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Karen Rønø Thingholm,1 Frederik Boëtius Hertz,1,2 Anders Løbner-Olesen,3 Niels Frimodt-Møller,1 Karen Leth Nielsen11Department of Clinical Microbiology, Rigshospitalet, Copenhagen 2100, Denmark; 2Department of Clinical Microbiology, Herlev and Gentofte Hospital, Herlev 2730, Denmark; 3Department of Biology, Section for Functional Genomics and Center for Bacterial Stress Response and Persistence, University of Copenhagen, Copenhagen, DenmarkBackground: Extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) causing urinary tract infections often belong to sequence type 131 (ST131), serotype O25, carrying blaCTX-M-15.Aim: The main aim of this study was to examine the conjugational frequencies of E. coli with plasmids carrying blaCTX-M-15 to E. coli isolates from the fecal flora of healthy humans to determine whether ST131 is more likely to uptake or donate ESBL resistance compared to other E. coli clones.Methods: Donors and recipients were all clinical isolates and did not harbor plasmids with identical incompatibility groups (Inc-groups) based on in silico analyses of Inc-groups and restriction/modification systems (R/M-systems). The in vitro conjugation experiments were performed as filter conjugation with verification of transconjugants by random amplified polymorphic DNA (RAPD) PCR and blaCTX-M-15 PCR.Results: The frequencies of conjugation with blaCTX-M-15-carrying plasmids were found to be very rare with detectable conjugation frequencies in the range of 4x10−9–7x10−7 transconjugants/recipient. Recipients of O25/ST131 type yielded significantly lower conjugation frequencies compared to recipients of other O-types (P=0.004). The applied ST131/O25 donors did not yield detectable levels of transconjugants regardless of the applied recipient. Presence of sub-MIC levels of ampicillin increased plasmid transfer frequencies x100 fold (P=0.07).Conclusion: The results indicate that blaCTX-M-15 is rarely transferred by conjugation to E. coli isolates of the intestinal flora, even when the gene is plasmid-borne.Keywords: horizontal gene transfer, antimicrobial resistance, Escherichia coli, molecular typing, ST131, faecal isolates

Filip Jansåker,1,2 Jonas Bredtoft Boel,3 Sara Thønnings,1,2 Frederik Boëtius Hertz,3 Katrine Hartung Hansen,2 Niels Frimodt-Møller,2 Jenny Dahl Knudsen1,21Department of Clinical Microbiology, Hvidovre Hospital, University of Copenhagen, 2650 Hvidovre, Denmark; 2Department of Clinical Microbiology, Rigshospitalet, University of Copenhagen, 2100 Copenhagen, Denmark; 3Department of Clinical Microbiology, Herlev and Gentofte Hospital, University of Copenhagen, 2730 Herlev, DenmarkObjectives: To compare the therapeutic effect of pivmecillinam and other common oral antibiotics for community-acquired urinary tract infections (UTIs) caused by Extended Spectrum Beta-Lactamase (ESBL)- or non-ESBL-producing Escherichia coli.Methods: Retrospective cohort study from 2010 to mid-2016 with data from the regional Laboratory Database and three national databases on antibiotic prescriptions, hospital admission, and mortality, respectively. Primary care patients (≥18 years) empirically treated for UTI caused by non-ESBL- or ESBL-producing E. coli (non-ESBL and ESBL E. coli) were included. Seven antibiotics, commonly used empirically for UTI, were investigated. Treatment failure measured as the redemption of a new antibiotic prescription or admission to hospital due to UTI. Cox proportional hazard ratios and adjusted risk differences along with 95% confidence intervals were calculated for 14 and 30 days, respectively.Results: Thirty-six thousand two hundred and ninety-three (95.7%) and 1624 (4.3%) cases were included in the non-ESBL and ESBL groups, respectively. Male sex, high age, ESBL production, and resistance to empirical therapy were found to independently increase the risk of treatment failure. Compared to pivmecillinam, ciprofloxacin had significantly lower treatment failure for non-ESBL E. coli, but significantly higher treatment failure in ESBL E. coli. There was no significant difference between nitrofurantoin and pivmecillinam.Conclusion: All antibiotics seem to have a higher risk of treatment failure for UTI caused by ESBL-producing E. coli as compared to non-ESBL-producing E. coli. At present, nitrofurantoin and pivmecillinam seem to be the most relevant orally available therapies for E. coli UTI. Local resistance data should guide which of the two that should be the contemporary first-line option. Keywords: UTI, ESBL, E. coli, pivmecillinam, nitrofurantoin, cohort

Filip Jansåker,1,2 Niels Frimodt-Møller,3 Thomas L Benfield,2,4 Jenny Dahl Knudsen1,3 1Department of Clinical Microbiology, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark; 2Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; 3Department of Clinical Microbiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; 4Department of Infectious Diseases, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark Purpose: The pharmacokinetic properties of mecillinam (MEC) for urinary tract infections are excellent, and the resistance rate in Enterobacteriaceae is low compared to other recommended antibiotics. The oral prodrug pivmecillinam (P-MEC) has been used successfully as first choice for cystitis in the Nordic countries for many years. Norwegian and Danish guidelines also recommend P-MEC for acute uncomplicated pyelonephritis (AUP) and intravenous (IV) MEC for suspected urosepsis (only in Denmark). Here, we wish to present an updated investigation on the clinical data behind these recommendations together with sparse but more current clinical data.Methods: Prospective clinical trials evaluating MEC as monotherapy or in polytherapy with one other beta-lactam (mostly ampicillin [AMP]) for pyelonephritis or bacteremia were reviewed. Outcomes of primary interest were clinical and bacteriological success and relapse, respectively. Search databases used were PubMed, Cochrane Library, and Embase.Results: Twelve clinical studies (1979–2015) were included in this integrated literature review. Clinical success was seen in 38/51 (75%) patients treated with MEC as monotherapy and in 152/164 (93%) patients treated with MEC and one other beta-lactam. Bacteriological success was seen in 35/47 (74%) and 117/167 (70%) patients treated with MEC alone and with one other beta-lactam, respectively. In complicated infections, bacteriological success was much lower. Clinical relapse rate was not well described. Several uropathogenic bacteremia cases were treated successfully with MEC alone (ie, 10/15 [67%] and 13/15 [87%] for clinical and bacteriological success, respectively) or with one other beta-lactam (ie, 57/65 [88%] and 53/63 [84%] for clinical and bacteriological success, respectively). However, data on bacteremia are very sparse. Adverse reactions were few and mild (73/406 [18%]) and primarily seen when AMP was co-administered (69/73 [95%]). No serious adverse reactions were reported.Conclusion: IV MEC or oral P-MEC for 14 days may be suitable for the treatment of AUP and pediatric pyelonephritis. Randomized controlled trials using a single standardized dose of P-MEC compared to other current recommendations are warranted. Similarly, more evidence is required before MEC should be recommended for bacteremia or sepsis due to Enterobacteriaceae. Keywords: pyelonephritis, mecillinam, review, pivmecillinam, amdinocillin

Background: The Danish Hospital-Acquired Infections Database (HAIBA) is an automated surveillance system using hospital administrative, microbiological, and antibiotic medication data.Aim: To define and evaluate the case definition for hospital-acquired urinary tract infection (HA-UTI) and to describe surveillance data from 2010 to 2014.Methods: The HA-UTI algorithm defined a laboratory-diagnosed UTI as a urine culture positive for no more than two micro-organisms with at least one at ≥10(4)cfu/mL, and a probable UTI as a negative urine culture and a relevant diagnosis code or antibiotic treatment. UTI was considered hospital-acquired if a urine sample was collected ≥48h after admission and <48h post discharge. Incidence of HA-UTI was calculated per 10,000 risk-days. For validation, prevalence was calculated for each day and compared to point prevalence survey (PPS) data.Findings: HAIBA detected a national incidence rate of 42.2 laboratory-diagnosed HA-UTI per 10,000 risk-days with an increasing trend. Compared to PPS the laboratory-diagnosed HA-UTI algorithm had a sensitivity of 50.0% (26/52) and a specificity of 94.2% (1842/1955). There were several reasons for discrepancies between HAIBA and PPS, including laboratory results being unavailable at the time of the survey, the results considered clinically irrelevant by the surveyor due to an indwelling urinary catheter or lack of clinical signs of infection, and UTIs being considered HA-UTI in PPS even though the first sample was taken within 48h of admission.Conclusion: The HAIBA algorithm was found to give valid and valuable information and has, among others, the advantages of covering the whole population and allowing continuous standardized monitoring of HA-UTI. [ABSTRACT FROM AUTHOR]

Summary Background & aims Inflammation is catabolic and causes muscle loss. It is unknown if amino acid supplementation reverses these effects during the acute phase of inflammation. The aim was to test whether amino acid supplementation counteracts endotoxin-induced catabolism. Methods Eight young, healthy, lean males were investigated three times in randomized order: (i) normal conditions (Placebo), (ii) endotoxemia (LPS), and (iii) endotoxemia with amino acid supplementation (LPS + A). Protein kinetics were determined using phenylalanine, tyrosine, and urea tracers. Each study day consisted of a four-hour non-insulin stimulated period and a two-hour hyperinsulinemic euglycemic clamp period. Muscle biopsies were collected once each period. Results Endotoxin administration created a significant inflammatory response (cytokines, hormones, and vital parameters) without significant differences between LPS and LPS + A. Whole body protein breakdown was elevated during LPS compared with Placebo and LPS + A (p < 0.05). Whole body protein synthesis was higher during LPS + A than both Placebo and LPS (p < 0.003). Furthermore, protein synthesis was higher during LPS than during Placebo (p < 0.02). Net muscle phenylalanine release was markedly decreased during LPS + A (p < 0.004), even though muscle protein synthesis and breakdown rates did not differ significantly between interventions. LPS + A increased mammalian target of rapamycin (mTOR) phosphorylation (p < 0.05) and eukaryotic translation factor 4E-binding protein 1 (4EBP1) phosphorylation (p = 0.007) without activating AMPK or affecting insulin signaling through Akt. During insulin stimulation net muscle phenylalanine release and protein degradation were further reduced. Conclusions Amino acid supplementation in the acute phase of inflammation reduces whole body and muscle protein loss, and this effect is associated with activation of mTOR and downstream signaling to protein synthesis through mTORC1, suggesting a therapeutic role for intravenous amino acids in inflammatory states. Clinical trial registry : The Central Denmark Region Ethics Commitee (1-10-71-410-12) www.clinicaltrials.gov (identification number NCT01705782 ). [ABSTRACT FROM AUTHOR]

Abstract: Objective: Growth hormone (GH) increases during exercise, but the response of the insulin-like growth factor (IGF) system has not been as definitive. Therefore, we investigated the effect of the exercise-induced GH response on the circulating IGF-system in GH-deficient (GHD) and intact adults. Design: Eight GHD adults were studied on 2 occasions, with (+GH) and without (−GH) GH administered (0.4 IU) during exercise (45 min of cycle ergometer exercise at the lactate threshold). Eight age-matched controls were only studied on one occasion. Blood samples were drawn at baseline, during and post-exercise. IGFBP-3 proteolysis was measured by an in vitro proteolytic activity assay, IGF-I bioactivity by novel IGF-I kinase receptor activation assay (KIRA) and other hormones by immunoassay. Results: GH administration to GHD adults resulted in a serum GH peak similar to the exercise-stimulated GH response in GH intact controls, but exercise had only a small impact on the IGF system. IGF-I concentration was lower in controls but was only significantly lower than the +GH day. Neither IGF-I nor -II levels changed over time. IGFBP-1 demonstrated a time effect (P <0.01) in all groups, and a time×group interaction (P <0.01) with a rise at 75 min post-exercise, which was greater in the GHD subjects than controls. IGFBP-2 and -3 increased significantly (P <0.01) over time in the GHD subjects, but not in the controls. No change in IGFBP-3 proteolysis or IGF-I bioactivity was found during exercise or recovery in either group. Conclusion: Submaximal exercise induced minor changes in IGFBP-1, -2 and -3, without affecting IGFBP-3 proteolysis and IGF-I bioavailability. Thus the metabolic status during submaximal exercise does not require a change in plasma IGF-I bioavailability. Administration of GH to GHD adults does not result in changes in proteolysis or bioavailability. [Copyright &y& Elsevier]

The formation of immune complexes was studied by analytical rate-zonal ultracentrifugation using isomolar solutions of rabbit anti-human serum albumin IgG and of the corresponding F(ab′)2 fragments. The F(ab′)2 fragments retained full ability to react with the antigenic determinants and to form genuine antigen antibody complexes. Thus, the difference between the IgG and the F(ab′)2 systems was supposed solely to reflect the lack of the Fc portion. The complexes formed with F(ab′)2 fragments in the zone of low and moderate antigen excess were found to be distinctively more soluble than those formed with intact IgG. The data indicated that there were two kinds of precipitating immune complexes, namely antibody-rich and antigen-rich complexes. In the antibody-excess zone and the first part of the equivalence zone the immune complexes precipitated due to their antibody richness. Antigen-rich complexes formed in the zone of low antigen excess precipitate only in the presence of antibody-rich insoluble complexes. It is believed that this type of precipitation was due to an Fc-Fc interaction. This new function of the Fc portion of IgG was designated Fc-mediated immune precipitation. [ABSTRACT FROM AUTHOR]

By comparing the amounts of precipitate formed by the reaction between human serum albumin (HSA) and isomolar solutions of rabbit anti-HSA F(ab′)2 fragments and of the corresponding intact rabbit anti-HSA IgG, respectively, it was found that the Fc portion of IgG was of great importance for the precipitin reaction. In the present antigen-antibody system, about half of the antigen was precipitated by this mechanism. This effect was called Fc-mediated precipitation and was most clearly expressed in the biologically important zone of low antigen excess and the zone of equivalence. It was found that the pepsin digestion did not change the ability of the F(ab′)2 fragments to bind the antigen in comparison to intact IgG. Furthermore, turbidimetric analyses indicated that the Fc portion was involved in the overall mechanism of the precipitin reaction throughout the range of the precipitin curve. [ABSTRACT FROM AUTHOR]