[Show abstract][Hide abstract] ABSTRACT: Evidence shows a high rate of unnecessary antibiotic prescriptions in primary care in Europe and the United States. Given the costs of widespread use and associated antibiotic resistance, reducing inappropriate use is a public health priority.
[Show abstract][Hide abstract] ABSTRACT: Background
Whole genome (optical) mapping (WGM), a state-of-the-art mapping technology based on the generation of high resolution restriction maps, has so far been used for typing clinical outbreak strains and for mapping de novo sequence contigs in genome sequencing projects. We employed WGM to assess the genomic stability of previously sequenced Staphylococcus aureus strains that are commonly used in laboratories as reference standards.
S. aureus strains (n = 12) were mapped on the ArgusTM Optical Mapping System (Opgen Inc, Gaithersburg, USA). Assembly of NcoI-restricted DNA molecules, visualization, and editing of whole genome maps was performed employing MapManager and MapSolver softwares (Opgen Inc). In silico whole genome NcoI-restricted maps were also generated from available sequence data, and compared to the laboratory-generated maps. Strains showing differences between the two maps were resequenced using Nextera XT DNA Sample Preparation Kit and Miseq Reagent Kit V2 (MiSeq, Illumina) and de novo assembled into sequence contigs using the Velvet assembly tool. Sequence data were correlated with corresponding whole genome maps to perform contig mapping and genome assembly using MapSolver. Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data. Resequencing of the USA300_FPR3757 identified the deleted fragment to be a 13kbp-long integrative conjugative element ICE6013.
Frequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains. WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.
[Show abstract][Hide abstract] ABSTRACT: Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTI), however, clinically significant resistance in E. coli is as yet uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p, ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole genome sequencing was performed on both susceptible isolates and on selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a, ST2747-a) and anaerobically (ST540-an, ST2747-an). ST540-a/-an and ST2747-a (aerobic MIC >64 μg/ml) harbored mutations in known nitrofurantoin resistance determinants, nfsA and/or nfsB that encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC 32 μg/ml) and exhibited remarkable growth deficits, however, did not harbor nfsA/B mutations. We identified a 12-nucleotide deletion in ribE encoding lumazine synthase, an essential enzyme involved in the biosynthesis of FMN, an important cofactor of NfsA/B. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli (NRCI-1 to -6) isolated from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB, but not in ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin-susceptibility (MIC range 16-48 μg/ml) also did not identify any relevant mutations. NRCI-1, -2, -5 exhibited upto four-fold higher anaerobic MICs compared to the in vitro mutants, presumably because of additional mutations in oxygen-sensitive nitroreductases.
Antimicrobial Agents and Chemotherapy 09/2014; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: De novo genome assembly can be challenging due to inherent properties of the reads, even when using current state-of-the-art assembly tools based on de Bruijn graphs. Often users are not bio-informaticians and, in a black box approach, utilise assembly parameters such as contig length and N50 to generate whole genome sequences, potentially resulting in mis-assemblies.
[Show abstract][Hide abstract] ABSTRACT: Rhinovirus infections occur frequently throughout life and have been reported in about one-third of asymptomatic cases. The clinical significance of sequential rhinovirus infections remains unclear. To determine the incidence and clinical relevance of sequential rhinovirus detections, nasopharyngeal samples from 2485 adults with acute cough/lower respiratory illness were analysed. Patients were enrolled prospectively by general practitioners from 12 European Union countries during three consecutive years (2007-2010). Nasopharyngeal samples were collected at the initial general practitioner consultation and 28 days thereafter and symptom scores were recorded by patients over that period. Rhinovirus RNA was detected in 444 (18%) out of 2485 visit one samples and in 110 (4.4%) out of 2485 visit two respiratory samples. 21 (5%) of the 444 patients had both samples positive for rhinovirus. Genotyping of both virus detections was successful for 17 (81%) out of 21 of these patients. Prolonged rhinovirus shedding occurred in six (35%) out of 21 and re-infection with a different rhinovirus in 11 (65%) out of 21. Rhinovirus re-infections were significantly associated with chronic obstructive pulmonary disease (p=0.04) and asthma (p=0.02) and appeared to be more severe than prolonged infections. Our findings indicate that in immunocompetent adults rhinovirus re-infections are more common than prolonged infections, and chronic airway comorbidities might predispose to more frequent rhinovirus re-infections.
The European respiratory journal. 07/2014; 44(1):169-77.
[Show abstract][Hide abstract] ABSTRACT: Infection with human cytomegalovirus (CMV) is a significant cause of morbidity and mortality in solid organ and hematopoietic stem cell transplant (HSCT) recipients.
[Show abstract][Hide abstract] ABSTRACT: While multiple rodent pre-clinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immune-competent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically-derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neo-angiogenesis, microglia/macrophage recruitment, astrogliosis and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the sub-ventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses, and will eventually allow for successful manipulation of this intervention.
[Show abstract][Hide abstract] ABSTRACT: Canuti and Martin Deijs have contributed equally to this work. Previously unknown or unexpected pathogens may be responsible for that proportion of respiratory diseases in which a causative agent cannot be identified. The application of broad-spectrum, sequence independent virus discovery techniques may be useful to reduce this proportion and widen our knowledge about respiratory pathogens. Thanks to the availability of high-throughput sequencing (HTS) technology, it became today possible to detect viruses which are present at a very low load, but the clinical relevance of those viruses must be investigated. In this study we used VIDISCA-454, a restriction enzyme based virus discovery method that utilizes Roche 454 HTS system, on a nasal swab collected from a subject with respiratory complaints. A γ-papillomavirus was detected (complete genome: 7142 bp) and its role in disease was investigated. Respiratory samples collected both during the acute phase of the illness and 2 weeks after full recovery contained the virus. The patient presented antibodies directed against the virus but there was no difference between IgG levels in blood samples collected during the acute phase and 2 weeks after full recovery. We therefore concluded that the detected γ-papillomavirus is unlikely to be the causative agent of the respiratory complaints and its presence in the nose of the patient is not related to the disease. Although HTS based virus discovery techniques proved their great potential as a tool to clarify the etiology of some infectious diseases, the obtained information must be subjected to cautious interpretations. This study underlines the crucial importance of performing careful investigations on viruses identified when applying sensitive virus discovery techniques, since the mere identification of a virus and its presence in a clinical sample are not satisfactory proofs to establish a causative link with a disease.
Frontiers in Microbiology 07/2014; 5:374. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purposes of this study were to investigate the intestinal carriage of extended-spectrum β-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 β-lactamases by plating on β-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.
[Show abstract][Hide abstract] ABSTRACT: Although implantation of cellular material in the central nervous system (CNS) is a key direction in CNS regenerative medicine, this approach is currently limited by the occurrence of strong endogenous immune cell responses. In a model of mesenchymal stem cell (MSC) grafting in the CNS of immune-competent mice, we previously described that MSC grafts become highly surrounded and invaded by Iba1(+) myeloid cells (microglia and/or macrophages). Here, following grafting of blue fluorescent protein (BFP)-expressing MSC in the CNS of CX3CR1(+/-) and CX3CR1(-/-) mice, our results indicate: (1) that the observed inflammatory response is independent of the fractalkine signalling axis, and (2) that a significant spatial distribution of Iba1(+) inflammatory cells occurs, in which Iba1(+) CX3CR1(+) myeloid cells mainly surround the MSC graft and Iba1(+) CX3CR1(-) myeloid cells mainly invade the graft at 10 days post transplantation. Although Iba1(+) CX3CR1(+) myeloid cells are considered to be of resident microglial origin, Iba1(+) CX3CR1(-) myeloid cells are most likely of peripheral monocyte/macrophage origin. In order to confirm the latter, we performed MSC-BFP grafting experiments in the CNS of eGFP(+) bone marrow chimeric C57BL/6 mice. Analysis of MSC-BFP grafts in the CNS of these mice confirmed our observation that peripheral monocytes/macrophages invade the MSC graft and that resident microglia surround the MSC graft site. Furthermore, analysis of major histocompatibility complex class II (MHCII) expression revealed that mainly macrophages, but not microglia, express this M1 pro-inflammatory marker in the context of MSC grafting in the CNS. These results again highlight the complexity of cell implantation immunology in the CNS.Immunology and Cell Biology advance online publication, 1 July 2014; doi:10.1038/icb.2014.49.
Immunology and Cell Biology 07/2014; · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.
Journal of Cellular and Molecular Medicine 06/2014; · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background / Purpose:
To study the effects of a human papillomavirus (HPV) vaccine on a natural existing dendritic cell (DC) subset and the subsequent effects on natural killer (NK) cell activity.
The HPV vaccine is able to maturate the interleukin 15 (IL-15) dendritic cells. In addition, the vaccine is capable of unlocking the cytotoxic capacity of these IL-15 dendritic cells and increases the dendritic cell-mediated NK cell cytotoxicity.
12th Cancer Immunotherapy (CIMT) Annual Meeting 2014; 05/2014
[Show abstract][Hide abstract] ABSTRACT: Infections remain a leading cause of morbidity and mortality in patients with reduced immunity caused by haematological disease and chemotherapy-induced neutropenia. We evaluated the clinical and microbiological impact of discontinuing fluoroquinolone prophylaxis in these patients.
We analysed 154 admissions in three sequential periods of 8 months: long-standing use, discontinuation and reintroduction of prophylaxis. Clinical endpoints were occurrence of febrile neutropenia, bacteraemia, severe sepsis, septic shock, response to antibiotic therapy, total antibiotic consumption and duration of hospital stay. Microbiological analysis included bacterial isolates from stool and blood cultures and their resistance pattern.
No significant increase in serious infectious complications was seen with the discontinuation of prophylaxis. The overall incidence of bacteraemia did not change, but a higher proportion of bacterial isolates were Gram-negative (22.2% versus 5.9% & 8.6%; p=0.030), more often multi-susceptible (50% versus 0%) and less fluoroquinolone-resistant (10% versus 100%). Screening of stools showed a higher prevalence of organisms in the discontinuation period (86.7% versus 37.3% & 55.2%; p=<0.001), but they were more frequently multi-susceptible (53.8% versus 10.5% & 6.3%; p=<0.001). After discontinuation of prophylaxis, fluoroquinolone resistance decreased rapidly from 73.7% to 7.7%, in association with a significant decrease in extended spectrum beta-lactamase (ESBL)-producing isolates from 42.1% to 10.3%. Resistance figures immediately returned to pre-discontinuation values after reinstitution of prophylaxis.
No clinically relevant short-term drawbacks were observed with the discontinuation of fluoroquinolone prophylaxis in patients with chemotherapy-induced prolonged profound neutropenia, which led to a significant decrease in fluoroquinolone resistance as well as occurrence of ESBL-producing isolates. This article is protected by copyright. All rights reserved.
European Journal Of Haematology 04/2014; · 2.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are no reliable data on antibiotic use in non-European Union (EU) southern and eastern European countries and newly independent states. We aimed to collect valid, representative, comparable data on systemic antimicrobial use in these non-EU countries of the WHO European region.
Validated 2011 total national wholesale antibiotic-use data of six southern and eastern European countries and regions and seven newly independent states were analysed in accordance with the WHO anatomical therapeutic chemical (ATC)/defined daily doses (DDD) method and expressed in DDD/1000 inhabitants per day (DID).
Total (outpatients and hospital care) antibiotic use ranged from 15·3 DID for Armenia to 42·3 DID for Turkey. Co-amoxiclav was mainly used in Georgia (42·9% of total antibiotic use) and Turkey (30·7%). Newly independent states used substantial quantities of ampicillin and amoxicillin (up to 55·9% of total antibiotic use in Azerbaijan). Montenegro and Serbia were the highest consumers of macrolides (15·8% and 19·5% of total antibiotic use, respectively), mainly azithromycin. Parenteral antibiotic treatment is common practice: 46·4% of total antibiotic use in Azerbaijan (mainly ampicillin; 5·3 DID) and 31·1% of total antibiotic use in Tajikistan (mainly ceftriaxone; 4·7 DID).
This study provides publicly available total antibiotic-use data for 13 non-EU countries and areas of the WHO European region. These data will raise awareness of inappropriate antibiotic use and stimulate policy makers to develop action plans. The established surveillance system provides a method to develop quality indicators of antibiotic use and to assess the effect of policy and regulatory actions.
Netherlands Ministry of Health, Welfare, and Sport, and EU.
The Lancet Infectious Diseases 03/2014; · 19.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To complement analyses of the linear trend and seasonal fluctuation of European outpatient antibiotic use expressed in defined daily doses (DDD) by analyses of data in packages, to assess the agreement between both measures and to study changes in the number of DDD per package over time.
Data on outpatient antibiotic use, aggregated at the level of the active substance (WHO version 2011) were collected from 2000 to 2007 for 31 countries and expressed in DDD and packages per 1000 inhabitants per day (DID and PID, respectively). Data expressed in DID and PID were analysed separately using non-linear mixed models while the agreement between these measurements was analysed through a joint non-linear mixed model. The change in DDD per package over time was studied with a linear mixed model.
Total outpatient antibiotic and penicillin use in Europe and their seasonal fluctuation significantly increased in DID, but not in PID. The use of combinations of penicillins significantly increased in DID and in PID. Broad-spectrum penicillin use did not increase significantly in DID and decreased significantly in PID. For all but one subgroup, country-specific deviations moved in the same direction whether measured in DID or PID. The correlations are not perfect. The DDD per package increased significantly over time for all but one subgroup.
Outpatient antibiotic use in Europe shows contrasting trends, depending on whether DID or PID is used as the measure. The increase of the DDD per package corroborates the recommendation to adopt PID to monitor outpatient antibiotic use in Europe.
Journal of Antimicrobial Chemotherapy 03/2014; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND Antibiotics are of limited overall clinical benefit for uncomplicated lower respiratory tract infection (LRTI) but there is uncertainty about their effectiveness for patients with features associated with higher levels of antibiotic prescribing. AIM To estimate the benefits and harms of antibiotics for acute LRTI among those producing coloured sputum, smokers, those with fever or prior comorbidities, and longer duration of prior illness. DESIGN AND SETTING Secondary analysis of a randomised controlled trial of antibiotic placebo for acute LRTI in primary care. METHOD Two thousand and sixty-one adults with acute LRTI, where pneumonia was not suspected clinically, were given amoxicillin or matching placebo. The duration of symptoms, rated moderately bad or worse (primary outcome), symptom severity on days 2-4 (0-6 scale), and the development of new or worsening symptoms were analysed in pre-specified subgroups of interest. Evidence of differential treatment effectiveness was assessed in prespecified subgroups by interaction terms. RESULTS No subgroups were identified that were significantly more likely to benefit from antibiotics in terms of symptom duration or the development of new or worsening symptoms. Those with a history of significant comorbidities experienced a significantly greater reduction in symptom severity between days 2 and 4 (interaction term -0.28, P = 0.003; estimated effect of antibiotics among those with a past history -0.28 [95% confidence interval = -0.44 to -0.11], P = 0.001), equivalent to three people in 10 rating symptoms as a slight rather than a moderately bad problem. For subgroups not specified in advance antibiotics provided a modest reduction in symptom severity for non-smokers and for those with short prior illness duration (<7 days), and a modest reduction in symptom duration for those with short prior illness duration. CONCLUSION There is no clear evidence of clinically meaningful benefit from antibiotics in the studied high-risk groups of patients presenting in general practice with uncomplicated LRTIs where prescribing is highest. Any possible benefit must be balanced against the side-effects and longer-term effects on antibiotic resistance.
British Journal of General Practice 02/2014; 64(619):e75-80. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The study aims were: 1) to define the prevalence of and risk factors for colonization by ESBL-producing Enterobacteriaceae (ESBL-Ent) among healthcare workers (HCWs) and family members (FMs) of ESBL-Ent-colonized patients in rehabilitation units (RUs) and 2) to compare ESBL-Ent isolates from these three groups. The study included 286 FMs of 194 ESBL-Ent-carrying patients identified in 5 RUs located in Israel, Italy, France and Spain. ESBL-Ent were detected by rectal swabs in 26 (9%) of 286 FMs screened. In multivariate analyses, older age of the FM, longer mean number of hours spent with the patient, being a daughter or a female spouse of a patient, and chronic lung disease of the patient were significantly associated with carriage in the FM. Escherichia coli was the most common organism (76%), followed by Klebsiella pneumoniae (19%). Isolates were typed by PFGE and MLST, and ESBLs were identified by PCR-sequencing. A comparison of paired species isolates from FMs and their respective patient showed that 17/23 strains were indistinguishable. ESBL-Ent was detected in 35 (3.5%, E. coli=34) of the 1001 HCWs screened. Feeding patients was associated with ESBL-Ent carriage of HCWs. Only 7 of 23 E. coli subclones cultured from HCWs were also represented among 376 patient-derived ESBL-producing E. coli isolates from the same RUs. In Spain, a higher proportion of HCW and FM were ESBL carriers than elsewhere (p<0.05). In conclusion, the molecular and epidemiological data suggest that FMs are at higher risk of ESBL-Ent acquisition from their relative patients than HCW workers. This article is protected by copyright. All rights reserved.
Clinical Microbiology and Infection 01/2014; · 4.58 Impact Factor