protect L. monocytogenes against the antimicrobial activity of FFAs, whereas the ccpA mutants remain sensitive toward FFA's PrfA inhibitory effect. A regulatory link involving CcpA, connecting the response toward the antimicrobial and anti-virulence activities of FFAs, is therefore unlikely. To further study how deletion of ccpA promotes FFA tolerance, we performed a transcriptomic analysis of the response to LA. Our data indicated that the FFA-tolerant phenotype of the ∆ccpA strain is not induced upon LA exposure but appears to be an inherent phenotypic trait of the ccpA deletion mutation. Interestingly, we found that the bacterial surface of L. monocytogenes becomes more hydrophilic upon deletion of ccpA, and we demonstrate that CcpA plays a role in the response of L. monocytogenes to other stress conditions, including low pH and antibiotics. Altogether, our study revealed that regulatory activities of CcpA lead to an increased hydrophobicity of the bacterial surface, which may confer sensitivity of L. monocytogenes against the antimicrobial activity of FFAs. Notably, CcpA is not involved in responding to the PrfA inhibitory effect of FFAs, showing that FFA-tolerant strains can still be targeted by the anti-virulent activity of FFAs.

Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: Comparison with automated systems.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.

A Chlamydia trachomatis 23S rRNA G1523A variant escaping detection in the Aptima Combo 2 assay (Hologic) was widespread across Denmark in July-September 2019.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection globally, and nucleic acid amplification tests (NAATs) are recommended for highly sensitive and specific diagnosis. In early 2019, the Finnish new variant of Chlamydia trachomatis (FI-nvCT) was identified. The FI-nvCT has a C1515T mutation in the 23S rRNA gene, making it escaping detection in the Aptima Combo 2 (AC2; Hologic) NAAT, and the FI-nvCT has been subsequently reported in Sweden and Norway. In the present study, we investigated the presence of the FI-nvCT and other AC2 diagnostic-escape CT mutants in July-September 2019 in Denmark. The FI-nvCT was present but rare in Denmark. However, another AC2 diagnostic-escape CT mutant (with a 23S rRNA G1523A mutation) was found to be widespread across Denmark, accounting for 95% (76/80) of AC2 diagnostic-escape nvCT samples from five Danish CT-diagnostic laboratories. This nvCT-G1523A has previously only been detected in one single sample in the United Kingdom and Norway, respectively. It is vital to monitor the continued stability of the NAAT targets in local, national and international settings and monitor as well as appropriately analyse incidence, unexplained shifts in diagnostics rates and/or annual collections of samples diagnosed as negative/equivocal using NAATs with different target(s). Furthermore, diagnostic CT NAATs with dual target sequences are crucial, and fortunately, an updated Hologic AC2 assay including one additional target sequence is in advanced development.

Three-decade epidemiological analysis of Escherichia coli O15:K52:H1.

The successful Escherichia coli O15:K52:H1 clonal group provides a case study for the emergence of multiresistant clonal groups of Enterobacteriaceae generally. Accordingly, we tested the hypotheses that, over time, the O15:K52:H1 clonal group has become increasingly (i) virulent and (ii) resistant to antibiotics. One hundred archived international E. coli O15:K52:[H1] clinical isolates from 100 unique patients (1975 to 2006) were characterized for diverse phenotypic and molecular traits. All 100 isolates derived from phylogenetic group D and, presumptively, sequence type ST393. They uniformly carried the F16 papA allele and papG allele II (P fimbria structural subunit and adhesin variants), iha (adhesin-siderophore), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iutA (aerobactin receptor), and kpsM II (group 2 capsule); 85% to 89% of them contained a complete copy of the pap operon and ompT (outer membrane protease). Slight additional virulence profile variation was evident, particularly within a minor diarrhea-associated subset (biotype C). However, in contrast to the clonal group's fairly stable virulence profiles over the past 30+ years, during the same interval the clonal group members' antimicrobial resistance profiles increased by a mean of 2.8 units per decade (P < 0.001). Moreover, the numbers of virulence genes and resistance markers were positively associated (P = 0.046), providing evidence against antimicrobial resistance and virulence being mutually exclusive in these strains. Thus, the O15:K52:H1 clonal group has become increasingly resistant to antimicrobials while maintaining (or expanding) its virulence potential, a particularly concerning trend if other emerging multiresistant enterobacterial clonal groups follow a similar pattern.

Use of Loop-Mediated Isothermal Amplification in a Resource-Saving Strategy for Primary Malaria Screening in a Non-Endemic Setting.

Malaria is traditionally diagnosed by blood smear microscopy, which requires continuous resource-demanding training. In areas with only a few cases of malaria, a simple and rapid test that can reliably exclude malaria could significantly reduce the need for microscopy and training. We evaluated whether loop-mediated isothermal amplification (LAMP) for screening malaria parasites could reduce the workload in the diagnosis of malaria. Loop-mediated isothermal amplification was used to analyze 38 ethylene-diamine-tetraacetic acid (EDTA) blood samples from 23 patients who had previously been tested for malaria by microscopy, antigen-based rapid diagnostic test (antigen-RDT), and in-house real-time polymerase chain reaction (RT-PCR). The samples included blood with low-level parasitaemia and samples with discrepancies between the results of the different methods. Loop-mediated isothermal amplification detected malaria parasites in 27 of 28 samples that were positive according to in-house RT-PCR. There were negative microscopy results in 10 of these and negative antigen-RDT results in 11. The sample with a negative LAMP result and positive in-house RT-PCR result was from a patient who had recently been treated for low-level Plasmodium falciparum malaria parasitaemia. We found LAMP to be reliable for malaria screening and suitable for replacing microscopy without loss of performance. The low number of LAMP-positive samples needing microscopy can be handled by a limited number of trained microscopists. The time saved on training and documentation was estimated to be 520 working hours yearly in our laboratory. Using LAMP for primary screening of patient samples, we have made a diagnostic workflow that ensures more reliable, faster, and less resource-demanding diagnosis of malaria.

Reversal of methicillin resistance in Staphylococcus aureus by thioridazine.

OBJECTIVES: Thioridazine has been shown to reverse oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) in vitro. The aim of this study was to investigate whether thioridazine alone or in combination with oxacillin affects the transcription of the methicillin resistance gene mecA and the protein level of the encoded protein PBP2a. METHODS: Viability of MRSA was determined in liquid media in the presence of oxacillin or thioridazine alone or in combination. Transcription of mecA was analysed by primer extension, and the protein level of PBP2a was analysed by western blotting in the presence of thioridazine and oxacillin. RESULTS: We observed an increased susceptibility of MRSA towards oxacillin in the presence of thioridazine compared with bacteria grown with oxacillin or thioridazine alone. Transcription of mecA was reduced with increasing concentrations of thioridazine in the presence of a fixed amount of oxacillin. Furthermore, the protein level of PBP2a was reduced when bacteria were treated with the combination of oxacillin and thioridazine. The two drugs also affected the mRNA level of the beta-lactamase gene, blaZ. CONCLUSIONS: The present study indicates that reversal of methicillin resistance by thioridazine in MRSA may be explained by a reduced transcription of mecA and blaZ, resulting in a reduced protein level of PBP2a.

[Microbe hunters are advancing within rapid diagnostics].

Rapid diagnostics within clinical microbiology is more required, as hospitals need to be more effective. Tests for multi-resistant organisms, influenza virus and life-threatening diseases such as malaria and meningitis are warranted. This review describes the advances within rapid diagnostics and the impact on patient care. To achieve the full potential of rapid diagnostics, logistics such as transportation and personnel around the clock is necessary. However, with the right set-up, clinical microbiology rapid diagnostics will contribute to better and more effective patient care.

Cryptosporidium Species are Frequently Present But Rarely Detected in Clinical Samples From Children with Diarrhea in a Developed Country.

Two studies were done on cryptosporidiosis in children. A retrospective survey showed that from 2005 to 2015, Cryptosporidium species was detected by microscopy of stool from 0.25% of children with diarrhea. In a subsequent prospective study, polymerase chain reaction detected Cryptosporidium species in 4 (1.3%) of 304 children. Cryptosporidium species is as frequent as other intestinal pathogens in childhood diarrhea. Testing is relevant.

Virulence Factors Associated with Enterococcus Faecalis Infective Endocarditis: A Mini Review.

INTRODUCTION: The enterococci are accountable for up to 20% of all cases of infective endocarditis, with Enterococcus faecalis being the primary causative isolate. Infective endocarditis is a life-threatening infection of the endocardium that results in the formation of vegetations. Based on a literature review, this paper provides an overview of the virulence factors associated with E. faecalis infective endocarditis. Furthermore, it reports the effects of active or passive immunization against some of these involved factors. INDIVIDUAL VIRULENCE FACTORS: Nine virulence factors have in particular been associated with E. faecalis infective endocarditis. Absence of these factors entailed attenuation of strains in both mixed- and mono-bacterial infection endocarditis models as well as in in vitro and ex vivo assays when compared to their virulence factor expressing parental strains. PATHOGENESIS: The virulence factors promote a broad spectrum of events that together allow for disease development and progression. The infection is initiated through bacterial binding to ligands present at the site of infection after which the colonization can be accelerated through inter-bacterial attachment and modulation of the host immune response. The formation and growth of the vegetation provide protection and promote growth. Controlled degeneration of the vegetation appears to increase the likelihood of embolization and dissemination, without exposing protected bacteria. PROPHYLACTIC IMMUNIZATION: In most cases, active and passive immunization against associated virulence factors provided partial protection. FUTURE PROSPECTS: There is a need for further evaluation of the known virulence factors. Immunization against two or more virulence factors might be an effective prophylactic tool.

An enhanced Enterovirus surveillance system allows identification and characterization of rare and emerging respiratory enteroviruses in Denmark, 2015-16.

BACKGROUND: The potential for outbreaks due to Enteroviruses (EV) with respiratory tropism, such as EV-D68, and the detection of new and rare EV species C is a concern. These EVs are typically not detected in stool specimens and may therefore be missed by standard EV surveillance systems. Following the North American outbreak of EV-D68 in 2014, Denmark piloted an enhanced EV surveillance system that included the screening of respiratory samples. OBJECTIVES: We aim to report clinical manifestations and phylogenetic descriptions from the rare and emerging EVs identified thereby demonstrating the usefulness of this system. STUDY DESIGN: Positive EV samples received through the enhanced non-polio EV pilot surveillance system were characterized by sequencing fragments of VP1, VP2 and VP4 capsid proteins and clinical observations were compiled. RESULTS: Between January 2015 and October 2016, six cases of rare genotypes EV-C104, C105 and C109 and nine cases of EV-D68 were identified. Patients presented with mild to moderately severe respiratory illness; no paralysis occurred. Distinct EV-C104, EV-C109 and EV-D68 sequences argue against a common source of introduction of these genotypes in the Danish population. CONCLUSIONS: The enhanced EV surveillance system enabled detection and characterization of rare EVs in Denmark. In order to improve our knowledge of and our preparedness against emerging EVs, public health laboratories should consider expanding their EV surveillance system to include respiratory specimens.