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The online monitoring of Oil-in-Water (OiW) concentration in dynamic flow-loops using UV-Fluorescence-based sensors is investigated. Though the OiW sensors can be carefully calibrated in the static condition, many dynamic flow conditions can still impact their real-time measurement. In order to examine the considered OiW sensor's capability in handling dynamic flows, an extensive experimental study has been committed, which considers the following aspects, such as (i) different flow-loop configurations, e.g., standalone-loops and cyclone-in-loop; (ii) different online sampling mechanisms, e.g., (direct) in-line measurement and (indirect) side-stream measurement; (iii) different flow-rates of total stream or side-stream under different configurations and sampling mechanisms; (iv) impacts of air bubbles and pressure-pumps. The experimental results show that the considered OiW sensor can provide quite reasonable dynamic measurements in general. However this sensor's measurement can be very sensitive to different flow regimes, impurities (incl. air) in the measured stream as well as upstream (nearby) operating conditions. These observations could inspire automation researchers and industrial operators to create some innovative sensor fusion method(s), to combine this OiW measurement with other available measurements in the produced water treatment processes for better control of the de-oiling hydrocyclone system.

Haemophilus influenzae is a common cause of mucosal infections that warrants accurate surveillance. We aimed to assess the prevalence of the species in clinical specimens, and characterise population structure and resistance to aminopenicillins by whole genome sequencing.We assessed the point prevalence by entering the database records of 1 day in Denmark and examined the genome sequences of nationwide, collected isolates from the same day. The prevalence of H. influenzae in clinical samples on the 10th of January 2018 was 1.78 per 100,000 person-days (all samples), and 2.47 per 1000 hospital bed-days (hospital samples). Of 2009 bacteria deemed clinically relevant and collected in a concerted action by the Danish departments of clinical microbiology, 62 (3.1%) were H. influenzae. All 62 isolates belonged to phylogenetic group I and were unencapsulated. Three strains from separate Danish regions had identical core genome sequences, but a small number of intergenic mutations testified to circulating clones, rather than individual cases of patient-to-patient transmission. The TEM-1 β-lactamase gene was present in 24 strains, while 13 strains were genetically categorised as ampicillin-resistant due to substitutions in penicillin-binding protein 3; shared patterns of amino acid substitutions in unrelated strains indicated putative lateral transfer of chromosomal resistance. Circulating clones of H. influenzae are frequent, and host factors, rather than direct transmission of epidemic strains, may be the primary cause of infection. The bleak presence of ampicillin resistance revealed by sequencing of point prevalence strains underscores the necessity for close examination of testing methods.

Candida albicans and Candida glabrata are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the Candida population in the country. The majority of the 30 C. albicans isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight C. glabrata isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all C. albicans isolates to be susceptible to both azoles and echinocandins. Two C. glabrata isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species.

E. coli ST131 is an important extraintestinal pathogenic lineage. A signature characteristic of ST131 is its ability to asymptomatically colonize the gastrointestinal tract and then opportunistically cause extraintestinal infections, such as cystitis, pyelonephritis, and urosepsis. In this study, we identified an ST131 H27 sublineage that has acquired the enteroaggregative diarrheagenic phenotype, spread across multiple continents, and caused multiple outbreaks of community-acquired ESBL-associated bloodstream infections in Denmark. The strain’s ability to both cause diarrhea and innocuously colonize the human gastrointestinal tract may facilitate its dissemination and establishment in the community., Escherichia coli sequence type 131 (ST131) is a major cause of urinary and bloodstream infections. Its association with extended-spectrum β-lactamases (ESBLs) significantly complicates treatment. Its best-described component is the rapidly expanding H30Rx clade, containing allele 30 of the type 1 fimbrial adhesin gene fimH. This lineage appears to have emerged in the United States and spread around the world in part due to the acquisition of the ESBL-encoding blaCTX-M-15 gene and resistance to fluoroquinolones. However, non-H30 ST131 sublineages with other acquired CTX-M-type resistance genes are also emerging. Based on whole-genome analyses, we describe here the presence of an (fimH) H27 E. coli ST131 sublineage that has recently caused an outbreak of community-acquired bacteremia and recurrent urinary tract infections (UTIs) in Denmark. This sublineage has acquired both a virulence plasmid (pAA) that defines the enteroaggregative E. coli (EAEC) diarrheagenic pathotype and multiple genes associated with extraintestinal E. coli (ExPEC); combined, these traits have made this particular ST131 sublineage successful at colonizing its human host and causing recurrent UTI. Moreover, using a historic World Health Organization (WHO) E. coli collection and publicly available genome sequences, we identified a global H27 EAEC ST131 sublineage that dates back as far as 1998. Most H27 EAEC ST131 isolates harbor pAA or pAA-like plasmids, and our analysis strongly implies a single ancestral acquisition among these isolates. These findings illustrate both the profound plasticity of this important pathogenic E. coli ST131 H27 sublineage and genetic acquisitions of EAEC-specific virulence traits that likely confer an enhanced ability to cause intestinal colonization.

Introduction. Cefuroxime is an important antibiotic to treat several serious infections. Rapid elimination through the kidneys and the variation in MICs of various susceptible pathogens such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pneumoniae give rise to dosing issues, especially in otherwise healthy patients. Aim. To investigate the probability of target attainment (PTA) for obtaining the optimal dosage regimens for cefuroxime in healthy young people. Methodology. Two weeks apart 750 and 1500 mg cefuroxime were administered as an intravenous bolus to 20 healthy volunteers (mean age: 27 years). Population modelling and simulation studies were done based on the obtained data for cefuroxime plasma concentration. Results. With a target value of time above MIC (T >MIC) greater than 50 % the simulations revealed that a PTA of >99 % is obtained for S. pneumoniae with a dosage regimen of 750 mg q12h. For E. coli and K. pneumoniae the PTA was S. aureus a dosage of 1500 mg q8h gave a PTA above 97 %. Conclusions. S. pneumoniae is most likely treatable with a two-daily dose of 750 mg cefuroxime. Not treatable are K. pneumoniae and E. coli . For S. aureus 1500 mg q8h constitutes an optimal dosing schedule.

Objectives Patients having previous contact with healthcare systems abroad are routinely screened for resistant bacteria on admission to hospitals in Copenhagen. This study aimed to present carriage prevalence and geographical risk stratification, as well as phenotypic and genotypic characterisation of resistant isolates. Methods This study included screening samples analysed at one department of clinical microbiology in Copenhagen from 2016–2019. Patients who had previous contact with healthcare systems abroad within 6 months were screened at admission for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and carbapenemase-producing organisms (CPO). Isolates were characterised phenotypically and by whole-genome sequencing. The relative frequency of positive findings stratified by geographical regions correlated with relative frequency of Danish residents’ travel destinations. Results Of 2849 screening sets included in the study, 103 (3.6%) were positive. A total of 120 resistant isolates were detected (36 MRSA, 31 VRE and 53 CPO). The carrier prevalence for MRSA was 1.3%, 1.1% for VRE and 1.5% for CPO. Southern and Western Asia were overrepresented travel destinations in positive screening sets (41%). For VRE, 40% were related to Southern Europe, which also represented 35% of travel destinations. Genotypic characterisation confirmed a heterogenous genomic background reflecting global distribution of resistant clones. Conclusions Exposure targeted screening identified a substantial number of asymptomatic carriers of MRSA, VRE and CPO with heterogenous genetic backgrounds. Although some geographical regions were overrepresented, the complex epidemiology of the different pathogens did not allow a restriction of the screening strategy to certain geographical regions.

Objectives: To evaluate a genome-based surveillance of all Danish third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec ) from bloodstream infections between 2014 and 2015, focusing on horizontally transferable resistance mechanisms.Methods: A collection of 552 3GC-R Ec isolates were whole-genome sequenced and characterized by using the batch uploader from the Center for Genomic Epidemiology (CGE) and automatically analysed using the CGE tools according to resistance profile, MLST, serotype and fimH subtype. Additionally, the phylogenetic relationship of the isolates was analysed by SNP analysis.Results: The majority of the 552 isolates were ESBL producers (89%), with bla CTX-M-15 being the most prevalent (50%) gene, followed by bla CTX-M-14 (14%), bla CTX-M-27 (11%) and bla CTX-M-101 (5%). ST131 was detected in 50% of the E. coli isolates, with the remaining isolates belonging to 73 other STs, including globally disseminated STs (e.g. ST10, ST38, ST58, ST69 and ST410). Five of the bloodstream isolates were carbapenemase producers, carrying bla OXA-181 (3) and bla OXA-48 (2). Phylogenetic analysis revealed 15 possible national outbreaks during the 2 year period, one caused by a novel ST131/ bla CTX-M-101 clone, here observed for the first time in Denmark. Additionally, the analysis revealed three individual cases with possible persistence of closely related clones collected more than 13 months apart.Conclusions: Continuous WGS-based national surveillance of 3GC-R Ec , in combination with more detailed epidemiological information, can improve the ability to follow the population dynamics of 3GC-R Ec , thus allowing for the detection of potential outbreaks and the effects of changing treatment regimens in the future.

Vancomycin-resistant enterococci (VRE) are increasingly important nosocomial pathogens and screening for colonization status is a mainstay in infection control. We implemented PCR-based screening during vanA-positive Enterococcus faecium outbreaks in four university hospitals in Copenhagen, Denmark. Xpert® vanA/vanB was performed directly on rectal swabs and the vanA PCR result was used to guide infection control measures. Concurrently, all samples were selectively cultured including an overnight enrichment step. Diagnostic accuracy was calculated as well as turnaround time and the impact of the earlier available PCR results on infection control decision making. In all, 1110 samples were analysed. The vanA PCR positivity rate was 13.8% and culture positivity rate was 15.2%. The diagnostic accuracy of the vanA part of the assay was high with a sensitivity of 87.1%, a specificity of 99.7%, and positive and negative predictive values of 98.0% and 97.7%, respectively. The vanB PCR had a considerably lower specificity of 77.6% and a positive predictive value of 0.4%. In 1067 (96.1%) samples, PCR results were reported within 1 day, whereas median culture turnaround time was 3 days. The saving of time to available results corresponded to 141 saved isolation days and 292 saved transmission risk days. False-negative or false-positive PCR results led to six additional transmission risk days and 13 additional isolation days, respectively. The vanA PCR had high diagnostic accuracy and the prompt availability of results gave a considerable benefit for infection control decision making.

Escherichia colisequence type 131 (ST131) is a major cause of urinary and bloodstream infections and its association with extended-spectrum β-lactamases (ESBL) significantly complicates treatment. Most notorious is its rapidly expandingH30-Rx clade (named for containing allele 30 of the type-1 fimbrial adhesin genefimHand extensive antimicrobial resistance), which appears to have emerged in the United States due in part due to the acquisition of the ESBL-encodingblaCTX-M-15gene and resistance to fluoroquinolones. However, non-H30 ST131 lineages with acquired CTX-M-type resistance genes also are emerging. Based on whole-genome analyses, we describe here the presence of an (fimH)H27E. coliST131 lineage that currently is causing an outbreak of community-acquired bacteremia and recurrent urinary tract infections (UTIs) in Denmark. This lineage has acquired both a virulence plasmid (pAA) that defines the enteroaggregativeE. coli(EAEC) diarrheagenic pathotype and multiple genes associated with extraintestinalE. coli(ExPEC) that combined has made this particular ST131 lineage highly successful at colonizing its human host and cause recurrent UTI. Moreover, using a historic World Health OrganizationE. colicollection and publically available genome sequences, we identify a globalH27 EAEC ST131 lineage dating back as far as 1998. MostH27 EAEC ST131 isolates harbor pAA or pAA-like plasmids, which analysis strongly imply was caused by a single ancestral acquisition. These findings illustrate the profound plasticity of this important pathogenicE. coli H27 lineage in general, and the genetic acquisitions of EAEC-specific virulence traits that likely confer an enhanced ability to cause intestinal colonization.ImportanceTheE. coliST131 lineage is a notorious extraintestinal pathogen. A signature characteristic of ST131 is its ability to asymptomatically colonize the gastrointestinal tract and then opportunistically cause extraintestinal infections, such as cystitis, pyelonephritis and urosepsis. In this study, we report a novel ST131 sublineage that has acquired the enteroaggregative diarrheagenic phenotype, spread across multiple continents and has been associated with multiple outbreaks of community-acquired bloodstream infections in Denmark. The strain’s ability to both cause diarrhea and colonize the human gastrointestinal tract may facilitate its dissemination and establishment in the community, whereas the strain’s clonal nature may facilitate targeted control strategies, such as vaccination.

Objectives This study compares the genome of an ST131 CMY-2-producing Escherichia coli isolate from a Danish patient with other ST131 CMY-2-producing E. coli isolates of both human and animal origin. Methods In 2016, an ST131 CMY-2-producing E. coli isolate (ESBL20160056) was obtained from a patient with a bloodstream infection. The genome of the ESBL20160056 isolate was compared with genomes from six ST131 CMY-2-producing E. coli isolates obtained from broiler meat imported to Denmark, 15 ST131 CMY-2-producing E. coli isolates obtained from Enterobase (http://enterobase.warwick.ac.uk) and two ST131 CMY-2-producing E. coli from European collaborators. The plasmid from ESBL20160056 was sequenced using a MinION Mk1B (Oxford Nanopore Technologies). Results The E. coli isolate from the Danish patient clustered together with 13 other fimH22 ST131 CMY-2-producing E. coli isolates in a distinct clade. The clade consisted of genomes from six E. coli isolates from humans collected in Denmark, Spain, Cambodia and the USA, six E. coli isolates obtained from broiler meat samples imported to Denmark from France, the Netherlands and Germany, and two E. coli isolates obtained from broilers in Belgium and Luxembourg. The 101.5 kb plasmid with blaCMY-2 from ESBL20160056 had an IncI1 replicon and belonged to ST12 using the plasmid MLST scheme. In total, 10 of the 14 ST131 E. coli isolates belonging to the fimH22 clade carried an IncI1 ST12 plasmid with blaCMY-2. Conclusions From our data, it seems plausible that the ST131 fimH22 CMY-2-producing E. coli isolate obtained from the Danish patient could have a zoonotic broiler origin.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the main cause of urinary tract infections and septicemia. Significant attention has been given to the ExPEC sequence type ST131, which has been categorized as a “high-risk” clone. High-risk clones are globally distributed clones associated with various antimicrobial resistance determinants, ease of transmission, persistence in hosts, and effective transmission between hosts. The high-risk clones have enhanced pathogenicity and cause severe and/or recurrent infections. We show that clones of the E. coli ST410 lineage persist and/or cause recurrent infections in humans, including bloodstream infections. We found evidence of ST410 being a highly resistant globally distributed lineage, capable of patient-to-patient transmission causing hospital outbreaks. Our analysis suggests that the ST410 lineage should be classified with the potential to cause new high-risk clones. Thus, with the clonal expansion over the past decades and increased antimicrobial resistance to last-resort treatment options, ST410 needs to be monitored prospectively., Escherichia coli sequence type 410 (ST410) has been reported worldwide as an extraintestinal pathogen associated with resistance to fluoroquinolones, third-generation cephalosporins, and carbapenems. In the present study, we investigated national epidemiology of ST410 E. coli isolates from Danish patients. Furthermore, E. coli ST410 was investigated in a global context to provide further insight into the acquisition of the carbapenemase genes blaOXA-181 and blaNDM-5 of this successful lineage. From 127 whole-genome-sequenced isolates, we reconstructed an evolutionary framework of E. coli ST410 which portrays the antimicrobial-resistant clades B2/H24R, B3/H24Rx, and B4/H24RxC. The B2/H24R and B3/H24Rx clades emerged around 1987, concurrently with the C1/H30R and C2/H30Rx clades in E. coli ST131. B3/H24Rx appears to have evolved by the acquisition of the extended-spectrum β-lactamase (ESBL)-encoding gene blaCTX-M-15 and an IncFII plasmid, encoding IncFIA and IncFIB. Around 2003, the carbapenem-resistant clade B4/H24RxC emerged when ST410 acquired an IncX3 plasmid carrying a blaOXA-181 carbapenemase gene. Around 2014, the clade B4/H24RxC acquired a second carbapenemase gene, blaNDM-5, on a conserved IncFII plasmid. From an epidemiological investigation of 49 E. coli ST410 isolates from Danish patients, we identified five possible regional outbreaks, of which one outbreak involved nine patients with blaOXA-181- and blaNDM-5-carrying B4/H24RxC isolates. The accumulated multidrug resistance in E. coli ST410 over the past two decades, together with its proven potential of transmission between patients, poses a high risk in clinical settings, and thus, E. coli ST410 should be considered a lineage with emerging “high-risk” clones, which should be monitored closely in the future. IMPORTANCE Extraintestinal pathogenic Escherichia coli (ExPEC) is the main cause of urinary tract infections and septicemia. Significant attention has been given to the ExPEC sequence type ST131, which has been categorized as a “high-risk” clone. High-risk clones are globally distributed clones associated with various antimicrobial resistance determinants, ease of transmission, persistence in hosts, and effective transmission between hosts. The high-risk clones have enhanced pathogenicity and cause severe and/or recurrent infections. We show that clones of the E. coli ST410 lineage persist and/or cause recurrent infections in humans, including bloodstream infections. We found evidence of ST410 being a highly resistant globally distributed lineage, capable of patient-to-patient transmission causing hospital outbreaks. Our analysis suggests that the ST410 lineage should be classified with the potential to cause new high-risk clones. Thus, with the clonal expansion over the past decades and increased antimicrobial resistance to last-resort treatment options, ST410 needs to be monitored prospectively.

Objectives: To examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations.Methods: Sixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs.Results: A triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (Conclusions: The EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs

CTX-M-1 is a common extended-spectrum β-lactamase (ESBL) in Escherichia coli from animals and is often detected among human clinical isolates. The objective of this study was to investigate the epidemiological relationship between CTX-M-1-producing E. coli isolated from patients and animals in Denmark between 2006 and 2010. In total, 65 CTX-M-1-producing isolates from patients (n=22), pigs (n=21), cattle (n=4), organic poultry layers (n=3) and horses (n=15) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harbouring blaCTX-M-1 were characterised by S1 PFGE, PCR-based replicon typing, plasmid multilocus sequence typing, restriction fragment length polymorphism, and sequencing. Human and animal strains were unrelated based on PFGE. IncI1 was more common in human isolates (13/22) than in animal isolates (7/43), whereas the opposite trend was observed for IncN (5/22 human isolates and 24/43 animal isolates). Full characterisation of the plasmids harbouring blaCTX-M-1 revealed host-specific patterns in the distribution of plasmid types, with specific IncI1, IncN and IncH1 plasmid subtypes being predominant in humans, livestock and horses, respectively. Three indistinguishable human, bovine and porcine IncI1/ST49 plasmids had high nucleotide sequence homology and differed by the presence of IS66 elements in the bovine plasmid and the absence of one gene within the microcin-encoding operon in the human plasmid. In conclusion, this work suggests a minor contribution by animals to the occurrence of CTX-M-1 in human E. coli infections in Denmark during the study period.

Escherichia hermanniiis an extremely rare etiological agent of invasive infection, and thus, the bacterium was initially considered non-pathogenic. However, in five previously reported case reportsE. hermanniihas been implicated as the sole pathogen. Our case report describes blood stream infection withE.hermanniiin a haemodialysis patient with persisting symptoms, high fever and inflammatory markers despite appropriate antibiotic treatment until replacement of the dialysis catheter. We suspect biofilm formation to be a crucial pathogenic feature forE. hermanniiin the maintenance of an infection, which stresses the necessity of antibiotic treatment along with catheter replacement in bloodstream- and catheter-related infection withE. hermannii.

To identify possible explanations for the recent global emergence of Escherichia coli sequence type (ST) 131 (ST131), we analyzed temporal trends within ST131 O25 for antimicrobial resistance, virulence genes, biofilm formation, and the H 30 and H 30-Rx subclones. For this, we surveyed the WHO E. coli and Klebsiella Centre's E. coli collection (1957 to 2011) for ST131 isolates, characterized them extensively, and assessed them for temporal trends. Overall, antimicrobial resistance increased temporally in prevalence and extent, due mainly to the recent appearance of the H 30 (1997) and H 30-Rx (2005) ST131 subclones. In contrast, neither the total virulence gene content nor the prevalence of biofilm production increased temporally, although non- H 30 isolates increasingly qualified as extraintestinal pathogenic E. coli (ExPEC). Whereas virotype D occurred from 1968 forward, virotypes A and C occurred only after 2000 and 2002, respectively, in association with the H30 and H30 -Rx subclones, which were characterized by multidrug resistance (including extended-spectrum-beta-lactamase [ESBL] production: H 30-Rx) and absence of biofilm production. Capsular antigen K100 occurred exclusively among H 30-Rx isolates (55% prevalence). Pulsotypes corresponded broadly with subclones and virotypes. Thus, ST131 should be regarded not as a unitary entity but as a group of distinctive subclones, with its increasing antimicrobial resistance having a strong clonal basis, i.e., the emergence of the H 30 and H 30-Rx ST131 subclones, rather than representing acquisition of resistance by diverse ST131 strains. Distinctive characteristics of the H 30-Rx subclone—including specific virulence genes ( iutA , afa and dra , kpsII ), the K100 capsule, multidrug resistance, and ESBL production—possibly contributed to epidemiologic success, and some (e.g., K100) might serve as vaccine targets.

BACKGROUND: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in older children. Pulmonary and extra-pulmonary symptoms associated with M. pneumoniae infection are reported. M. pneumoniae is mainly epidemic in Denmark with the recurrence every 4-7th year.AIMS: Retrospectively, to describe the epidemiology and clinical features, in infants and children, during the M. pneumoniae epidemic in 2010 and 2011.METHODS: All children under the age of 16 that were tested for M. pneumoniae during the period 01.02.2010-31.01.2012 were included. Medical charts, as well as radiological findings, were reviewed for all children with M. pneumoniae. A post-hoc analysis of viral co-infections was done on part of the cohort.RESULTS: 134 of 746 children were tested positive for M. pneumoniae by PCR or serology. Positive tests were found in 65% of children seven years and older, in 30% of 2-6-year-olds and 4% of infants (less than two years of age). Viral co-infection was found in 27% of the tested samples. The clinical presentation was a cough, asthma-like symptoms and low-grade fever. Extra-pulmonary symptoms were common and presented as nausea/vomiting by 33% of the children and skin manifestations by 25%. 84% of the children had a chest x-ray taken, and there were positive radiological findings in 94% of these.CONCLUSION: M. pneumoniae also affected infants and young children and symptoms were similar to infections with respiratory viruses, but severe LRTI were also seen. During an up-coming epidemic, assessment of extra-pulmonary manifestations can be helpful when diagnosing M. pneumoniae infections.

The plasmid-mediated colistin resistance gene, mcr-1, was detected in an Escherichia coli isolate from a Danish patient with bloodstream infection and in five E. coli isolates from imported chicken meat. One isolate from chicken meat belonged to the epidemic spreading sequence type ST131. In addition to IncI2*, an incX4 replicon was found to be linked to mcr-1. This report follows a recent detection of mcr-1 in E. coli from animals, food and humans in China.

Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.

Erm(A) is rarely reported as erythromycin resistance determinant in pneumococci. One invasive erm(A) isolate was initially tested intermediate erythromycin resistant using E-test. However, upon re-testing it was resistant, thus close to the breakpoint value. This may be a reason why erm(A) only rarely is reported to cause resistance in pneumococci.

The aim of the study was to investigate the potential spread of gentamicin resistant (GENR) Escherichia coli isolates or GENR determinants from a Danish university hospital to the waste water environment. Waste water samples were collected monthly from the outlets of the hospital bed wards and the inlet of the related waste water treatment plant (WWTP) from October 2002 to August 2003. Waste water samples were also collected monthly from a residential area in the same period to be able to compare the prevalence of GENRE. coli isolates from hospital related and residential waste water. The waste water isolates were compared to GENRE. coli isolates obtained consecutively from September 2002 to September 2003 from patients mainly with urinary tract infections at the hospital with respect to Pulsed Field Gel Electrophoresis (PFGE) profiles. All isolates were investigated for GENR mechanisms (aac(3)-II, aac(3)-IV, ant(2″)-I, armA), phenotypic resistance pattern, and virulence genes (hlyA, chuA, sfaS, fogG, malX, traT, iutA, fyuA, iroN, cnf1) to investigate if the hospital and waste water could be reservoirs of antimicrobial resistance and virulence. The ability for GENR determinants to transfer horizontally was investigated by mating experiments.A total of 38, 15, 21, and two GENRE. coli were isolated from patients, the hospital outlets, the inlet of the WWTP, and the residential area, respectively. GENRE. coli were more prevalent in waste water from the hospital and the WWTP than in waste water from the residential area. PFGE profiling revealed no spread of specific patient isolates to the waste water. The aac(3)-II gene was detected both in patient and waste water isolates. Furthermore horizontal transfer of the aac(3)-II gene of patient origin to a recipient was shown in vitro, indicating a potential spread of the gene from patient isolates to waste water isolates. Regardless of origin, most isolates exhibited multi-resistance and contained several virulence genes.In conclusion, our study showed a possible spread of aac(3)-II from the hospital to the waste water. Most of the GENRE. coli isolates from both patients and waste water had a multi-resistant phenotype and contained virulence genes and should therefore be considered reservoirs of antimicrobial resistance and virulence genes. Keywords: Escherichia coli, Gentamicin, Dissemination, Antimicrobial resistance, Virulence

This study evaluated a novel peptide nucleic acid (PNA) probe targeting a region of the 23S rRNA gene of Klebsiella pneumoniae by fluorescence in situ hybridization (FISH). Analytical performance was determined using 39 reference strains and other well-characterized strains of Klebsiella spp. and Enterobacter aerogenes. The probe was found to be specific for the K. pneumoniae complex (K. pneumoniae including Klebsiella ozaenae and Klebsiella variicola). The diagnostic accuracy was evaluated with 264 blood cultures containing Gram-negative rods. Using conventional identification as the reference, performance specifications were as follows: sensitivity 98.8 %, specificity 99.5 %, positive predictive value 98.8 % and negative predictive value 99.5 %. Discrepancies were resolved by PNA FISH retest and phenotypic tests. In conclusion, the K. pneumoniae probe provided an accurate diagnosis within 3 h and may supplement other methods for direct identification of Gram-negative bacteria.

Please cite this article as: Hammerum AM, Hansen F, Olesen B, Struve C, Holzknecht BJ, Andersen PS, Thye A-M, Jakobsen L, Roder BL, Stegger M, Hansen DS, Investigation of a possible outbreak of NDM-5-producing ST16 Klebsiella pneumoniae among patients in Denmark with no history of recent travel using whole-genome sequencing, Journal of Global Antimicrobial Resistance (2010), http://dx.doi.org/10.1016/j.jgar.2015.05.003

From January 1st 2011 through June 30th 2011, 116 nonreplicate, noncystic fibrosis-related Pseudomonas aeruginosa isolates with reduced carbapenem susceptibility were collected from 12 out of 13 Danish departments of clinical microbiology. The presence of acquired β-lactamases was assessed with combination tablet-diffusion methodology and polymerase chain reaction. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay, and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing acquired β-lactamases were further investigated by serotyping and multi locus sequence typing. Eight isolates produced the metallo-β-lactamase (MBL) VIM-2, and one isolate produced OXA-10 and VEB-1-like extended-spectrum beta-lactamase (ESBL). Phenotypic indications of derepressed AmpC and efflux pump were seen in 56 and 43 isolates, respectively. Overall, the results indicate that mutational factors related to permeability--often combined with derepressed, chromosomal AmpC--is the main factor behind carbapenem nonsusceptibility in Danish P. aeruginosa isolates. The ESBL producer and all the VIM producers belonged to international clones. PFGE revealed that most of the isolates were unrelated, but clonal spread was seen; the 116 isolates distributed in 97 PFGE types, with the largest cluster consisting of 4 isolates (including three isolates from the same hospital with 100% similarity). Thirty-two isolates were pair-wise related, while the remaining isolates were clonally unrelated, as were all nine ESBL/MBL producers.

We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates ( n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH , fyuA , malX , and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.

In 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according to these traits and resistance profiles, are unknown. Accordingly, we extensively characterized 51 archived E. coli O78:H10 isolates (48 human isolates from seven countries, including 19 Copenhagen outbreak isolates, and 1 each of calf, avian, and unknown-source isolates), collected from 1956 through 2000. E. coli O78:H10 was clonally heterogeneous, comprising one dominant clonal group (61% of isolates, including all 19 outbreak isolates) from ST10 (phylogenetic group A) plus several minor clonal groups (phylogenetic groups A and D). All ST10 isolates, versus 25% of non-ST10 isolates, were identified by molecular methods as enteroaggregative E. coli (EAEC) ( P < 0.001). Genes present in >90% of outbreak isolates included fimH (type 1 fimbriae; ubiquitous in E. coli ); fyuA , traT , and iutA (associated with extraintestinal pathogenic E. coli [ExPEC]); and sat , pic , aatA , aggR , aggA , ORF61, aaiC , aap , and ORF3 (associated with EAEC). An outbreak isolate was lethal in a murine subcutaneous sepsis model and exhibited characteristic EAEC “stacked brick” adherence to cultured epithelial cells. Thus, the 1991 Copenhagen outbreak was caused by a tight, non-animal-associated subset within a broadly disseminated O78:H10 clonal group (ST10; phylogenetic group A), members of which exhibit both ExPEC and EAEC characteristics, whereas O78:H10 isolates overall are phylogenetically diverse. Whether ST10 O78:H10 EAEC strains are both uropathogenic and diarrheagenic warrants further investigation.

Three cases of meningitis caused by the fastidious Gram-negative rod Capnocytophaga canimorsus have been observed at a regional hospital in 1 y. The difficulties connected with the correct diagnosis by classical culturing methods in contrast to molecular methods, as well as possible reasons for the accumulation of cases, are discussed.

Taxonomy and identification of fastidious Gram negatives are evolving and challenging. We compared identifications achieved with the Vitek 2 Neisseria-Haemophilus (NH) card and partial 16S rRNA gene sequence (526 bp stretch) analysis with identifications obtained with extensive phenotypic characterization using 100 fastidious Gram negative bacteria. Seventy-five strains represented 21 of the 26 taxa included in the Vitek 2 NH database and 25 strains represented related species not included in the database. Of the 100 strains, 31 were the type strains of the species. Vitek 2 NH identification results: 48 of 75 database strains were correctly identified, 11 strains gave `low discrimination´, seven strains were unidentified, and nine strains were misidentified. Identification of 25 non-database strains resulted in 14 strains incorrectly identified as belonging to species in the database. Partial 16S rRNA gene sequence analysis results: For 76 strains phenotypic and sequencing identifications were identical, for 23 strains the sequencing identifications were either probable or possible, and for one strain only the genus was confirmed. Thus, the Vitek 2 NH system identifies most of the commonly occurring species included in the database. Some strains of rarely occurring species and strains of non-database species closely related to database species cause problems. Partial 16S rRNA gene sequence analysis performs well, but does not always suffice, additional phenotypical characterization being useful for final identification.

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum β-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla (TEM), bla (SHV), and bla (CTX-M), and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla (CTX-M15), bla (SHV-28), and bla (TEM-1) positive by PCR. Ten out of 52 were ST16 and tested positive for bla (CTX-M15), bla (SHV-1), and bla (TEM-1). Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for bla (CTX-M15), bla (SHV-28), and bla (TEM-1), and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing bla (CTX-M15) and bla (SHV-28) constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.

The main purpose of the study was to investigate the frequency of ESBL-producing E. coli and Klebsiella strains in the Greater Copenhagen area. Four collections of strains were investigated: A) 380 consecutive E. coli and Klebsiella isolates primarily from urine, B) 200 gentamicin-resistant E. coli and Klebsiella isolates primarily from urine, C) 210 consecutive E. coli isolates from blood cultures, and D) 68 cefuroxime-resistant E. coli and Klebsiella isolates primarily from urine. Only one strain per patient was included. Strains with a zone diameter for cefpodoxime

Klebsiella pneumoniae and Klebsiella oxytoca are the two most frequently encountered Klebsiella species giving rise to infections in humans, but other Klebsiella species can also be found in clinical specimens: Klebsiella ozaenae , Klebsiella rhinoscleromatis , Klebsiella terrigena , Klebsiella planticola , Klebsiella ornithinolytica , and Enterobacter aerogenes ( Klebsiella mobilis ). However, many of these species are indistinguishable by the conventional methods employed routinely in the clinical microbiological laboratory. Several investigators have suggested various additional tests, but as yet there is no standardized test panel for identifying all Klebsiella species and subspecies. In the present study, performed in three national Klebsiella reference laboratories, we have evaluated a test panel consisting of 18 biochemical tests on 242 strains comprising all Klebsiella species and subspecies. The test panel was designed to identify organisms preliminarily identified as belonging to the genus Klebsiella on the basis of conventional methods or automated identification systems. With the described test panel it is possible to find one or more positive test results differentiating any Klebsiella species, except Klebsiella rhinoscleromatis , from its closest relative.

The ability of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing Klebsiella pneumoniae strains to induce a respiratory burst in polymorphonuclear leukocytes (PMNLs) was investigated. Ninety ESBL-producing and 178 non-ESBL-producing Klebsiella pneumoniae isolates were serotyped and their ability to induce a respiratory burst in PMNLs tested by monitoring the cells' chemiluminescence (CL) response. The percentage of isolates inducing high levels of CL response (CL75%) was significantly higher among non-ESBL producers (52%) than among ESBL producers (32.2%) ( P0.0001; OR=3.396; 95%CI=2.036-5.664). The median CL response was significantly higher among the non-ESBL producers (76.9%) than among the ESBL producers (52.6%) ( P=0.034). The two groups did not differ in their ability to resist intracellular killing by PMNLs ( P0.05), with strains inducing high levels of CL response having significantly lower survival rates (31.8% vs. 42.4%) than strains inducing low levels of CL response (164% vs. 200%) ( P0.01). The frequencies of the K2 and the K25 serotypes were significantly higher among ESBL-producing strains (17.8% and 22.2%, respectively) than among the non-ESBL producers (6.2% and 1.7%, respectively) ( P=0.0057 and P0.0001). Of the 77 Klebsiella K serotypes, 71 were detectable among the non-ESBL producers, but only 24 were detectable among the ESBL producers. ESBL-producing Klebsiella pneumoniae strains might have a greater pathogenic potential by virtue of their ability to escape the phagocytic activity of PMNLs.

The 16S rDNAs of 20 strains of Klebsiella were sequenced and used for construction of a phylogenetic tree together with already published Enterobacteriaceae 16S rDNA sequences. The taxonomy within the Klebsiella genus, as reflected by the 16S rDNA tree, was in agreement with existing DNA-DNA hybridisation and numerical taxonomy data, indicating that for Klebsiella, 16S rDNA sequencing is a valid method for identification and taxonomical purposes. Five closely related clusters were found in the Klebsiella genus; Cluster I, K. oxytoca; Cluster II, K. terrigena, Cluster III, K. planticola and K. ornithinolytica; Cluster IV, Enterobacter aerogenes (K. mobilis); and Cluster V, K. pneumoniae. The position of Calymmatobacterium granulomatis within the genus and closest to K. pneumoniae was confirmed. For the species K. oxytoca, data seem to indicate a subdivision into two subspecies. In addition, a biochemically aberrant Klebsiella strain (BEC441) that was included in the analysis could not be assigned to any of the known species, but was found to be closest related to K. oxytoca. Furthermore, the high sequence similarity between the two environmental species K. planticola and K. ornithinolytica does not justify a distinction of the two species. Finally, within a 165-bp stretch of the 16S rDNA sequences, species-specific nucleotides were found.

Objective To compare pulsed-field gel electrophoresis (PFGE) typing and O:K-serotyping of Klebsiella in two different epidemiological settings. Methods One hundred and four bacteremia isolates without known epidemiological relation and 47 isolates from an outbreak in a neonatal intensive care unit (NICU) were K-typed by countercurrent immunoelectrophoresis (CCIE), O-typed by an inhibition enzyme-linked immunosorbent assay method, and typed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Results Typing data for the 104 bacteremia isolates were compared with regard to typability, number of types, maximum number of isolates per type, and the Discriminative Index (DI). O-typing combined with K-typing (DI 0.98) as O:K-serotyping (DI 0.99) gave a very discriminative typing system, whereas O-typing alone was not very discriminative (DI 0.76). PFGE (DI 1.00) was a more discriminative typing method than O:K-serotyping, as it could subdivide 13/22 O:K-serotypes into smaller groups. Isolates with the same PFGE-type had the same O:K-serotype, indicating that isolates with different O- and/or K-types could be expected to be of different PFGE-types. Typing of the 47 isolates from the outbreak in the NICU showed that 38 isolates belonged to a single clone, and that during an epidemic limited in time and space, differences in the electrophoretic patterns of up to five bands between a parental pattern type and a subtype may be found in the PFGE profiles. Conclusions Both O:K-serotyping and PFGE typing are highly discriminative typing methods. PFGE is the most discriminative method and is excellent for typing outbreaks with few isolates. If large numbers of isolates are to be typed, a more convenient strategy might be first to K- or O:K-serotype isolates followed by PFGE typing of possible identical isolates. Since K- or O:K-serotyping is a definitive typing method, while PFGE typing is a comparative one, PFGE cannot, for the time being, replace O:K-serotyping for surveillance purposes.