A comparative study between real-time PCR and loop-mediated isothermal amplification to detect carbapenemase and/or ESBL genes in Enterobacteriaceae directly from bronchoalveolar lavage fluid samples.

OBJECTIVES: To evaluate and compare the efficacy of real-time PCR (Xpert Carba-R) and loop-mediated isothermal amplification (Eazyplex® SuperBug CRE) for detecting carbapenemase carriage in Enterobacteriaceae directly from bronchoalveolar lavage (BAL). METHODS: Negative BAL samples were spiked with 21 well-characterized carbapenemase-producing Enterobacteriaceae strains to a final concentration of 102-104 cfu/mL. Xpert Carba-R (Cepheid, Sunnyvale, CA, USA), which detects five targets (blaKPC, blaNDM, blaVIM, blaOXA-48 and blaIMP-1), and the Eazyplex® SuperBug CRE system (Amplex-Diagnostics GmbH, Germany), which detects seven genes (blaKPC, blaNDM, blaVIM, blaOXA-48, blaOXA-181, blaCTXM-1 and blaCTXM-9), were evaluated for the detection of these genes directly from BAL samples. RESULTS: Xpert Carba-R showed 100% agreement with carbapenemase characterization by PCR and sequencing for all final bacteria concentrations. Eazyplex® SuperBug CRE showed 100%, 80% and 27% agreement with PCR and sequencing when testing 104, 103 and 102 cfu/mL, respectively. False negative results for Eazyplex® SuperBug CRE matched the highest cycle threshold values for Xpert Carba-R. Hands-on time for both assays was about 15 min, but Eazyplex® SuperBug CRE results were available within 30 min, whereas Xpert Carba-R took around 50 min. CONCLUSIONS: We here describe the successful use of two commercial diagnostic tests, Xpert Carba-R and Eazyplex® SuperBug CRE, to detect bacterial carbapenem resistance genes directly in lower respiratory tract samples. Our results could be used as proof-of-concept data for validation of these tests for this indication.

Point Prevalence Survey of Antimicrobial Prescribing in a Nigerian Hospital: Findings and Implications on Antimicrobial Resistance.

BACKGROUND: Antimicrobial resistance is a global health challenge. There is inadequate information on antimicrobial prescribing practices in many sub-Saharan African countries including Nigeria. A standardized method for surveillance of antimicrobial use in hospitals was employed to assess the antimicrobial prescribing practices in UCH, Ibadan, Nigeria. METHODS: A point prevalence survey (PPS) was conducted in December 15, 2017 at the UCH Ibadan. The survey included all in-patients receiving an antimicrobial on the day of PPS. Data collected included details on the antimicrobial agents, reasons and indications for treatment as well as a set of quality indicators. A web-based application was used for data-entry, validation and reporting as designed by the University of Antwerp (www.global-pps.be). RESULTS: This survey included 451 patients from 38 different wards of which 59.6% received at least one antimicrobial. The neonatal medical wards contributed the highest number of patients who received antibiotics. A total of 172 therapeutic antibiotic prescriptions were issued, mainly for Community Acquired Infections (n=119; 69.2%). Most prescriptions for Healthcare Associated Infections (n=53) were intervention related (47.2%). Frequently used antibiotics include third generation cephalosporins (23.9%; mainly ceftriaxone); followed by combination of penicillin's (17.4%; mainly amoxicillin with enzyme inhibitor) and fluoroquinolones (16.6%). Majority, 312(69.9%)of the patients had parenteral antibiotics and only 95 (21.3%) of all antibiotic prescriptions had a documented stop or review date. Although the reason for antibiotic prescription was indicated for 413 (92.4%) prescriptions, targeted therapy was the basis for only 17 (3.8%)of these prescriptions. For surgical prophylaxis, 98.7% of all prescriptions were given for more than one day. Compliance to guidelines was non-existent. CONCLUSION: Our findings showed high broad spectrum prescribing, high number of intervention related health care infections, high use of prolonged surgical prophylaxis, inexistence of local guidelines; and low utilization of laboratory facilities. Hospital related intervention should include development of antibiotic guideline and increased enlightenment on rational prescribing practices.

THE GLOBAL POINT PREVALENCE SURVEY (PPS) OF ANTIMICROBIAL USE AND ANTIMICROBIAL RESISTANCE AMONG HOSPITALIZED CHILDREN IN GEORGIA.

The aims of our study were to determine antibiotic prescribing rates for prevention and treatment of infections in pediatric units, to evaluate the number and type of antimicrobial agents and administration route, reveal commonly used antibiotic subgroups and identify targets for improving the quality of antimicrobial prescribing. A 1-day PPS (Point Prevalence Study) on antibiotic use in hospitalized children was performed in Georgia from 2015 to 2019. 18 clinics in different regions of Georgia were included in the survey. Antimicrobial prevalence rates increased over the years from 60.1% in 2015 to 92.6% in 2018. The most commonly, antibiotics were prescribed for lower respiratory tract infections (LRTI). In 2015 25.1% of LRTI were treated by ampicillin-sulbactam but the next year it replaced with ceftriaxone (37.1% in 2017 and 38.2% in 2018). In pediatric surgical ward, the antibiotics were commonly prescribed for surgical prevention (54.1% in 2015, 32.3% in 2018). The most common conditions treated with antibiotics in neonates were sepsis (30.1%) and LRTI (45.3%). The most used antibiotic was ceftriaxone (33.3% in 2015). Ampicilin-sulbactam was prescribed in 28.1% of pneumonia case in neonates in 2018. In 2015 antibiotics were mainly prescribed empirically (98.0%). In 2018 resistance of MRSA was 8.1%, and resistance to the third-generation cephalosporin 17.3%. Prevalence rate of antibiotics for prevention and treatment of infection disease in pediatric units increased in 2018. Main feasible targets for optimization of antibiotic prescribing have been identified: high use of broad-spectrum antibiotics in hospitals, high frequency of empirical treatment, rarely performed culture tests, prolonged antibiotic prophylaxis in surgery patients and an alarming raise of resistant strains. The implementation of disease-specific clinical pathways associated with annual PPSs could be a good way to monitor and improve antibiotic prescription patterns in neonatal and pediatric inpatients over time.

Longitudinal point prevalence survey of antibacterial use in Northern Ireland using the European Surveillance of Antimicrobial Consumption (ESAC) PPS and Global-PPS tool.

Antimicrobial resistance is a limiting factor for the success of the treatment of infectious diseases and is associated with increased morbidity and cost. The present study aims to evaluate prescribing patterns of antimicrobials and quantify progress in relation to targets for quality improvement in the prescription of antimicrobials in Northern Ireland's secondary care sector using three repetitive point prevalence surveys (PPS) over a 6-year period: the European Surveillance of Antimicrobial Consumption (ESAC-PPS) in 2009 and 2011 and the Global-PPS on Antimicrobial Consumption and Resistance in 2015. Out of 3605 patients surveyed over the three time points, 1239 (34.4%) were treated with an antibiotic, the most frequently prescribed antibiotic groups were a combination of penicillins, including ß-lactamase inhibitors. Compliance with hospital antibiotic policies in 2009, 2011 and 2015 were 54.5%, 71.5% and 79.9%, respectively. Likewise, an indication for treatment was recorded in patient notes 88.5%, 87.7% and 90.6% in 2009, 2011 and 2015, respectively, and surgical prophylactic antibiotic prescriptions for >24 h was 3.9%, 3.2% and 0.7% in 2009, 2011 and 2015, respectively. Treatment based on biomarker data was used in 61.5% of cases. In conclusion, a general trend in the improvement of key antimicrobial-related quality indicators was noted. The PPS tool provided a convenient, inexpensive surveillance system of antimicrobial consumption and should be considered an essential component to establish and maintain informed antibiotic stewardship in hospitals.

Molecular epidemiology of MRSA in 13 ICUs from eight European countries.

OBJECTIVES: The European epidemiology of MRSA is changing with the emergence of community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA). In this study, we investigated the molecular epidemiology of MRSA during 2 years in 13 ICUs in France, Greece, Italy, Latvia, Luxemburg, Portugal, Slovenia and Spain. METHODS: Surveillance cultures for MRSA from nose and wounds were obtained on admission and twice weekly from all patients admitted to an ICU for ≥3 days. The first MRSA isolate per patient was genotyped in a central laboratory by MLST, spa typing, agr typing and SCCmec (sub)typing. Risk factors for patients with an unknown history of MRSA colonization were identified. RESULTS: Overall, 14 390 ICU patients were screened, of whom 8519 stayed in an ICU for ≥3 days. Overall MRSA admission prevalence was 3.9% and ranged from 1.0% to 7.0% for individual ICUs. Overall MRSA acquisition rate was 2.5/1000 patient days at risk and ranged from 0.2 to 8/1000 patient days at risk per ICU. In total, 557 putative MRSA isolates were submitted to the central laboratory for typing, of which 511 (92%) were confirmed as MRSA. Each country had a distinct epidemiology, with ST8-IVc (UK-EMRSA-2/-6, USA500) being most prevalent, especially in France and Spain, and detected in ICUs in five of eight countries. Seventeen (3%) and three (<1%) isolates were categorized as CA-MRSA and LA-MRSA, respectively. Risk factors for MRSA carriage on ICU admission were age >70 years and hospitalization within 1 year prior to ICU admission. CONCLUSIONS: The molecular epidemiology of MRSA in 13 European ICUs in eight countries was homogeneous within, but heterogeneous between, countries. CA-MRSA and LA-MRSA genotypes and Panton-Valentine leucocidin-producing isolates were detected sporadically.

The Innovative Medicines Initiative's New Drugs for Bad Bugs programme: European public-private partnerships for the development of new strategies to tackle antibiotic resistance.

Antibiotic resistance (ABR) is a global public health threat. Despite the emergence of highly resistant organisms and the huge medical need for new drugs, the development of antibacterials has slowed to an unacceptable level worldwide. Numerous government and non-government agencies have called for public-private partnerships and innovative funding mechanisms to address this problem. To respond to this public health crisis, the Innovative Medicines Initiative Joint Undertaking programme has invested more than €660 million, with a goal of matched contributions from the European Commission and the European Federation of Pharmaceutical Industries and Associations, in the development of new antibacterial strategies. The New Drugs for Bad Bugs (ND4BB) programme, an Innovative Medicines Initiative, has the ultimate goal to boost the fight against ABR at every level from basic science and drug discovery, through clinical development to new business models and responsible use of antibiotics. Seven projects have been launched within the ND4BB programme to achieve this goal. Four of them will include clinical trials of new anti-infective compounds, as well as epidemiological studies on an unprecedented scale, which will increase our knowledge of ABR and specific pathogens, and improve the designs of the clinical trials with new investigational drugs. The need for rapid concerted action has driven the funding of seven topics, each of which should add significantly to progress in the fight against ABR. ND4BB unites expertise and provides a platform where the commitment and resources required by all parties are streamlined into a joint public-private partnership initiative of unprecedented scale.

Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. METHODS: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). RESULTS: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). CONCLUSIONS: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

Systemic antifungal prescribing in neonates and children: outcomes from the Antibiotic Resistance and Prescribing in European Children (ARPEC) Study.

The appropriate use of systemic antifungals is vital in the prevention and treatment of invasive fungal infection (IFI) in immunosuppressed children and neonates. This multicenter observational study describes the inpatient prescribing practice of antifungal drugs for children and neonates and identifies factors associated with prescribing variability. A single-day point prevalence study of antimicrobial use in hospitalized neonates and children was performed between October and December 2012. The data were entered through a study-specific Web-based portal using a standardized data entry protocol. Data were recorded from 17,693 patients from 226 centers. A total of 136 centers recorded data from 1,092 children and 380 neonates receiving at least one antifungal agent. The most frequently prescribed systemic antifungals were fluconazole (n=355) and amphotericin B deoxycholate (n=195). The most common indications for antifungal administration in children were medical prophylaxis (n=325), empirical treatment of febrile neutropenia (n=122), and treatment of confirmed or suspected IFI (n=100 [14%]). The treatment of suspected IFI in low-birthweight neonates accounted for the majority of prescriptions in the neonatal units (n=103). An analysis of variance (ANOVA) demonstrated no significant effect of clinical indication (prophylaxis or treatment of systemic or localized infection) on the total daily dose (TDD). Fewer than one-half of the patients (n=371) received a TDD within the dosing range recommended in the current guidelines. Subtherapeutic doses were prescribed in 416 cases (47%). The predominance of fluconazole and high incidence of subtherapeutic doses in participating hospitals may contribute to suboptimal clinical outcomes and an increased predominance of resistant pathogenic fungi. A global consensus on antifungal dosing and coordinated stewardship programs are needed to promote the consistent and appropriate use of antifungal drugs in neonates and children.

KPC-Like Carbapenemase-Producing Enterobacteriaceae Colonizing Patients in Europe and Israel.

In a 2008-2011 survey, 17,945 patients in 18 hospital units in Europe and Israel were screened for carriage of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae, resulting in identification of 124 positive patients. The isolates were dominated by Klebsiella pneumoniae sequence type 258 (ST258) KPC-2 and ST512 KPC-3, mainly from Greece and Italy, respectively, whereas Israeli isolates were of diverse species, clones, and KPC variants. Various blaKPC platforms were observed, among which IncFIIK-FIBK plasmids with blaKPC-2/-3 genes in the Tn4401a transposon prevailed.

MLST reveals potentially high-risk international clones of Enterobacter cloacae.

OBJECTIVES: To perform the first multinational Enterobacter cloacae clonality study, using the MLST scheme newly developed in Japan. METHODS: The analysis included 195 rectal carriage E. cloacae isolates resistant to expanded-spectrum cephalosporins (ESCs), collected from patients in 12 hospital units across Europe and Israel. All of the isolates were typed by PFGE and 173 isolates were subjected to MLST. ESC resistance was analysed phenotypically; genes encoding ESBLs and carbapenemases were identified by PCR and sequencing. RESULTS: MLST distinguished 88 STs, which correlated with the PFGE data. PFGE was more discriminatory, producing 129 pulsotypes (169 patterns). Numerous STs were observed in several countries each. The most widespread were ST66, ST78, ST108 and ST114, each having at least 10 isolates from three to five countries, diversified into multiple pulsotypes, with clusters of related isolates in one or more centres. Analysis of the STs against the MLST database revealed several epidemic clonal complexes, such as those with central genotypes ST74 (including ST78) or ST114 (including ST66). ESC resistance was equally related to overexpression of the AmpC cephalosporinase and to ESBL production. Among ESBL producers some spreading subclones were identified, including specific ST66, ST78 and ST114 pulsotypes, associated with CTX-M-15 production. Several isolates produced carbapenemase VIM-1 or KPC-2. CONCLUSIONS: Together with the information available in the MLST database, our results suggest that, like Escherichia coli and Klebsiella pneumoniae, E. cloacae harbours clonal lineages of increased epidemic potential that may be associated with resistance spread.

Survey of metallo-ß-lactamase-producing Enterobacteriaceae colonizing patients in European ICUs and rehabilitation units, 2008-11.

OBJECTIVES: The objective of this study was to perform a multinational survey of patients' colonization by metallo-ß-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. METHODS: Patients in 18 hospital units across Europe and Israel (n = 17 945) were screened between mid-2008 and mid-2011. MBL-producing isolates were typed by PFGE and MLST. MBL genes were amplified and sequenced within their integrons. Plasmids with MBL genes were analysed by nuclease S1 plus hybridization profiling, mating and transformation assays, and by PCR-based replicon typing. RESULTS: Ninety-one patients in nine centres (six countries), including 62 patients in two Greek ICUs, carried 94 non-duplicate MBL-producing organisms. Klebsiella pneumoniae isolates from Greece dominated (n = 57) and belonged mainly to ST147, ST36 and ST383. All but one of the isolates expressed VIM-1-type MBLs. Isolates of Greek origins produced five enzymes, including new VIM-39, encoded by class 1 integrons of four types. In-e541-like elements prevailed, comprising six variants located on IncR, IncFIIK, IncR + FIIK, IncR + A/C or non-typeable plasmids. The other group were new In4873 and In4863, being the first In416-like elements identified in Greece, which were present on IncA/C or non-typeable plasmids. Isolates from other countries produced only VIM-1 and the major integron was In916, identified in 16 organisms from France, Italy and Spain. In916 was carried by four plasmid types, including IncA/C, IncFIIK and IncHI2. Other integrons included a new element, In3103, in Spain and In110 identified only in Latvia. CONCLUSIONS: This study provided fully comparable data on the occurrence and molecular characteristics of VIM-producing Enterobacteriaceae in a group of hospital units across Europe, documenting recent changes in their epidemiology.

Phylogenetic lineages, clones and ß-lactamases in an international collection of Klebsiella oxytoca isolates non-susceptible to expanded-spectrum cephalosporins.

OBJECTIVES: The objective of this study was to examine Klebsiella oxytoca clonal and phylogenetic diversity, based on an international collection of carriage isolates non-susceptible to expanded-spectrum cephalosporins (ESCs). METHODS: The study material comprised 68 rectal carriage K. oxytoca isolates non-susceptible to ESCs recovered in 2008-11 from patients in 14 hospitals across Europe and Israel. ESC resistance was tested phenotypically; genes encoding ESBLs, AmpC cephalosporinases and carbapenemases were amplified and sequenced. The isolates were typed by PFGE and MLST, followed by sequencing of blaOXY genes. RESULTS: MLST and PFGE distinguished 34 STs and 47 pulsotypes among the isolates, respectively. Six STs were split into several pulsotypes each. Five STs were more prevalent (n = 2-9) and occurred in several countries each, including ST2, ST9 and ST141, which belong to a growing international clonal complex (CC), CC2. Four phylogenetic lineages were distinguished, each with another type of chromosomal OXY-type ß-lactamase. Three of these, with OXY-1/-5, OXY-2 types and OXY-4, corresponded to previously described phylogroups KoI, KoII and KoIV, respectively. A single isolate from Israel represented a distinct lineage with a newly defined OXY-7 type. The phylogroups showed interesting differences in mechanisms of ESC resistance; KoI strains rarely overexpressed the OXY enzymes but commonly produced ESBLs, whereas KoII strains often were OXY hyperproducers and carried ESBLs much less frequently. AmpCs (DHA-1) and carbapenemases (VIM-1) occurred sporadically. CONCLUSIONS: The study confirmed the high genetic diversity of the collection of K. oxytoca ESC-non-susceptible isolates, composed of phylogroups with distinct types of OXY-type ß-lactamases, and revealed some STs of broad geographical distribution.

Beyond the Diketopiperazine Family with Alternatively Bridged Brevianamide F Analogues.

A method for the preparation of 3,5-bridged piperazin-2-ones from a tryptophan-proline-based diketopiperazine is described using diphosgene to induce the ring closure. Density functional theory calculations were conducted to study the mechanism of this C-C bond formation. Several derivatives of the thus obtained α-chloroamine were synthesized by substitution of the chlorine atom using a range of O-, N-, S-, and C-nucleophiles. This novel class of brevianamide F analogues possess interesting breast cancer resistance protein inhibitory activity.

Analytic laboratory performance of a point of care urine culture kit for diagnosis and antibiotic susceptibility testing.

Currently available point-of-care (POC) diagnostic tests for managing urinary tract infections (UTIs) in general practice are limited by poor performance characteristics, and laboratory culture generally provides results only after a few days. This laboratory evaluation compared the analytic performance of the POC UK Flexicult(™) (Statens Serum Institut) (SSI) urinary kit for quantification, identification and antibiotic susceptibility testing and routine UK National Health Service (NHS) urine processing to an advanced urine culture method. Two hundred urine samples routinely submitted to the Public Health Wales Microbiology Laboratory were divided and: (1) analysed by routine NHS microbiological tests as per local laboratory standard operating procedures, (2) inoculated onto the UK Flexicult(™) SSI urinary kit and (3) spiral plated onto Colorex Orientation UTI medium (E&O Laboratories Ltd). The results were evaluated between the NHS and Flexicult(™ )methods, and discordant results were compared to the spiral plating method. The UK Flexicult(™) SSI urinary kit was compared to routine NHS culture for identification of a pure or predominant uropathogen at ≥ 10(5) cfu/mL, with a positive discordancy rate of 13.5% and a negative discordancy rate of 3%. The sensitivity and specificity were 86.7% [95% confidence interval (CI) 73.8-93.7] and 82.6% (95% CI 75.8-87.7), respectively. The UK Flexicult(™) SSI urinary kit was comparable to routine NHS urine processing in identifying microbiologically positive UTIs in this laboratory evaluation. However, the number of false-positive samples could lead to over-prescribing of antibiotics in clinical practice. The Flexicult(™) SSI kit could be useful as a POC test for UTIs in primary care but further pragmatic evaluations are necessary.

Assessing the risk of measles resurgence in a highly vaccinated population: Belgium anno 2013.

Despite long-standing two-dose measles-mumps-rubella (MMR) vaccination, measles outbreaks still occur in highly vaccinated European populations. For instance, large measles outbreaks occurred in France (2008­13), the United Kingdom (2012­13) and the Netherlands (2012). Based on a multicohort model approach, using spatial serological survey data, MMR vaccination coverage data and data on social contacts, we found effective reproduction numbers significantly higher than 1 for measles in Belgium. This indicates that at one of the expected re-introductions, a measles outbreak is likely to spread, especially when it occurs during school term. The predicted average effective reproduction number increased over a 30-year time span from 1.3 to 2.2 and from 1.9 to 3.2 for basic reproduction numbers of 12 and 18, respectively. The expected relative measles incidence was highest in infants under one year of age, in adolescents and young adults. In conclusion, gradually increasing proportions of susceptible adolescents and young adults provide through their highly active social life an avenue for measles to resurge in large outbreaks upon re-introduction in Belgium, especially during school terms. Infants form an important vulnerable group during future measles outbreaks.

Determinants of between-country differences in ambulatory antibiotic use and antibiotic resistance in Europe: a longitudinal observational study.

OBJECTIVES: To identify key determinants explaining country-year variations in antibiotic use and resistance. METHODS: Ambulatory antibiotic use data [in defined daily doses per 1000 inhabitants per day (DIDs)] for 19 European countries from 1999 to 2007 were collected, along with 181 variables describing countries in terms of their agriculture, culture, demography, disease burden, education, healthcare organization and socioeconomics. After assessing data availability, overlap and relevance, multiple imputation generalized estimating equations were applied with a stepwise selection procedure to select significant determinants of global antibiotic use (expressed in DIDs), relative use of subgroups (amoxicillin and co-amoxiclav) and resistance of Escherichia coli and Streptococcus pneumoniae. RESULTS: Relative humidity, healthcare expenditure proportional to gross domestic product, feelings of distrust, proportion of population aged >65 years and availability of treatment guidelines were associated with higher total antibiotic use expressed in DIDs. Restrictions on marketing activities towards prescribers, population density, number of antibiotics, educational attainment and degree of atheism were associated with a lower number of total DIDs used. Relative prescribing of amoxicillin and co-amoxiclav was mainly determined by healthcare system choices [e.g. general practitioner (GP) registration and restricted marketing]. Specific antibiotic use was found to be a significant determinant of resistance for some but not all drug/organism combinations. Incentives to stimulate GP gatekeeping were associated with lower levels of resistance, and life expectancy at age 65+ and atheism were associated with more resistance. CONCLUSIONS: Myriad factors influence antibiotic use and resistance at the country level and an important part of these can be modified by policy choices.

High rates of intestinal colonisation with fluoroquinolone-resistant ESBL-harbouring Enterobacteriaceae in hospitalised patients with antibiotic-associated diarrhoea.

The purposes of this study were to investigate the intestinal carriage of extended-spectrum ß-lactamase-harbouring Enterobacteriaceae (ESBL-EN) and associated fluoroquinolone resistance (FQ-R) in 120 hospitalised patients with antibiotic-associated diarrhoea, and to investigate a correlation between Clostridium difficile (C. difficile) infection and intestinal colonisation with ESBL-EN in these patients. Stool samples were screened for C. difficile infection by toxin A/B enzyme-linked immunosorbent assay (ELISA) and for the presence of enterobacterial isolates producing ß-lactamases by plating on ß-lactamase screening (BLSE) agar. Recovered isolates were confirmed pheno- and genotypically for the presence of ESBL genes (bla CTX-M, bla TEM, bla SHV) by the double-disc synergy test and polymerase chain reaction (PCR) sequencing, and tested for the presence of topoisomerase mutations (gyrA, parC) and plasmid-mediated quinolone resistance (PMQR) determinants [qnrA, qnrB, qnrS, qepA, aac(6')-Ib-cr] by PCR sequencing. ESBL-EN were detected in 44/120 (37 %) stool samples. C. difficile-infected patients showed a significantly higher frequency of intestinal colonisation with ESBL-EN compared to C. difficile non-infected patients (62 % vs. 31 %, p = 0.008). Of the 73 ESBL-EN recovered, 46 (63 %) showed high-level FQ-R [ciprofloxacin minimum inhibitory concentration (MIC) ≥32 mg/L]. The largest group consisted of 21 bla CTX-M-15-harbouring Enterobacteriaceae (ciprofloxacin MIC ≥64 mg/L) with multiple topoisomerase mutations in gyrA and parC, in combination with co-carriage of aac(6')-Ib-cr. Most of them were Escherichia coli isolates belonging to sequence types ST131 and ST410. We found remarkably high rates of intestinal colonisation with high-level FQ-R ESBL-EN in hospitalised patients with antibiotic-associated diarrhoea, especially among C. difficile-infected patients. These data underscore the need for stringent infection control to contain this potentially infectious and multidrug-resistant reservoir.

European Antibiotic Awareness Day: a five-year perspective of Europe-wide actions to promote prudent use of antibiotics.

Following the European Union (EU) Council Recommendation on prudent use of antimicrobial agents in human medicine in 2001, and the success of national campaigns, i.e. Belgium and France, the European Centre for Disease Prevention and Control (ECDC) decided to establish the European Antibiotic Awareness Day (EAAD) on 18 November as platform to support national campaigns across Europe. This article provides an overview of EAAD tools, materials, and activities developed during the first five years. It shows that EAAD has been successful due to good cooperation between ECDC and national institutions, strong political and stakeholder support and evidence-based development of campaign materials. EAAD has provided a platform for pre-existing national campaigns and encouraged similar campaigns to develop where neither political support had been secured, nor financial support had been available. As a result, participating countries have continuously expressed strong support for ECDC to continue its work on EAAD. This has been endorsed by a steadily increasing number of countries participating and the growing interest of varied professional and stakeholder organisations. We conclude that EAAD should continue to act as catalyst for discussion and as mechanism to raise awareness of the public and prescribers about prudent use of antibiotics.

Preparedness for admission of patients with suspected Ebola virus disease in European hospitals: a survey, August-September 2014.

In response to the Ebola virus disease (EVD) outbreak in West Africa, the World Health Organization has advised all nations to prepare for the detection, investigation and management of confirmed and suspected EVD cases in order to prevent further spread through international travel. To gain insights into the state of preparedness of European hospitals, an electronic survey was circulated in August­September 2014 to 984 medical professionals representing 736 hospitals in 40 countries. The survey addressed the willingness and capacity to admit patients with suspected EVD as well as specific preparedness activities in response to the current Ebola crisis. Evaluable responses were received from representatives of 254 (32%) hospitals in 38 countries, mostly tertiary care centres, of which 46% indicated that they would admit patients with suspected EVD. Patient transfer agreements were in place for the majority of hospitals that would not admit patients. Compared with non-admitting hospitals, admitting hospitals were more frequently engaged in various preparedness activities and more often contained basic infrastructural characteristics such as admission rooms and laboratories considered important for infection control, but some gaps and concerns were also identified. The results of this survey help to provide direction towards further preparedness activities and prioritisation thereof.

Long-term effects of ciprofloxacin treatment on the gastrointestinal and oropharyngeal microbiome are more pronounced after longer antibiotic courses.

BACKGROUND: Urinary tract infections (UTIs) are one of the main reasons for antibiotic prescriptions in primary care. Recent studies demonstrate similar clinical outcomes with short vs. long antibiotics courses. The aim of this study was to investigate the differential collateral effect of ciprofloxacin treatment duration on the gastrointestinal and oropharyngeal microbiome in patients presenting with uncomplicated UTI to primary care practices in Switzerland, Belgium and Poland. METHODS: Stool and oropharyngeal samples were obtained from 36 treated patients and 14 controls at the beginning of antibiotic therapy, end of therapy and one month after the end of therapy. Samples underwent shotgun metagenomics. RESULTS: At the end of therapy, patients treated with both short (≤7 days) and long (>7 days) ciprofloxacin courses showed similar changes in the gastrointestinal microbiome compared to non-treated controls. After one month, most changes in patients receiving short courses were reversed; however, long courses led to increased abundance of the genera Roseburia, Faecalicatena and Escherichia. Changes in the oropharynx were minor and reversed to baseline levels within one month. Ciprofloxacin resistance encoding mutations in gyrA/B and parC/E reads were observed in both short and long treatment groups but decreased to baseline levels after one month. An increased abundance of resistance genes was observed in the gastrointestinal microbiome after longer treatment, and correlated to increased prevalence of aminoglycoside, ß-lactam, sulphonamide, and tetracycline resistance genes. CONCLUSION: Collateral effects on the gastrointestinal community, including an increased prevalence of antimicrobial resistance genes, persists for up to at least one month following longer ciprofloxacin therapy. These data support the use of shorter antimicrobial treatment duration.